WO2016094873A2 - Receptor tyrosine kinase-like orphan receptor 1 binding proteins and related compositions and methods - Google Patents

Receptor tyrosine kinase-like orphan receptor 1 binding proteins and related compositions and methods Download PDF

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WO2016094873A2
WO2016094873A2 PCT/US2015/065395 US2015065395W WO2016094873A2 WO 2016094873 A2 WO2016094873 A2 WO 2016094873A2 US 2015065395 W US2015065395 W US 2015065395W WO 2016094873 A2 WO2016094873 A2 WO 2016094873A2
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seq
amino acid
ror1
acid sequence
set forth
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WO2016094873A3 (en
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John W. Blankenship
Lynda Misher
Philip Tan
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Emergent Product Development Seattle, Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure relates to molecules that specifically bind to receptor tyrosine kinase-like orphan receptor 1 (ROR1), which may have at least one humanized ROR1 - binding domain. These molecules are useful for the characterization or treatment of disorders characterized by expression of ROR1 , such as cancer.
  • a protein therapeutic binding to ROR1 may be a monospecific protein therapeutic or a multispecific protein therapeutic.
  • a multspecific protein therapeutic may bind both ROR1 -expressing cells and the T-cell receptor complex on T-cells to induce target-dependent T-cell cytotoxicity, activation and proliferation.
  • Receptor tyrosine kinase-like orphan receptor 1 is a glycosylated type I membrane protein normally expressed in early embryonic development and attenuated during fetal development. Normal non-embryonic and non-fetal tissues do not express R OR1 at detectable levels, except for hematogones, which are unusual CD5 precursor B cells. However, ROR1 surface expression has been reported to be upregulated in many hematologic and solid cancers. For example, ROR1 has been found to be expressed in chronic lymphocytic leukemia (CLL) and mantle cell leukemia (MCL) (Baskar et al., Clin. Cancer Res., 14: 396-404 (2008); Hudecek et al., Blood, 1 16: 4532-4541 (2010)).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell leukemia
  • ROR1 acts as a receptor for Wnt ligands and can activate non-canonical Wnt signaling, which can provide a survival advantage to cancer cells.
  • the disclosure encompasses a receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding domain that specifically binds to human ROR1 , wherein the ROR1 -binding polypeptide comprises (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12 or a sequence that differs from SEQ ID NO: 12 by at least one amino acid substitution; (b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14 or a sequence that differs from SEQ ID NO: 14 by at least one amino acid substitution; (c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or
  • the disclosure relates to a ROR1 - binding domain that binds an epitope near the junction of the Frizzled and Kringle domains of ROR1 (e.g., human ROR1).
  • this epitope is a discontinuous and/or conformational epitope.
  • a ROR1 -binding domain binds residues in stretches 259-273 (LCQTEYIFARSNPMI (residues 259-273 of SEQ ID NO: 128)) and 389- 403 (PACDSKDSKEKNKME (residues 389-403 of SEQ ID NO: 128)) of the ROR1 ectodomain.
  • the ROR1 -binding domain of a ROR1 -binding polypeptide comprises (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 ,
  • the LCDR1 has an amino acid sequence set forth in SEQ ID NO:47 or a sequence that differs from SEQ ID NO:47 by at least one amino acid substitution
  • the LCDR2 has an amino acid sequence set forth in SEQ ID NO:49 or a sequence that differs from SEQ ID NO:49 by at least one amino acid substitution
  • the LCDR3 has an amino acid sequence set forth in SEQ ID NO:51 , or a sequence that differs from SEQ ID NO:51 by at least one amino acid substitution
  • the HCDR1 has an amino acid sequence set forth in SEQ ID NO:41 or a sequence that differs from SEQ ID NO:41 by at least one amino acid substitution
  • the HCDR2 has an amino acid sequence set forth in SEQ ID NO:43 or a sequence that differs from SEQ ID NO:43 by at least one amino acid substitution
  • the HCDR3 has an amino acid sequence set forth in SEQ ID NO:45 or
  • the disclosure relates to a ROR1 -binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12; (b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14; (c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268; (d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:266; (e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:8; and (f) the HCDR3 has an amino acid sequence set forth
  • the at least one amino acid substitution may be a conservative or a non-conservative amino acid substitution.
  • an LCDR1 , LCDR2, LCDR3, HCDR1 , HCDR2, and/or HCDR3 differs from a recited sequence by 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • a CDR of the present disclosure contains about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g. , conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared to the CDR sequence of a known monoclonal antibody.
  • the disclosure encompasses a ROR1 -binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; (ii) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, S
  • a ROR1 -binding domain comprises (i) an immunoglobulin light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; (ii) an immunoglobulin heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252; (iii) the immunoglobulin light chain comprises an amino acid sequence set forth in SEQ ID NO:333 or SEQ ID NO:337; or (iv) the immunoglobulin light chain comprises an amino acid sequence set forth in SEQ ID NO:335 or SEQ ID NO:339.
  • the disclosure relates to a ROR1 -binding domain wherein (i) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:23 and the
  • immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27;
  • the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27;
  • the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set
  • the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:210; (viii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid
  • immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232; (xii) the
  • immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232; (xiii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the
  • the disclosure encompasses a ROR1 -binding domain wherein (i) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27; (ii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27; (iii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59; (iv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 ; (v) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in
  • immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232; (xii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232; (xiii) the
  • immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:244; (xiv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:250 and the
  • immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:252; (xv) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:333 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:335; or (xvi) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:337 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:339.
  • a ROR1-binding domain is humanized.
  • a ROR1- binding domain is a single chain variable fragment (scFv).
  • the light chain variable region of said scFv may be carboxy-terminal or amino-terminal to the heavy chain variable region of said scFv.
  • a ROR1 -binding domain specifically binds to human ROR1 and comprises SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69.
  • the disclosure encompasses a ROR1 - binding polypeptide comprising a ROR1 -binding domain as described herein and an immunoglobulin constant region.
  • a ROR1 -binding polypeptide may specifically bind to human ROR1 and comprise SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91 .
  • a ROR1 -binding domain binds to human ROR1 with a dissociation constant that is lower than the dissociation constant of a ROR1 -binding domain comprising SEQ ID NO:73.
  • Some ROR1 -binding domains have a reduced isoelectric point compared to the isoelectric point of a ROR1 -binding domain comprising an immunoglobulin light chain variable region of SEQ ID NO:220 and an immunoglobulin heavy chain variable region of SEQ ID NO:232.
  • a ROR1 -binding domain having a lower dissociation constant and/or a reduced isolectric point comprises one or more CDR amino acid sequences set forth in Table 3.
  • a ROR1 -binding domain (or a ROR1 -binding polypeptide comprising this domain) is conjugated to a drug or a toxin.
  • an anti-ROR1 x anti-CD3 molecule as described herein is conjugated to a drug or a toxin.
  • ROR248, ROR246, ROR252, ROR250, or ROR243 is conjugated to a drug or a toxin.
  • the disclosure encompasses an isolated nucleic acid molecule encoding a ROR1 - binding domain described herein or a portion of said ROR1 -binding domain.
  • this isolated nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, S
  • the disclosure further encompasses a ROR1 -binding polypeptide comprising: (i) a ROR1 -binding domain and (ii) a second binding domain.
  • a ROR1 - binding polypeptide comprises, in order from amino-terminus to carboxyl-terminus or in order from carboxyl-terminus to amino-terminus, (i) a ROR1 -binding domain, (ii) a hinge region and (iii) an immunoglobulin constant region.
  • a ROR1 -binding polypeptide comprises, in order from amino-terminus to carboxyl-terminus, (i) a ROR1 - binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl- terminus linker, and (v) a second binding domain.
  • a ROR1 -binding polypeptide comprises, in order from carboxyl-terminus to amino-terminus, (i) a ROR1 - binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) an amino- terminus linker, and (v) a second binding domain.
  • the first and/or the second binding domain is an scFv.
  • carboxyl-terminus and amino- terminus linkers include flexible linkers comprising glycine-serine (e.g., (Gly 4 Ser)) repeats and linkers derived from (i) a stalk region of a type II C lectin or (ii) an immunoglobulin hinge region.
  • a carboxyl-terminus linker (or an amino-terminus linker) comprises or consists of SEQ ID NO:265, SEQ ID NO:301 , SEQ ID NO:302, or SEQ ID NO:303.
  • the disclosure relates to a ROR1 -binding polypeptide (e.g., multispecific), wherein (i) the ROR1 -binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3; and (ii) the second binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3.
  • the ROR1 -binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3.
  • a ROR1 -binding polypeptide comprises, in order from amino-terminus to carboxyl-terminus or in order from carboxyl- terminus to amino-terminus, (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) a CD3-binding domain; wherein said ROR1 -binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 12; (b) the LCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 14; (c) the
  • the HCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:266;
  • the HCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:8; and
  • the HCDR3 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:267; and wherein said CD3-binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a ' ) the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1
  • a ROR1 -binding polypeptide comprises an immunoglobulin constant region that comprises immunoglobulin CH2 and CH3 domains of lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 or IgD.
  • an immunoglobulin constant region comprises a human lgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A, according to the EU numbering system.
  • a ROR1 -binding polypeptide of the disclosure does not exhibit or exhibits minimal antibody- dependent cell-mediated cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDC) activity.
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ROR248, ROR246, ROR252, ROR250, or ROR243 does not exhibit or exhibits minimal ADCC activity or CDC activity.
  • a ROR1 -binding polypeptide comprises a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex, including TCR alpha/beta chains.
  • the ROR1 - binding polypeptide may induce redirected T-cell cytotoxicity (RTCC).
  • RTCC T-cell cytotoxicity
  • a ROR1 -binding polypeptide that also binds a T-cell, CD3, CD3z or a TCR complex may induce T-cell activation, T-cell proliferation and/or T-cell-dependent lysis of ROR1 - expressing cells (e.g., tumor or cancer cells).
  • a ROR1 -binding polypeptide that also binds a T-cell, CD3, CD3z or a TCR complex does not induce or induces a minimally detectable cytokine release from said T cell.
  • the second binding domain of a ROR1 -binding polypeptide described herein competes for binding to CD3z with a monoclonal antibody selected from CRIS-7, HuM291 and I2C.
  • the second binding domain specifically binds CD3 and comprises an immunoglobulin light chain variable region and an
  • the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:260; or at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:261 ; and wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:259.
  • the second binding domain of a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) described herein is a humanized binding domain.
  • the second binding domain of a ROR1 -binding polypeptide (e.g., an anti- ROR1 x anti-CD3 molecule) described herein is a single-chain variable fragment (scFv).
  • the second binding domain of a ROR1 -binding polypeptide described herein comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region derived from a monoclonal antibody selected from CRIS-7, HuM291 and I2C.
  • the second binding domain of a ROR1 -binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 10, 1 1 1 and 1 12, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 272, 273 and 274, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 269, 270 and 271 , respectively.
  • the second binding domain of a ROR1 -binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 278, 279 and 280, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 275, 276 and 277, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 284, 285 and 286, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 281 , 282 and 283, respectively.
  • the second binding domain of a ROR1 - binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 290, 291 and
  • the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 287, 288 and 289, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 296, 297 and 298, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs:
  • the second binding domains comprising the CDR sequences recited in this paragraph are humanized.
  • a ROR1 -binding domain competes for binding to human ROR1 with a single chain variable fragment (scFv) having the amino acid sequence set forth in SEQ ID NO: 18, SEQ I D NO:21 or SEQ ID NO:53.
  • scFv single chain variable fragment
  • the ROR1 -binding domain of a ROR1 -binding polypeptide (e.g. , an anti-ROR1 x anti- CD3 molecule) described herein may be a humanized binding domain.
  • the ROR1 -binding domain of a ROR1 -binding polypeptide (e.g. , an anti-ROR1 x anti-CD3 molecule) described herein is a single chain variable fragment (scFv).
  • a ROR1 -binding domain that is an scFv comprises SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69.
  • the disclosure encompasses a ROR1 -binding polypeptide, wherein the light chain variable region of the ROR1 -binding domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:39, SEQ I D NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; and the heavy chain variable region of the ROR1 -binding domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:2; SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ I D NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252.
  • the disclosure relates to a ROR1 -binding polypeptide, wherein the light chain variable region of the ROR1 -binding domain comprises the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; and the heavy chain variable region of the ROR1 -binding domain comprises an amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252.
  • the ROR1 -binding domain may be an scFv and may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO:21 , SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 or SEQ ID NO:69.
  • a ROR1 -binding polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:71 , SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91 , SEQ ID NO:214, SEQ ID NO:218, or SEQ ID NO:222.
  • a ROR1 -binding polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:
  • a ROR1 -binding polypeptide may comprise an amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321
  • a ROR1 -binding polypeptide comprises in order from amino- terminus to carboxyl-terminus (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker, and (v) a second binding domain, wherein said ROR1 -binding polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:2
  • the disclosure relates to a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) that is a dimer of two identical polypeptides, wherein each polypeptide may be a ROR1 -binding polypeptide comprising the sequences disclosed herein.
  • the disclosure encompasses a dimer of two identical polypeptides, wherein each polypeptide in the dimer is ROR248, ROR246, ROR252, ROR250, or ROR243.
  • a ROR1 -binding polypeptide may further comprise an immunoglobulin heterodimerization domain.
  • the immunoglobulin heterodimerization domain may optionally comprise an immunoglobulin CH1 domain or an immunoglobulin CL domain.
  • a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) is a heterodimeric ROR1 -binding protein comprising (i) a first polypeptide chain comprising, in order from amino-terminus to carboxyl-terminus or from carboxyl-terminus to amino-terminus, (a) a ROR1 -binding domain that specifically binds human ROR1 , (b) a first hinge region, (c) a first immunoglobulin constant region, and (d) a first immunoglobulin heterodimerization domain; and (ii) a second polypeptide chain comprising, in order from amino-terminus to carboxyl-terminus or from carboxyl-terminus to amino-terminus, (a') a second hinge region, (b') a second immunoglobulin constant region, and (c') a second immunoglobulin heterodimerization domain that is
  • the first immunoglobulin heterodimerization domain may comprise an immunoglobulin CH1 domain and the second immunoglobulin heterodimerization domain may comprise an immunoglobulin CL domain, or the first immunoglobulin heterodimerization domain may comprise an immunoglobulin CL domain and the second immunoglobulin heterodimerization domain may comprise an immunoglobulin CH1 domain.
  • At least one of the first and second immunoglobulin constant regions may comprise immunoglobulin CH2 and CH3 domains of lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD or any combination thereof; an immunoglobulin CH3 domain of lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM or any combination thereof; or immunoglobulin CH3 and CH4 domains of IgE, IgM or a combination thereof.
  • the ROR1 -binding polypeptide heterodimer may comprise a second polypeptide chain that further comprises a second binding domain. The second binding domain may be amino-terminal to the second hinge region.
  • a heterodimer may comprise ROR248, ROR246, ROR252, ROR250, or ROR243.
  • a ROR1 -binding polypeptide is a bispecific single chain antibody molecule comprising a ROR1 binding domain and a CD3 binding domain, wherein the binding domains are arranged in the order VH ROR1 -VL ROR1 -VH CD3-VL CD3 or VL ROR1 -VH ROR1 -VH CD3-VL CD3 or VH CD3-VL CD3-VH ROR1 -VL ROR1 or VH CD3-VL CD3-VL ROR1-VH ROR1 or VH ROR1 -VL ROR1 -VL CD3-VH CD3 or VL ROR1 -VH ROR1 - VL CD3-VH CD3 or VL ROR1 -VH ROR1 - VL CD3-VH CD3 or VL ROR1 -VH ROR1 - VL CD3-VH CD3 or VL ROR1 -VH ROR1 -
  • these VH and VL regions are linked, such as in an scFv.
  • the scFvs are joined by a linker with or without an immunoglobulin CH1 CH2 region.
  • the two scFvs are joined as described in, for example, US20130295121 , US 20130129730, WO 2010/037836, WO 2004/106381 or WO 201 1/121 1 10, each of which is incorporated herein by reference in its entirety.
  • the disclosure encompasses an isolated nucleic acid molecule encoding a ROR1 - binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) described herein or a portion of said ROR1 -binding polypeptide.
  • a ROR1 - binding polypeptide e.g., an anti-ROR1 x anti-CD3 molecule
  • the isolated nucleic acid molecule may comprise a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106,
  • the disclosure relates to an expression vector comprising a nucleic acid segment encoding a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) described herein, wherein the nucleic acid segment is operatively linked to regulatory sequences suitable for expression of the nucleic acid segment in a host cell.
  • a ROR1 -binding polypeptide e.g., an anti-ROR1 x anti-CD3 molecule
  • the nucleic acid segment of the expression vector may comprise a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:
  • the disclosure encompasses an expression vector comprising first and second expression units, wherein the first and second expression units respectively comprise first and second nucleic acid segments encoding the first and second polypeptide chains of a heterodimeric ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule) described herein, and wherein the first and second nucleic acid segments are operably linked to regulatory sequences suitable for expression of the nucleic acid segments in a host cell.
  • a heterodimeric ROR1 -binding protein e.g., an anti-ROR1 x anti-CD3 molecule
  • the disclosure includes a recombinant host cell comprising an expression vector described herein.
  • the disclosure relates to a method for producing a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243), the method comprising culturing a recombinant host cell comprising an expression vector described herein under conditions whereby the nucleic acid segment of the vector is expressed, thereby producing the ROR1-binding polypeptide.
  • the method may further comprise recovering the ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein).
  • the disclosure encompasses a method for producing a heterodimeric ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule), the method comprising culturing a recombinant host cell comprising first and second expression units, wherein the first and second expression units respectively comprise first and second nucleic acid segments encoding the first and second polypeptide chains of a heterodimeric ROR1 -binding protein, wherein the first and second nucleic acid segments are operably linked to regulatory sequences suitable for expression of the nucleic acid segments in a host cell, and wherein said culturing is under conditions whereby the first and second nucleic acid segments are expressed and the encoded polypeptide chains are produced as the heterodimeric ROR1 - binding protein.
  • the method may further comprise recovering the heterodimeric ROR1 - binding protein (e.g., an anti-ROR1 x anti-CD3 molecule).
  • the disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a ROR1-binding polypeptide or ROR1 -binding protein described herein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243), and a pharmaceutically acceptable carrier, diluent, or excipient.
  • a pharmaceutical composition comprises a ROR1-binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) that is a homodimer or a heterodimer.
  • a “homodimer” may be a dimer formed from two identical polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243).
  • a pharmaceutical composition may be formulated in a dosage form selected from the group consisting of: an oral unit dosage form, an intravenous unit dosage form, an intranasal unit dosage form, a suppository unit dosage form, an intradermal unit dosage form, an intramuscular unit dosage form, an intraperitoneal unit dosage form, a
  • the pharmaceutical composition formulated as an oral unit dosage form may be selected from the group consisting of: tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained-release formulations, aerosols, and sprays.
  • the disclosure also relates to a method for inducing redirected T-cell cytotoxicity (RTCC) against a cell expressing ROR1 , the method comprising: contacting said ROR1 - expressing cell with a ROR1-binding polypeptide or ROR1 -binding protein (e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein, wherein the second binding domain specifically binds a T-cell, CD3, CD3£ or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; and wherein said contacting is under conditions whereby RTCC against the ROR1 - expressing cell is induced.
  • a ROR1-binding polypeptide or ROR1 -binding protein e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, R
  • the disclosure relates to a method for inducing redirected T-cell cytotoxicity (RTCC) against a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 - expressing cell with a ROR1-binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; wherein said contacting is under conditions whereby RTCC against the ROR1 -expressing cell is induced.
  • a ROR1-binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and a second binding domain that specifically binds a T-
  • the disclosure encompasses a method for inducing T-cell dependent lysis of a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide or ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein, wherein the second binding domain specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; and wherein said contacting is under conditions whereby T-cell dependent lysis of the ROR1 -expressing cell is induced.
  • a ROR1 -binding polypeptide or ROR1 -binding protein e.g., an anti-ROR1 x anti-CD3 molecule; for example R
  • the disclosure relates to a method for inducing T-cell dependent lysis of a cell expressing receptor tyrosine kinase- like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; wherein said contacting is under conditions whereby T-cell dependent lysis of the ROR1 -expressing cell is induced.
  • ROR1 -binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and a second binding domain that specifically binds a T-cell, CD3, CD
  • the disclosure encompasses a method for treating a disorder (e.g., cancer) in a subject, wherein said disorder is characterized by expression of ROR1 , the method comprising administering to the subject a therapeutically effective amount of a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein.
  • a ROR1 -binding polypeptide e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243
  • the disclosure relates to a method for treating a disorder in a subject, wherein said disorder is characterized by expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising administering to the subject a therapeutically effective amount of a ROR1 -binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR.
  • ROR1 receptor tyrosine kinase-like orphan receptor 1
  • the disclosure also relates to a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein for the manufacture of a medicament for treatment of a disorder (e.g., cancer) in a subject, wherein said disorder is characterized by expression of ROR1 .
  • a ROR1 -binding polypeptide e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243
  • the disclosure relates to a receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 for the manufacture of a medicament for treatment of a disorder in a subject, wherein said disorder is characterized by expression of ROR1 .
  • RORI receptor tyrosine kinase-like orphan receptor 1
  • the disclosure includes a ROR1 -binding polypeptide or ROR1 -binding protein (e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein for use in treating a disorder (e.g., cancer) in a subject, wherein said disorder is characterized by expression of ROR1 .
  • a ROR1 -binding polypeptide or ROR1 -binding protein e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243
  • the disclosure relates to a receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 for use in treating a disorder in a subject, wherein said disorder is characterized by expression of ROR1 .
  • RORI receptor tyrosine kinase-like orphan receptor 1
  • the cancer treated by the ROR1 -binding polypeptides may be breast cancer (e.g., triple negative breast cancer (TNBC)), pancreatic cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), acute
  • ALL lymphoblastic leukemia
  • melanoma adrenal cancer
  • bladder cancer prostate cancer
  • Figure 1 is a graph showing the results of a flow cytometry study measuring the binding of four humanized R12 molecules (ROR033, ROR034, ROR035 and ROR036), the chimeric parental R12 molecule ROR016 and the chimeric R1 1 molecule ROR015 to the ROR1 (+) cell line Kasumi-2.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table shows the EC 50 values obtained from the data in the graph.
  • Figure 2 is a graph showing the results of a flow cytometry study measuring the binding of two chimeric R1 1 molecules (ROR015, ROR063) to the ROR1 (+) cell line MDA-MB-231 .
  • PBS Phosphate buffered saline
  • MFI Mean fluorescence intensity
  • FIG. 1 is a graph showing the results of a flow cytometry study measuring the binding of the hemi-humanized R1 1 molecule ROR091 , the chimeric molecule ROR063 and the fully humanized molecule ROR101 to the ROR1 (+) cell line MDA- MB-231 .
  • PBS Phosphate buffered saline
  • MFI Mean fluorescence intensity
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 4A is a graph showing the results of a flow cytometry study measuring the binding of the parental R1 1 antibody ROR066 and the chimeric R1 1 molecule with the scFv in the VL-VH orientation (ROR063) to the ROR1 (+) cell line MDA-MB-231 .
  • MFI fluorescence intensity
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • Figure 4B is a graph showing the results of a flow cytometry study measuring the binding of the parental R12 antibody ROR069 and the chimeric R12 molecule ROR016 to the ROR1 (+) cell line MDA-MB-231 .
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • Figure 5 is a graph showing the results of a flow cytometry study measuring the binding of the chimeric bispecific R12 molecule ROR021 , the humanized bispecific R12 molecule ROR050, the chimeric bispecific R1 1 molecule ROR070 and the humanized bispecific R1 1 molecule ROR084 to the ROR1 (+) cell line Kasumi-2.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • Figure 6 is a graph showing the results of a chromium-51 release assay measuring the effectiveness of the humanized bispecific R12 molecule ROR050 and the parental chimeric bispecific R12 molecule ROR021 at inducing target-dependent T-cell cytotoxicity in 4 hours against MDA-MB-231 cells. Percent specific lysis relative to a total lysis control is shown on the y-axis. Concentration (pM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 7 is a graph showing the results of a chromium-51 release assay measuring the effectiveness of the the chimeric bispecific R1 1 molecule ROR070 and the chimeric bispecific R1 1 molecule ROR020 at inducing target-dependent T-cell cytotoxicity in 4 hours against MDA-MB-231 cells. Percent specific lysis relative to a total lysis control is shown on the y-axis. Concentration (pM) of the ROR1 -binding molecules is shown on the x- axis. The table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 8 is a graph showing the results of a flow cytometry study measuring the effectiveness of the bispecific R1 1 molecules ROR070 and ROR084 and the bispecific R12 molecules ROR021 and ROR050 at inducing target-dependent T-cell cytotoxicity in 18 hours against Kasumi-2 cells. Percentage of live target cells remaining after 18 hours is shown on the y-axis. Concentration (pM) of the ROR1 -binding molecules is shown on the x-axis.
  • Figure 9A is a graph showing the results of a flow cytometry study measuring the effectiveness of the bispecific R1 1 molecules ROR070 and ROR084 and the bispecific R12 molecules ROR021 and ROR050 at inducing CD4 + (CD8 " ) T-cell proliferation in the presence of Kasumi-2 cells. Percentage of dividing (proliferating) cells is shown on the y- axis. Concentration (pM) of the ROR1 -binding molecules is shown on the x-axis.
  • Figure 9B is a graph showing the results of a flow cytometry study measuring the effectiveness of the bispecific R1 1 molecules ROR070 and ROR084 and the bispecific R12 molecules ROR021 and ROR050 at inducing CD8 + T-cell proliferation in the presence of Kasumi-2 cells. Percentage of dividing (proliferating) cells is shown on the y-axis.
  • Figure 10 is a graph showing the results of a flow cytometry study measuring the binding of the chimeric molecule ROR063 and the fully humanized molecules ROR101 , ROR1 1 1 , and ROR1 12 to the ROR1 (+) cell line Kasumi-2.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table shows the EC 50 values obtained from the data in the graph.
  • Figure 11 is a graph showing the results of a flow cytometry study measuring the binding of the hemi-humanized molecule ROR091 and the fully humanized molecules ROR1 19, ROR1 1 1 , and ROR1 12 to the ROR1 (+) cell line Kasumi-2.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 12 is a graph showing the results of a study measuring the serum stability in human serum of anti-ROR1 x anti-CD3 bispecific molecules ROR133 and ROR193 over the number of days specified on the x-axis.
  • Figure 13 is a graph showing the results of a study measuring the ability of the ROR1 -directed bispecific molecules ROR182 and ROR192 to inhibit subcutaneous tumor outgrowth of MDA-MB-231 tumors co-implanted with human T cells in NOD/SCID mice.
  • the mean tumor volume (mm 3 ) is shown on the y-axis. Mice were treated with 3 of anti- ROR1 molecules on day 0, 4 and 8 post tumor challenge.
  • Figure 14 is a graph showing the results of a study measuring the effect of treatment with ROR1 -directed bispecific molecules ROR1 82 and ROR192 on body weight of
  • NOD/SCID mice co-implanted with MDA-MB-231 tumors and human T cells. Percentage of maximum body weight is shown on the y-axis. Mice were treated with 3 ⁇ g of anti-ROR1 molecules on day 0, 4 and 8 post tumor challenge.
  • Figure 15A is a graph (top panel) showing the results of a study measuring the ability of codon-optimized anti-ROR1 molecules ROR238, ROR242, ROR243, and ROR244 to induce redirected T-cell cytotoxicity (RTCC) of PC-3 cells which were stably transfected with GFP. Percentage specific mortality is shown on the y-axis. Concentration (pM) of the anti- ROR1 molecules is shown on the x-axis. The table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 15B is a graph (top panel) showing the results of a study measuring the ability of codon-optimized anti-ROR1 molecules ROR241 , ROR250, ROR251 , ROR252, and ROR253 to induce redirected T-cell cytotoxicity (RTCC) of PC-3 cells which were stably transfected with GFP. Percentage specific mortality is shown on the y-axis. Concentration (pM) of the anti-ROR1 molecules is shown on the x-axis. The table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 16A is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR241 , ROR250, ROR251 , ROR252, and ROR253) and the parental anti-ROR1 molecule ROR185 to the ROR1 (+) cell line Kasumi-2.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 16B is a graph (top panel) showing the results of a flow cytometry study measuring the measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR241 , ROR250, ROR251 , ROR252, and ROR253) and the parental anti-ROR1 molecule ROR185 to the CD3(+) cell line Jurkat-60.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 - binding molecules is shown on the x-axis.
  • the table (bottom panel) shows the EC 5 o values obtained from the data in the graph.
  • Figure 17A is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR241 , ROR238, ROR239, ROR242, ROR243, and ROR244) and the parental anti-ROR1 molecules ROR185 and ROR192 to the ROR1 (+) cell line Kasumi-2.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 17B is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR241 , ROR238, ROR239, ROR242, ROR243, and ROR244) and the parental anti-ROR1 molecules ROR185 and ROR192 to the CD3(+) cell line Jurkat-60.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 18A is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR239, ROR246, ROR247, ROR248, and ROR249) and the parental anti-ROR1 molecule ROR192 to the ROR1 (+) cell line Kasumi-2.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • Figure 18B is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR239, ROR246, ROR247, ROR248, and ROR249) and the parental anti-ROR1 molecule ROR192 to the CD3(+) cell line Jurkat-60.
  • Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
  • Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
  • the table (bottom panel) shows the EC 50 values obtained from the data in the graph.
  • the disclosure provides binding domains that specifically bind to receptor tyrosine kinase-like orphan receptor 1 (ROR1) and binding molecules (e.g. polypeptides and proteins) that specifically bind to ROR1 . These binding molecules may bind specifically to ROR1 and to another target.
  • ROR1 -binding molecule described herein binds to ROR1 more effectively than a parent (non-humanized) antibody used to construct the humanized molecule.
  • Administration of a therapeutically effective amount of a ROR1 -binding polypeptide or protein to a patient in need thereof is useful for treatment of certain disorders associated with the expression of ROR1 , including certain cancers.
  • a ROR1 -binding polypeptide or protein binds both a target cell expressing ROR1 and a T-cell, thereby "cross-linking" the target cell expressing ROR1 and the T-cell.
  • the binding of both domains to their targets elicits potent target-dependent redirected T-cell cytotoxicity (RTCC) (e.g., induces target-dependent T-cell cytotoxicity, T-cell activation and/or T-cell proliferation).
  • RTCC target-dependent redirected T-cell cytotoxicity
  • the ROR1 -binding therapeutics of the disclosure offer various advantages in treating patients, for example, effective binding to ROR1 , efficient induction of RTCC activity, reduced levels of cytokine release and/or a lower risk of adverse events (e.g., toxicity).
  • ROR1 -binding proteins bind to ROR1 more effectively in certain formats (e.g., scFv compared to parent antibody) and/or certain orientations (e.g., VL-VH compared to VH-VL), leading to higher potency and improved utility in treating disorders associated with expression of ROR1 .
  • a target cell expresses ROR1 at a higher level than a non-target cell (e.g., normal cell or non-cancerous cell in the same subject, organ, or tissue) expresses ROR1 .
  • a target cell expresses ROR1 while a non-target cell (e.g., normal cell or non-cancerous cell in the same subject, organ, or tissue) does not express ROR1 .
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated.
  • the use of the alternative e.g. , "or” should be understood to mean either one, both, or any combination thereof of the alternatives.
  • the terms “include” and “comprise” are used synonymously. In addition, it should be understood that the
  • polypeptides comprising the various combinations of the components (e.g. , domains or regions) and substituents described herein, are disclosed by the present application to the same extent as if each polypeptide was set forth individually. Thus, selection of particular components of individual polypeptides is within the scope of the present disclosure.
  • binding domain refers to the domain, region, portion, or site of a protein, polypeptide, oligopeptide, or peptide or antibody or binding domain derived from an antibody that possesses the ability to specifically recognize and bind to a target molecule, such as an antigen, ligand, receptor, substrate, or inhibitor (e.g. , ROR1 , CD3).
  • exemplary binding domains include single-chain antibody variable regions (e.g. , domain antibodies, sFv, scFv, scFab), receptor ectodomains, and ligands (e.g. , cytokines, chemokines).
  • the binding domain comprises or consists of an antigen binding site (e.g. , comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g. , human FRs optionally comprising one or more amino acid substitutions).
  • an antigen binding site e.g. , comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g. , human FRs optionally comprising one or more amino acid substitutions).
  • FRs alternative framework regions
  • assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, including Western blot, ELISA, phage display library screening, and BIACORE® interaction analysis.
  • a ROR1 -binding polypeptide can have a "first binding domain” and, optionally, a "second binding domain.”
  • the "first binding domain” is a ROR1 -binding domain and the format is an antibody or antibody-like protein or domain.
  • the second binding domain is a T-cell binding domain such as a scFv derived from a mouse monoclonal antibody (e.g. , CRIS-7) or phage display (e.g., I2C) that binds to a T-cell surface antigen (e.g. , CD3).
  • the second binding domain is a second ROR1 -binding domain.
  • the second binding domain is a binding domain other than a ROR1 -binding domain or a T-cell binding domain.
  • a binding domain or protein "specifically binds" a target if it binds the target with an affinity or K a (i.e. , an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 s M "1 , while not significantly binding other components present in a test sample. Binding domains can be classified as “high affinity” binding domains and "low affinity” binding domains.
  • “High affinity” binding domains refer to those binding domains with a K a of at least 10 7 M “1 , at least 10 8 M “1 , at least 10 9 M “1 , at least 10 10 M “1 , at least 10 11 M “1 , at least 10 12 M “1 , or at least 10 13 M “1 .
  • “Low affinity” binding domains refer to those binding domains with a K a of up to 10 7 M “1 , up to 10 6 M “1 , up to 10 s M "
  • affinity can be defined as an equilibrium dissociation constant (K d ) of a particular binding interaction with units of M (e.g. , 10 "5 M to 10 "13 M).
  • binding domain polypeptides and single chain polypeptides can be readily determined using conventional techniques (see, e.g. , Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283, 173, 5,468,614, or the equivalent).
  • CD3 is known in the art as a multi-protein complex of six chains (see, e.g. , Abbas and Lichtman, 2003; Janeway et al. , p. 172 and 178, 1999), which are subunits of the T-cell receptor complex.
  • the CD3 subunits of the T-cell receptor complex are a CD3Y chain, a CD35 chain, two CD3z chains, and a homodimer of ⁇ 3 chains.
  • the CD3Y, CD35, and CD3z chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3v, CD35, and CD3z chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T-cell receptor chains.
  • the intracellular tails of the CD3v, CD35, and CD3z chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each ⁇ 3 chain has three. It is believed the ITAMs are important for the signaling capacity of a TCR complex.
  • CD3 as used in the present disclosure can be from various animal species, including human, monkey, mouse, rat, or other mammals.
  • a "conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • a conservative substitution includes a leucine to serine substitution.
  • derivative refers to a modification of one or more amino acid residues of a peptide by chemical or biological means, either with or without an enzyme, e.g. , by glycosylation, alkylation, acylation, ester formation, or amide formation.
  • a polypeptide or amino acid sequence "derived from” a designated polypeptide or protein refers to the origin of the polypeptide.
  • the polypeptide or amino acid sequence which is derived from a particular sequence (sometimes referred to as the "starting" or “parent” or “parental” sequence) has an amino acid sequence that is essentially identical to the starting sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or at least 50-150 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
  • a binding domain can be derived from an antibody, e.g., a Fab, F(ab')2, Fab', scFv, single domain antibody (sdAb), etc.
  • a ROR1 -binding domain sequence is derived from a ROR1 -binding antibody or protein by means of a computer algorithm or in silico.
  • Polypeptides derived from another polypeptide can have one or more mutations relative to the starting polypeptide, e.g. , one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions.
  • the polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variations necessarily have less than 100% sequence identity or similarity with the starting polypeptide. In one embodiment, the variant will have an amino acid sequence from about 60% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.
  • the variant will have an amino acid sequence from about 75% to less thant 100%, from about 80% to less than 100%, from about 85% to less than 100%, from about 90% to less than 100%, from about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.
  • a position of an amino acid residue in a variable region of an immunoglobulin molecule is numbered according to the IMGT criteria (Brochet, X, et al, Nucl. Acids Res. (2008) 36, W503-508), and a position of an amino acid residue in a constant region of an immunoglobulin molecule is numbered according to EU nomenclature (Ward et al. , 1995 Therap. Immunol. 2:77-94).
  • the Kabat numbering convention Kabat numbering convention (Kabat, Sequences of Proteins of Immunological Interest, 5 th ed.
  • the term "dimer” refers to a biological entity that consists of two subunits associated with each other via one or more forms of intramolecular forces, including covalent bonds (e.g. , disulfide bonds) and other interactions (e.g. , electrostatic interactions, salt bridges, hydrogen bonding, and hydrophobic interactions), and is stable under appropriate conditions (e.g. , under physiological conditions, in an aqueous solution suitable for expressing, purifying, and/or storing recombinant proteins, or under conditions for non- denaturing and/or non-reducing electrophoresis).
  • covalent bonds e.g. , disulfide bonds
  • other interactions e.g. , electrostatic interactions, salt bridges, hydrogen bonding, and hydrophobic interactions
  • heterodimer or “heterodimeric protein,” as used herein, refers to a dimer formed from two different polypeptides.
  • a heterodimer may comprise an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243.
  • a heterodimer does not include an antibody formed from four polypeptides (i.e. , two light chains and two heavy chains).
  • a “homodimer” or “homodimeric protein,” as used herein, refers to a dimer formed from two identical polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243).
  • a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) comprises, in order from amino-terminus to carboxyl-terminus or in order from carboxyl-terminus to amino-terminus, (i) the ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) a second binding domain.
  • the ROR1 -binding domain comprises, in order from amino-terminus to carboxyl-terminus or in order from carboxyl-terminus to amino-terminus, (i) the ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) a second binding domain.
  • a "hinge region” or a “hinge” refers to a polypeptide region between a binding domain (e.g., a ROR1 -binding domain) and an immunoglobulin constant region.
  • a "linker” may refer to (1) a polypeptide region between V H and V L regions in a single-chain Fv (scFv) or (2) a polypeptide region between an immunoglobulin constant region and a second binding domain in a ROR1 -binding polypeptide comprising two binding domains (e.g., an anti-ROR1 x anti-CD3 molecule as described herein).
  • a polypeptide region between an immunoglobulin constant region and a second binding domain in a ROR1 -binding polypeptide comprising two binding domains may also be referred to as a "carboxyl-terminus linker" or an "amino-terminus linker.”
  • carboxyl-terminus and amino-terminus linkers include flexible linkers comprising glycine-serine repeats, and linkers derived from (a) an interdomain region of a
  • transmembrane protein e.g. , a type I transmembrane protein
  • a stalk region of a type II C-lectin or (c) an immunoglobulin hinge.
  • hinges and linkers are provided in Tables 1 and 2.
  • a "linker” provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions.
  • a linker is comprised of five to about 35 amino acids, for instance, about 15 to about 25 amino acids.
  • a wild-type immunoglobulin hinge region refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.
  • a wild type immunoglobulin hinge region sequence is human, and can comprise a human IgG hinge region.
  • an "altered wild-type immunoglobulin hinge region” or “altered immunoglobulin hinge region” refers to (a) a wild type immunoglobulin hinge region with up to 30% amino acid changes (e.g. , up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (b) a portion of a wild type immunoglobulin hinge region that has a length of about 5 amino acids (e.g.
  • amino acids up to about 120 amino acids (for instance, having a length of about 10 to about 40 amino acids or about 15 to about 30 amino acids or about 15 to about 20 amino acids or about 20 to about 25 amino acids), has up to about 30% amino acid changes (e.g. , up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1 % amino acid substitutions or deletions or a combination thereof), and has an IgG core hinge region as disclosed in WO 201 1/090762 (US
  • the term "humanized” refers to a process of making an antibody or immunoglobulin binding proteins and polypeptides derived from a non-human species (e.g. , mouse or rat) less immunogenic to humans, while still retaining antigen-binding properties of the original antibody, using genetic engineering techniques.
  • the binding domain(s) of an antibody or immunoglobulin binding proteins and polypeptides e.g. , light and heavy chain variable regions, Fab, scFv
  • Non-human binding domains can be humanized using techniques known as CDR grafting (Jones et al.
  • an "immunoglobulin dimerization domain” or “immunoglobulin heterodimerization domain”, as used herein, refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts or associates with a different immunoglobulin domain of a second polypeptide chain, wherein the interaction of the different immunoglobulin heterodimerization domains substantially contributes to or efficiently promotes heterodimerization of the first and second polypeptide chains (i.e. , the formation of a dimer between two different polypeptide chains, which is also referred to as a "heterodimer").
  • immunoglobulin heterodimerization domains "substantially contributes to or efficiently promotes" the heterodimerization of first and second polypeptide chains if there is a statistically significant reduction in the dimerization between the first and second polypeptide chains in the absence of the immunoglobulin heterodimerization domain of the first polypeptide chain and/or the immunoglobulin heterodimerization domain of the second polypeptide chain.
  • the first and second polypeptide chains when the first and second polypeptide chains are co-expressed, at least 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least 80% to about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second polypetpide chains form heterodimers with each other.
  • immunoglobulin heterodimerization domains include an immunoglobulin CH1 domain, an immunoglobulin CL domain (e.g. , CK or CA isotypes), or derivatives thereof, including wild type immunoglobulin CH1 and CL domains and altered (or mutated) immunoglobulin CH1 and CL domains, as provided therein.
  • immunoglobulin CH1 domain an immunoglobulin CH1 domain
  • immunoglobulin CL domain e.g. CK or CA isotypes
  • derivatives thereof including wild type immunoglobulin CH1 and CL domains and altered (or mutated) immunoglobulin CH1 and CL domains, as provided therein.
  • an "immunoglobulin constant region” or “constant region” is a term defined herein to refer to a peptide or polypeptide sequence that corresponds to or is derived from part or all of one or more constant region domains.
  • the immunoglobulin constant region corresponds to or is derived from part or all of one or more constant region domains, but not all constant region domains of a source antibody.
  • the constant region comprises IgG CH2 and CH3 domains, e.g., lgG 1 CH2 and CH3 domains.
  • the constant region does not comprise a CH1 domain.
  • the constant region domains making up the constant region are human.
  • the constant region domains of a fusion protein of this disclosure lack or have minimal effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation and complement-dependent cytotoxicity (CDC), while retaining the ability to bind some F c receptors (such as F c Rn, the neonatal Fc receptor) and retaining a relatively long half life in vivo.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement activation and complement-dependent cytotoxicity
  • a fusion protein of this disclosure includes constant domains that retain such effector function of one or both of ADCC and CDC.
  • a binding domain of this disclosure is fused to a human lgG1 constant region, wherein the lgG 1 constant region has one or more of the following amino acids mutated: leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamate at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (numbering according to EU). For example, any one or more of these amino acids can be changed to alanine.
  • an lgG 1 Fc domain has each of L234, L235, G237, E318, K320, and K322 (according to EU numbering) mutated to an alanine (i.e. , L234A, L235A, G237A, E318A, K320A, and K322A, respectively), and optionally an N297A mutation as well (i.e. , essentially eliminating glycosylation of the CH2 domain).
  • Fc region or “Fc domain” refers to a polypeptide sequence corresponding to or derived from the portion of a source antibody that is responsible for binding to antibody receptors on cells and the C1 q component of complement.
  • Fc stands for "fragment crystalline," the fragment of an antibody that will readily form a protein crystal. Distinct protein fragments, which were originally described by proteolytic digestion, can define the overall general structure of an immunoglobulin protein. As originally defined in the literature, the Fc fragment consists of the disulfide-linked heavy chain hinge regions, CH2, and CH3 domains.
  • the term has been applied to a single chain consisting of CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide-linked dimer with a second such chain.
  • Fc includes variants of naturally occuring sequences.
  • a CD3-binding domain comprises a protein scaffold as generally disclosed in, for example, in US Patent Application Publication Nos.
  • a CD3-binding domain may comprise, in order from amino-terminus to carboxyl-terminus: a first binding domain, a hinge region, and an immunoglobulin constant region.
  • a CD3-binding domain comprises a protein scaffold as generally disclosed in, for example, in US Patent Application Publication No. 2009/0148447.
  • a CD3-binding domain may comprise, in order from amino- terminus to carboxyl-terminus: an immunoglobulin constant region, a hinge region and a first binding domain.
  • CD3-binding domains disclosed herein may incorporate a multi-specific binding protein scaffold.
  • Multi-specific binding proteins and polypeptides using scaffolds are disclosed, for instance, in PCT Application Publication No. WO 2007/146968, U.S. Patent Application Publication No. 2006/0051844, PCT Application Publication No. WO
  • a CD3-binding domain may comprise two binding domains (the domains can be designed to specifically bind the same or different targets), a hinge region, a linker (e.g., a carboxyl-terminus or an amino-terminus linker), and an immunoglobulin constant region.
  • a CD3-binding protein may be a homodimeric protein comprising two identical, disulfide- bonded polypeptides.
  • a CD3-binding protein may comprise ROR248, ROR246, ROR252, ROR250, or ROR243.
  • the "stalk region" of a type II C-lectin refers to the portion of the extracellular domain of the type II C-lectin that is located between the C-type lectin-like domain (CTLD; e.g. , similar to CTLD of natural killer cell receptors) and the transmembrane domain.
  • C-type lectin-like domain C-type lectin-like domain
  • the extracellular domain corresponds to amino acid residues 34- 179
  • the CTLD corresponds to amino acid residues 61 -176.
  • the stalk region of the human CD94 molecule includes amino acid residues 34-60, which is found between the membrane and the CTLD (see Boyington et al. , Immunity 10:75, 1999; for descriptions of other stalk regions, see also Beavil et al. , Proc. Nat'l. Acad. Sci. USA 89:753, 1992; and Figdor et al. , Nature Rev. Immunol. 2:77, 2002).
  • These type II C-lectins can also have from six to 10 junction amino acids between the stalk region and the transmembrane region or the CTLD.
  • the 233 amino acid human NKG2A protein GenBank Accession No.
  • P26715.1 has a transmembrane domain ranging from amino acids 71 -93 and an extracellular domain ranging from amino acids 94- 233.
  • the CTLD is comprised of amino acids 1 19-231 , and the stalk region comprises amino acids 99-1 16, which is flanked by junctions of five and two amino acids.
  • Other type II C- lectins, as well as their extracellular ligand-bind domains, interdomain or stalk regions, and CTLDs are known in the art (see, e.g. , GenBank Accession Nos. NPJD01993.2;
  • the "interdomain region" of a transmembrane protein refers to a portion of the extracellular domain of the transmembrane protein that is located between two adjacent domains.
  • interdomain regions include regions linking adjacent Ig domains of immunoglobulin superfamily members (e.g. , an immunoglobulin hinge region from IgG, IgA, IgD, or IgE; the region linking the IgV and lgC2 domains of CD2; or the region linking the IgV and IgC domains of CD80 or CD86).
  • Another example of an interdomain region is the region linking the non-lg and lgC2 domain of CD22, a type I sialic acid-binding Ig-like lectin.
  • a polypeptide region "derived from” a stalk region of a type II C-lectin, or “derived from” a transmembrane protein interdomain region refers to an about five to about 150 amino acid sequence, an about 5 to about 100 amino acid sequence, an about 5 to about 50 amino acid sequence, an about 5 to about 40 amino acid sequence, an about 5 to about 30 amino acid sequence, an about 5 to about 25 amino acid sequence, an about 5 to about 20 amino acid sequence, an about 10 to about 25 amino acid sequence, an about 10 to about 20 amino acid sequence, about 8 to about 20 amino acid sequence, about 9 to about 20 amino acid sequence, about 10 to about 20 amino acid sequence, about 1 1 to about 20 amino acid sequence, about 12 to about 20 amino acid sequence, about 13 to about 20 amino acid sequence, about 14 to about 20 amino acid sequence, about 15 to about 20 amino acid sequence, or an about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid sequence, wherein all or
  • a derivative of a stalk region is more resistant to proteolytic cleavage as compared to the wild-type stalk region sequence, such as those derived from about eight to about 20 amino acids of NKG2A, NKG2D, CD23, CD64, CD72, or CD94.
  • junction amino acids refers to one or more (e.g. , about 2-10) amino acid residues between two adjacent regions or domains of a polypeptide, such as between a hinge and an adjacent immunoglobulin constant region or between a hinge and an adjacent binding domain or between a peptide linker and an adjacent immunoglobulin variable domain or an adjacent immunoglobulin constant region.
  • Junction amino acids can result from the construct design of a polypeptide (e.g. , amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a polypeptide).
  • a "linker between CH3 and CH1 or CL” refers to one or more (e.g. , about 2-12, about 2-10, about 4-10, about 5-10, about 6-10, about 7-10, about 8- 10, about 9-10, about 8-12, about 9-12, or about 10-12) amino acid residues between the C- terminus of a CH3 domain (e.g. , a wild type CH3 or a mutated CH3) and the N-terminus of a CH1 domain or CL domain (e.g. , Ck).
  • the term "patient in need” refers to a patient at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a ROR1 -binding protein or polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243) or a composition thereof provided herein.
  • a ROR1 -binding protein or polypeptide e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243
  • a composition thereof provided herein.
  • the term "pharmaceutically acceptable” refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions 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 are considered to be “pharmaceutically acceptable.”
  • promoter refers to a region of DNA involved in binding RNA polymerase to initiate transcription.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • nucleic acid As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” are intended to include DNA molecules (e.g. , cDNA or genomic DNA), RNA molecules (e.g. , mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • DNA molecules e.g. , cDNA or genomic DNA
  • RNA molecules e.g. , mRNA
  • analogs of the DNA or RNA generated using nucleotide analogs e.g. , mRNA
  • expression refers to the biosynthesis of a product encoded by a nucleic acid.
  • expression involves transcription of the nucleic acid segment into mRNA and the translation of mRNA into one or more polypeptides.
  • expression unit and "expression cassette” are used interchangeably herein and denote a nucleic acid segment encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell.
  • An expression unit typically comprises a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in operable configuration.
  • an expression unit can further include other nucleic acid segments such as, e.g. , an enhancer or a polyadenylation signal.
  • expression vector refers to a nucleic acid molecule, linear or circular, comprising one or more expression units.
  • an expression vector can also include additional nucleic acid segments such as, for example, one or more origins of replication or one or more selectable markers.
  • Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both.
  • sequence identity refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparator sequence, the sequences are said to be “identical” at that position. The percentage “sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of "identical” positions.
  • the number of "identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of "sequence identity.” Percentage of "sequence identity” is determined by comparing two optimally aligned sequences over a comparison window.
  • the comparison window for nucleic acid sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids in length.
  • the comparison windon for polypeptide sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length.
  • the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant.
  • An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of "identical" positions between the reference and comparator sequences.
  • Sequence identity between two sequences can be determined using the version of the program "BLAST 2 Sequences" which was available from the National Center for Biotechnology Information as of September 1 , 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).
  • BLASTN for nucleotide sequence comparison
  • BLASTP for polypeptide sequence comparison
  • nucleotide or amino acid sequences are considered to have "substantially similar sequence identity” or “substantial sequence identity” if the two sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to each other.
  • polypeptide or “polypeptide chain” is a single, linear and contiguous arrangement of covalently linked amino acids. It does not include two polypeptide chains that link together in a non-linear fashion, such as via an interchain disulfide bond (e.g. , a half immunoglobulin molecule in which a light chain links with a heavy chain via a disulfide bond). Polypeptides can have or form one or more intrachain disulfide bonds. With regard to polypeptides as described herein, reference to amino acid residues corresponding to those specified by SEQ ID NO includes post-translational modifications of such residues.
  • ROR1 -binding protein may be used interchangeably with “ROR1 - binding polypeptide.”
  • ROR1 receptor tyrosine kinase-like orphan receptor 1 (ROR1) (e.g. , human ROR1), also known as neurotrophic tyrosine kinase, receptor-related 1 or NTRKR1 .
  • ROR1 is a type I membrane protein, with an extracellular domain comprising an Ig-like domain, a Frizzled (cysteine-rich) domain and a Kringle domain.
  • the ROR1 -binding proteins of the disclosure bind to the extracellular domain of ROR1 .
  • ROR1 may refer to any isoform of ROR1.
  • Exemplary human ROR1 nucleotide and amino acid sequences are provided in SEQ ID NO: 127 and SEQ ID NO: 128, respectively.
  • a ROR1 -binding protein is an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243.
  • a ROR1 -binding protein is a humanized or a chimeric antibody.
  • a ROR1 -binding protein is a construct that induces redirected T-cell cytotoxicity.
  • a ROR1 -binding protein may comprise a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex.
  • a ROR1 - binding protein is an anti-ROR1 x anti-CD3 molecule in the format of an scFv-Fc-scFv molecule, an scFv-scFv molecule, or a diabody.
  • a ROR1 -binding protein comprises from amino-terminus to carboxyl-terminus (or from carboxyl-terminus to amino-terminus), (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker, and (v) a second binding domain (e.g, a CD3-binding domain).
  • a ROR1 -binding protein is a homodimer or a heterodimer.
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein can also comprise non-peptidic components, such as carbohydrate groups.
  • Carbohydrates and other non-peptidic substituents can be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • a protein may be an antibody or an antigen-binding fragment of an antibody. In some embodiments, a protein may also be an scFv-Fc-scFv molecule, scFv-scFv dimer, or a diabody.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl-terminus of the reference sequence, but is not necessarily at the carboxyl-terminus of the complete polypeptide.
  • T-cell receptor is a molecule found on the surface of T-cells that, along with CD3, is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It consists of a disulfide-linked heterodimer of the highly variable a and ⁇ chains in most T-cells. In other T-cells, an alternative receptor made up of variable Y and ⁇ chains is expressed. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin variable domain, one
  • TCR as used in the present disclosure can be from various animal species, including human, mouse, rat, or other mammals.
  • TCR complex refers to a complex formed by the association of CD3 chains with other TCR chains.
  • a TCR complex can be composed of a CD3v chain, a CD35 chain, two CD3z chains, a homodimer of ⁇ 3 chains, a TCRa chain, and a TCRp chain.
  • a TCR complex can be composed of a CD3v chain, a CD35 chain, two CD3z chains, a homodimer of ⁇ 3 chains, a TCRv chain, and a TCR5 chain.
  • a component of a TCR complex refers to a TCR chain (i.e. , TCRa, TCRp, TCRv or TCR5), a CD3 chain (i.e. , CD3v, CD35, CD3z or CD3Q, or a complex formed by two or more TCR chains or CD3 chains (e.g. , a complex of TCRa and TCRp, a complex of TCRv and TCR5, a complex of CD3z and CD35, a complex of CD3v and CD3£, or a sub-TCR complex of TCRa, TCRp, CD3v, CD35, and two CD3z chains).
  • Antibody-dependent cell-mediated cytotoxicity and "ADCC,” as used herein, refer to a cell-mediated process in which nonspecific cytotoxic cells that express FcyRs (e.g., monocytic cells such as Natural Killer (NK) cells and macrophages) recognize bound antibody (or other protein capable of binding FcyRs) on a target cell and subsequently cause lysis of the target cell.
  • FcyRs e.g., monocytic cells such as Natural Killer (NK) cells and macrophages
  • NK Natural Killer
  • any effector cell with an activating FcyR can be triggered to mediate ADCC.
  • the primary cells for mediating ADCC are NK cells, which express only FcyRIII, whereas monocytes, depending on their state of activation, localization, or differentiation, can express FcyRI, FcyRII, and FcyRIII.
  • NK cells which express only FcyRIII
  • monocytes depending on their state of activation, localization, or differentiation, can express FcyRI, FcyRII, and FcyRIII.
  • immunoglobulin hinge region and an immunoglobulin constant region having CH2 and CH3 domains is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) through binding of a cytolytic Fc receptor (e.g., FcyRIII) on a cytolytic immune effector cell expressing the Fc receptor (e.g., an NK cell).
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • a ROR1 -binding protein e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243
  • null effector function e.g., null ADCC activity
  • Complement-dependent cytotoxicity and “CDC,” as used herein, refer to a process in which components in normal serum (“complement”), together with an antibody or other C1 q-complement-binding protein bound to a target antigen, exhibit lysis of a target cell expressing the target antigen.
  • Complement consists of a group of serum proteins that act in concert and in an orderly sequence to exert their effect.
  • classical complement pathway and “classical complement system,” as used herein, are synonymous and refer to a particular pathway for the activation of complement.
  • the classical pathway requires antigen-antibody complexes for initiation and involves the activation, in an orderly fashion, of nine major protein components designated C1 through C9.
  • the product is an enzyme that catalyzes the subsequent step. This cascade provides amplification and activation of large amounts of complement by a relatively small initial signal.
  • polypeptide or protein having CDC activity
  • polypeptide or protein for example, one comprising an amino acid sequence, amino acid sequence, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino
  • a ROR1 -binding protein e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243
  • null effector function e.g., null CDC activity
  • Redirected T-cell cytotoxicity and "RTCC,” as used herein, refer to a T-cell- mediated process in which a cytotoxic T-cell is recruited to a target cell using a multi-specific protein that is capable of specifically binding both the cytotoxic T-cell and the target cell, and whereby a target-dependent cytotoxic T-cell response is elicited against the target cell.
  • Polypeptides and proteins comprising anti-ROR1 and anti-CD3 binding domains are, in some embodiments, capable of RTCC.
  • a ROR1 -binding protein that induces RTCC is an anti-ROR1 x anti-CD3 molecule in the format of an scFv-Fc-scFv molecule, an scFv-scFv molecule, or a diabody.
  • a ROR1 -binding protein that induces RTCC comprises from amino-terminus to carboxyl-terminus (or from carboxyl-terminus to amino-terminus), (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker, and (v) a second binding domain (e.g, a CD3-binding domain).
  • a ROR1 -binding protein that induces RTCC is a homodimer or a heterodimer.
  • treatment refers to either a therapeutic treatment or prophylactic/preventative treatment.
  • a treatment is therapeutic if at least one symptom of disease in an individual receiving treatment improves or a treatment can delay worsening of a progressive disease in an individual, or prevent onset of additional associated diseases.
  • the term "therapeutically effective amount (or dose)" or “effective amount (or dose)” of a specific binding molecule or compound refers to that amount of the compound sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner or a statistically significant improvement in organ function.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously (in the same formuation or concurrently in separate formulations).
  • the term “transformation,” “transfection,” and “transduction” refer to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell.
  • the term “genetic transformation” refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell.
  • the transferred nucleic acid can be introduced into a cell via an expression vector.
  • variants refers to a nucleic acid or polypeptide differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide. For instance, a variant may exhibit at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity compared to the active portion or full length reference nucleic acid or polypeptide.
  • variable region also referred to as “light chain variable domain” or “VL” or V L
  • heavy chain variable region also referred to as “heavy chain variable domain” or “VH” or V H
  • CDRs complementarity determining regions
  • FRs frame regions
  • CL refers to an "immunoglobulin light chain constant region” or a "light chain constant region,” i.e., a constant region from an antibody light chain.
  • CH refers to an antibody light chain constant region
  • immunoglobulin heavy chain constant region or a “heavy chain constant region,” which is further divisible, depending on the antibody isotype into CH1 , CH2, and CH3 (IgA, IgD, IgG), or CH1 , CH2, CH3, and CH4 domains (IgE, IgM).
  • a "Fab” fragment antigen binding is the part of an antibody that binds to antigens and includes the variable region and CH1 domain of the heavy chain linked to the light chain via an inter-chain disulfide bond.
  • the present disclosure describes binding domains that specifically bind ROR1 (e.g., human ROR1), as well as polypeptides and proteins comprising these binding domains.
  • ROR1 e.g., human ROR1
  • the ROR1 -binding proteins and polypeptides comprise a second binding domain, which may bind to ROR1 or to a different target.
  • the polypeptides and proteins comprising binding domains of this disclosure can further comprise immunoglobulin constant regions, linker peptides, hinge regions, immunoglobulin
  • dimerization/heterodimerization domains junctional amino acids, tags, etc. These components of the disclosed polypeptides and proteins are described in further detail below.
  • the ROR1 -binding polypeptides and proteins disclosed herein can be in the form of an antibody or a fusion protein of any of a variety of different formats (e.g. , the fusion protein can be in the form of a ROR1 -binding bispecific or multispecific molecule).
  • bispecific molecules include a scFv-Fc-scFv molecule.
  • Some bispecific molecules comprise or consist of an anti-ROR1 scFv linked to a second binding domain scFv and do not include other sequences such as an immunoglobulin constant region.
  • a ROR1 -binding protein is a diabody.
  • a ROR1 -binding protein in accordance with the present disclosure generally includes at least one ROR1 -binding polypeptide chain comprising (a) a ROR1 -binding domain as set forth herein.
  • the ROR1 -binding polypeptide further includes (b) a hinge region carboxyl-terminal to the ROR1 -binding domain, and (c) an immunoglobulin constant region.
  • the ROR1 -binding polypeptide further includes (d) a carboxyl-terminus linker carboxyl-terminal to the immunoglobulin constant region, and (e) a second binding domain carboxyl-terminal to the carboxyl-terminus linker.
  • a ROR1 -binding polypeptide comprises (b) a hinge region amino-terminal to the ROR1 -binding domain, and (c) an immunoglobulin sub-region amino- terminal to the hinge region.
  • ROR1 -binding polypeptides are capable of
  • two identical single chain ROR1 -binding polypeptides e.g., an anti-ROR1 x anti- CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243
  • homodimerize to form a dimeric ROR1 -binding protein e.g., an anti-ROR1 x anti- CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243
  • a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) includes a heterodimerization domain that is capable of heterodimerization with a different heterodimerization domain in a second, non-identical polypeptide chain.
  • the second polypeptide chain for heterodimerization includes a second binding domain. Accordingly, in certain embodiments of the present disclosure, two non-identical polypeptide chains, one comprising the ROR1 -binding domain and the second optionally comprising a second binding domain, dimerize to form a heterodimeric ROR1 -binding protein.
  • a heterodimer comprises ROR248, ROR246, ROR252, ROR250, or ROR243.
  • Examples of types of heterodimers include those described in International Appl. Publ. No. WO 201 1/090754 (US 2013/0095097) and in US
  • a ROR1 -binding domain, protein or polypeptide is conjugated to a drug or a toxic moiety.
  • an anti-ROR1 x anti-CD3 molecule as described herein is conjugated to a drug or a toxic moiety.
  • ROR248, ROR246, ROR252, ROR250, or ROR243 is conjugated to a drug or a toxic moiety.
  • ROR1 -binding polypeptides, proteins, and their various components used in the therapeutics of the present disclosure are further described below.
  • a ROR1 -binding domain is capable of competing for binding to ROR1 with an antibody having V L and V H regions having amino acid sequences as shown in SEQ ID NO:4 and SEQ ID NO:2, respectively (e.g. , R1 1), or with a single-chain Fv (scFv) having an amino acid sequence as shown in SEQ ID NO: 18, SEQ ID NO:21 , SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33 or SEQ ID NO:35.
  • a ROR1 -binding domain is capable of competing for binding to ROR1 with an antibody having V L and V H regions having amino acid sequences as shown in SEQ ID NO:39 and SEQ ID NO:37, respectively (e.g. , R12), or with a single-chain Fv (scFv) having an amino acid sequence as shown in SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 or SEQ ID NO:69.
  • Monoclonal rabbit anti-ROR1 antibodies R1 1 and R12 are described in, for example, U.S. Patent Application Publication No. 2013/0251642 and Yang et al., PLoS ONE 6(6): e21018 (201 1).
  • a ROR1 -binding domain binds an epitope near the junction of the Frizzled and Kringle domains of ROR1 (e.g., human ROR1). In certain aspects, this epitope is a discontinuous and/or conformational epitope. In some embodiments, a ROR1 - binding domain binds residues in stretches 259-273 (LCQTEYIFARSNPMI (residues 259- 273 of SEQ ID NO: 128)) and 389-403 (PACDSKDSKEKNKME (residues 389-403 of SEQ ID NO: 128)) of the ROR1 ectodomain. For example, a ROR1 -binding domain may bind at least one, at least two, at least three, at least four, at least five, at least six, or at least seven residues in these stretches of the ROR1 ectodomain.
  • a ROR1-binding domain may comprise sequences shown in Table 3.
  • a ROR1 -binding domain comprises at least one sequence shown in Table 3 and binds an epitope near the junction of the Frizzled and Kringle domains of ROR1 (e.g., human ROR1).
  • the sequences shown in the disclosure, including in Table 3 contain amino acid substitutions relative to a parent sequence.
  • ROR1 19 has the amino acid sequence of the L6H15 construct with the asparagine residue at position 94 substituted with alanine.
  • a ROR1 -binding domain comprises (i) an immunoglobulin light chain variable region (V L ) comprising CDRs LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region (V H ) comprising CDRs HCDR1 , HCDR2, and HCDR3.
  • V L immunoglobulin light chain variable region
  • V H immunoglobulin heavy chain variable region
  • Suitable ROR1 -binding domains include those having V L and V H regions derived from rabbit antibody R1 1 or R12.
  • the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12 or a sequence that differs from SEQ ID NO: 12 by at least one amino acid substitution
  • the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14 or a sequence that differs from SEQ ID NO: 14 by at least one amino acid substitution
  • the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268, or a sequence that differs from SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268 by at least one amino acid substitution
  • the HCDR1 has an amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:266 or a sequence that differs from SEQ ID NO:6 or SEQ ID NO:266 by at least one amino acid
  • HCDR2 has an amino acid sequence set forth in SEQ ID NO:8 or a sequence that differs from SEQ ID NO:8 by at least one amino acid substitution
  • the HCDR3 has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO:267 or a sequence that differs from SEQ ID NO: 10 or SEQ ID NO:267 by at least one amino acid substitution.
  • the amino acid substitution described above may be a conservative or a non-conservative amino acid substitution.
  • the LCDR1 has an amino acid sequence set forth in SEQ ID NO:47 or a sequence that differs from SEQ ID NO:47 by at least one amino acid substitution
  • the LCDR2 has an amino acid sequence set forth in SEQ ID NO:49 or a sequence that differs from SEQ ID NO:49 by at least one amino acid substitution
  • the LCDR3 has an amino acid sequence set forth in SEQ ID NO:51 , or a sequence that differs from SEQ ID NO:51 by at least one amino acid substitution
  • the HCDR1 has an amino acid sequence set forth in SEQ ID NO:41 or a sequence that differs from SEQ ID NO:41 by at least one amino acid substitution
  • the HCDR2 has an amino acid sequence set forth in SEQ ID NO:43 or a sequence that differs from SEQ ID NO:43 by at least one amino acid substitution
  • the HCDR3 has an amino acid sequence set forth in SEQ ID NO:45 or a
  • a ROR1 -binding domain comprises the CDR sequences listed in this paragraph and binds an epitope near the junction of the Frizzled and Kringle domains of ROR1 (e.g., human ROR1).
  • a ROR1 -binding domain comprises the CDR sequences listed in this paragraph and binds residues in stretches 259- 273 (LCQTEYI FARSN PM I (residues 259-273 of SEQ ID NO: 128)) and 389-403
  • the disclosure relates to a ROR1 -binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12; (b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14; (c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268; (d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:266; (e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:8; and (f) the HCDR3 has an
  • a ROR1 -binding domain comprises humanized
  • immunoglobulin V L and/or V H regions Techniques for humanizing immunoglobulin V L and V H regions are known in the art and are discussed, for example, in U.S. Patent Application Publication No. 2006/0153837.
  • "Humanization" is expected to result in an antibody that is less immunogenic, with complete retention of the antigen-binding properties of the original molecule. In order to retain all of the antigen-binding properties of the original antibody, the structure of its antigen binding site should be reproduced in the "humanized” version. This can be achieved by grafting only the nonhuman CDRs onto human variable framework domains and constant regions, with or without retention of critical framework residues (Jones et al.
  • humanization by CDR grafting involves recombining only the CDRs of a non-human antibody onto a human variable region framework and a human constant region. Theoretically, this should substantially reduce or eliminate immunogenicity (except if allotypic or idiotypic differences exist). However, it has been reported that some framework residues of the original antibody also may need to be preserved (Reichmann et al. , Nature, 332:323 (1988); Queen et al. , Proc. Natl. Acad. Sci. USA, 86: 10,029 (1989)).
  • framework residues that need to be preserved are amenable to identification through computer modeling.
  • critical framework residues can potentially be identified by comparing known antigen-binding site structures (Padlan, Molec. Immunol. , 31 (3): 169-217 (1994), incorporated herein by reference).
  • the residues that potentially affect antigen binding fall into several groups.
  • the first group comprises residues that are contiguous with the antigen site surface, which could therefore make direct contact with antigens. These residues include the amino-terminal residues and those adjacent to the CDRs.
  • the second group includes residues that could alter the structure or relative alignment of the CDRs, either by contacting the CDRs or another peptide chain in the antibody.
  • the third group comprises amino acids with buried side chains that could influence the structural integrity of the variable domains.
  • the residues in these groups are usually found in the same positions (Padlan, 1994, supra) although their positions as identified may differ depending on the numbering system (see Kabat et al. , "Sequences of proteins of immunological interest, 5th ed., Pub. No. 91 -3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md., 1991).
  • a ROR1 -binding domain of the disclosure is a humanized V L and/or V H region derived from the variable region sequences of rabbit monoclonal antibody R1 1 (V L is SEQ ID NO:4; V H is SEQ ID NO:2) or R12 (V L is SEQ ID NO:39; V H is SEQ ID NO:37).
  • Monoclonal rabbit antibodies R1 1 and R12 are described in US 2013/0251642.
  • a ROR1 -binding domain comprises or is a sequence that is at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (V L ) (e.g.
  • V H heavy chain variable region
  • a ROR1 -binding domain or polypeptide comprises or is a sequence that is at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence of a light chain (HC) (e.g. , SEQ ID NO:333 or SEQ ID NO:337) or to a heavy chain (HC) (e.g. , SEQ ID NO:335 or SEQ ID NO:339).
  • HC light chain
  • HC heavy chain
  • the disclosure relates to ROR1 -binding domains wherein (i) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:23 and the
  • immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27;
  • the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27;
  • the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set
  • immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232; (xii) the
  • immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232; (xiii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the
  • the disclosure relates to ROR1 -binding domains or polypeptides wherein (i) the immunoglobulin light chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:333 and the immunoglobulin heavy chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:335; or (ii) the immunoglobulin light chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:337 and the immunoglobulin heavy chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least
  • the disclosure also relates to ROR1 -binding domains wherein (i) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27; (ii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27; (iii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59; (iv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the
  • immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 ;
  • the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59;
  • the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 ,
  • the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:210;
  • the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:210;
  • the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the
  • immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232; (xii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232; (xiii) the
  • immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:244; or (xiv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:250 and the
  • the disclosure also relates to ROR1 -binding domains and polypeptides wherein (i) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:333 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:335; or (ii) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:337 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:339.
  • each CDR comprises no more than one, two, or three substitutions, insertions or deletions, as compared to that from a monoclonal antibody or fragment or derivative thereof that specifically binds to a target of interest (e.g. , ROR1).
  • a target of interest e.g. , ROR1
  • a ROR1 -binding domain may specifically bind to human ROR1 and comprise SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69.
  • a ROR1 -binding polypeptide may specifically bind to human ROR1 and comprise SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91 .
  • a binding domain V L and/or V H region of the present disclosure is derived from a V L and/or V H of a known monoclonal antibody (e.g. , R1 1 or R12) and optionally contains about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g.
  • the binding domain containing the modified V L and/or V H region can still specifically bind its target with an affinity similar to the wild type binding domain.
  • a ROR1 -binding protein can comprise one or more additional binding domains (e.g. , second binding domain) that bind a target other than ROR1 .
  • additional binding domains e.g. , second binding domain
  • These other binding domains can comprise, for example, a particular cytokine or a molecule that targets the binding domain polypeptide to a particular cell type, a toxin, an additional cell receptor, an antibody, etc.
  • a ROR1 -binding molecule or protein can comprise a T-cell binding domain for recruitment of T-cells to target cells expressing ROR1 .
  • a ROR1 -binding protein as described herein can comprise (i) a binding domain that specifically binds a TCR complex or a component thereof (e.g., TCRa, TCRp, CD3Y, CD35, and CD3£) and (ii) another binding domain that specifically binds to ROR1.
  • a ROR1-binding protein can utilize essentially any binding domain that binds a T- cell, e.g., an antibody derived binding domain.
  • Exemplary anti-CD3 antibodies from which the CD3 binding domain can be derived include the CRIS-7 monoclonal antibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II., Springer Verlag, New York, (1986); V L and V H amino acid sequences respectively shown in SEQ ID NO: 131
  • BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172:1691); OKT3 monoclonal antibody (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (also referred to as OKT3 AA-FL or OKT3 FL), a humanized, Fc variant with alanine substitutions at positions 234 and 235 (Herold et al. (2003) J. Clin. Invest. 1 1 :409); visilizumab (Carpenter et al.
  • a CD3 binding domain may comprise a CD3 binding domain disclosed in U.S. Patent Application
  • Publication No. 20120244162 including a CD3 binding domain comprising a VL region selected from SEQ ID NO: 17, 21 , 35, 39, 53, 57, 71 , 75, 89, 83, 107, 1 1 1 , 125, 129, 143, 147, 161 , 165, 179 and 183 of US20120244162 and/or a VH region selected from SEQ ID NO:15, 19, 33, 37, 51 , 55, 69, 73, 87, 91. 105, 109, 123, 127, 141 , 145, 159, 163, 177 and 181 of US20120244162.
  • a CD3 binding domain comprises an amino acid sequence selected from SEQ ID NO: 23, 25, 41 , 43, 59, 61 , 77, 79, 95, 97, 1 13, 1 15, 131 , 133, 149, 151 , 167, 169, 185, and 187 of US20120244162.
  • a CD3 binding domain is one described in WO2004/106380, WO2005/040220A1 , US
  • An exemplary anti-TCR antibody is the BMA031 monoclonal antibody (Borst et al. (1990) Human Immunology 29: 175-188).
  • the CD3 binding domain may be derived from any of the antibodies or sequences described in WO 2013/158856 (incorporated herein by reference in its entirety).
  • the CD3 binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 10, 1 1 1 and 1 12, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 272, 273 and 274, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 269, 270 and 271 , respectively.
  • the second binding domain of a ROR1 -binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 278, 279 and 280, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 275, 276 and 277, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 284, 285 and 286, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 281 , 282 and 283, respectively.
  • the second binding domain of a RORI - binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 290, 291 and 292, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 287, 288 and 289, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 296, 297 and 298, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 293, 294 and 295, respectively.
  • a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) comprises, in order from amino-terminus to carboxyl-terminus (or in order from carboxyl-terminus to amino-terminus), (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) a CD3-binding domain; wherein said ROR1 -binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 12; (b) the LCDR2 of said R
  • the HCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:266;
  • the HCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:8; and
  • the HCDR3 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:267; and wherein said CD3-binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a ' ) the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1
  • the second binding domain competes for binding to CD3£ with the CRIS-7, HuM291 or I2C monoclonal antibody.
  • the CD3- binding domain comprises an immunoglobulin light chain variable region (V L ) and an immunoglobulin heavy chain variable region (V H ) derived from the CRIS-7, HuM291 or I2C monoclonal antibody (e.g. , the V L and V H of the second binding domain can be humanized variable regions comprising, respectively, the light chain CDRs and the heavy chain CDRs of the monoclonal antibody).
  • a second binding domain may comprise the light chain variable region, the heavy chain variable region, or both, of the DRA222, TSC455, or TSC456 CD3- binding domains.
  • the amino acid sequences of DRA222, TSC455, and TSC456 are provided in Table 3.
  • TSC455 is also referred to herein as TSC394 F87Y.
  • TSC455 is also referred to herein as TSC394 E86D F87Y or TSC394 DY.
  • the second binding domain specifically binds CD3 and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; wherein the
  • immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:260; or at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:261 ; and wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:259.
  • a ROR1 -binding polypeptide or protein further comprising a CD3-binding domain (e.g., ROR
  • ROR243 may have a low level of high molecular weight aggregates produced during recombinant expression of the polypeptide or protein.
  • a ROR1-binding polypeptide or protein further comprising a CD3-binding domain e.g., ROR248, ROR246, ROR252, ROR250, or ROR243
  • the second binding domain of a ROR1 -binding polypeptide described herein is a CD3-binding domain and comprises one or more of the CD3-binding sequences (e.g., CDRs or variable regions) disclosed in US 2013/0129730, US
  • a CD3-binding domain comprises one or more of the following sequences: LCDR1 LCDR2 LCDR3
  • a CD3-binding domain comprises one or more of the following sequences:
  • a ROR1 -binding polypeptide used in the methods and compositions described herein is a bispecific single chain molecule comprising a ROR1 binding domain and a CD3 binding domain.
  • a ROR1 - and/or a CD3- binding domain is derived from an antibody and comprises a variable heavy chain (VH) and a variable light chain (VL).
  • VH variable heavy chain
  • VL variable light chain
  • an scFv comprises a VH and VL.
  • variable domains may be arranged in the order such as VH ROR1 -VL ROR1 -VH CD3-VL CD3; VL ROR1 -VH ROR1 -VH CD3-VL CD3; VH ROR1 -VL ROR1 -VL CD3-VH CD3; VL ROR1 -VH ROR1 -VL CD3-VH CD3; VH CD3-VL CD3-VH ROR1 -VL ROR1 ; VL CD3-VH CD3-VL ROR1-VH ROR1 ; VH CD3-VL CD3-VL ROR1 -VH ROR1 ; VH CD3-VL CD3-VL ROR1 -VH ROR1 ; or VL CD3-VH CD3-VH ROR1 -VL ROR1 .
  • the pairs of VH regions and VL regions in the binding domain binding to CD3 may be in the format of
  • the scFv may bind to ROR1 more effectively than the antibody comprising the same VH and VL region sequences in the same orientation (see, e.g., Example 3). In certain embodiments, the scFv may bind more effectively to ROR1 in the VL-VH orientation than in the VH-VL orientation, or vice versa (see, e.g., Example 4).
  • the VH-region may be positioned N-terminally to a linker sequence.
  • the VL region may be positioned C-terminally to the linker sequence.
  • the domain arrangement in the CD3 binding domain of the bispecific single chain molecule may be VH-VL, with said CD3 binding domain located C-terminally to the ROR1 binding domain.
  • a bispecific molecule may comprise an scFv binding to ROR1 linked to an scFv binding to CD3. These scFvs may be linked, for example, with a short peptide.
  • bispecific single chain molecules do not comprise a hinge region or a constant region (see, for example, US 2013/0295121 , US 2013/0129730, WO 2010/037836, WO 2004/106381 and WO 201 1/121 1 10; each incorporated herein by reference in its entirety).
  • a binding domain is a single-chain Fv fragment (scFv) that comprises V H and V L regions specific for a target of interest.
  • scFv single-chain Fv fragment
  • the V H and V L regions are human or humanized.
  • a ROR1 -binding domain comprises or is an scFv that is at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence of a scFv of SEQ ID NO: 18, 21 , 29, 31 , 33, 35, 53, 63, 65, 67 or 69.
  • a binding domain is a single-chain Fv (scFv) comprising immunoglobulin V L and V H regions joined by a peptide linker.
  • scFv single-chain Fv
  • peptide linker is a 15mer consisting of three repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence ((Gly 4 Ser) 3 ) (SEQ ID NO: 135).
  • linkers have been used, and phage display technology, as well as selective infective phage technology, has been used to diversify and select appropriate linker sequences (Tang et a/. , J. Biol. Chem. 271 , 15682-15686, 1996; Hennecke et a/. , Protein Eng. 1 1 , 405-410, 1998).
  • Other suitable linkers can be obtained by optimizing a simple linker (e.g. ,
  • a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) comprises, in order from amino-terminus to carboxyl-terminus (or in order from carboxyl-terminus to amino-terminus), (i) the ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) the second binding domain.
  • the ROR1 -binding domain comprises, in order from amino-terminus to carboxyl-terminus (or in order from carboxyl-terminus to amino-terminus), (i) the ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) the second binding domain.
  • a “hinge region” or a “hinge” refers to a polypeptide region between the first binding domain and the Fc region.
  • a “carboxyl-terminus linker” or “an amino-terminus linker” refers to a polypeptide region between the Fc region and the second binding domain.
  • a carboxyl- terminus (or an amino-terminus linker) linker comprises or consists of SEQ ID NO:265, SEQ ID NO:301 , SEQ ID NO:302, or SEQ ID NO:303.
  • a hinge is a wild- type human immunoglobulin hinge region.
  • one or more amino acid residues can be added at the amino- or carboxyl-terminus of a wild type
  • immunoglobulin hinge region as part of a fusion protein construct design.
  • additional junction amino acid residues at the hinge amino-terminus can be "RT,” “RSS,” “TG,” or “T,” or at the hinge carboxyl-terminus can be "SG”, or a hinge deletion can be combined with an addition, such as ⁇ with "SG” added at the carboxyl-terminus.
  • a hinge, a carboxyl-terminus linker, or an amino-terminus linker is an altered immunoglobulin hinge in which one or more cysteine residues in a wild type immunoglobulin hinge region is substituted with one or more other amino acid residues (e.g. , serine or alanine).
  • Exemplary altered immunoglobulin hinges, carboxyl-terminus linkers, and amino- terminus linkers include an immunoglobulin human lgG1 hinge region having one, two or three cysteine residues found in a wild type human lgG1 hinge substituted by one, two or three different amino acid residues (e.g. , serine or alanine).
  • An altered immunoglobulin hinge can additionally have a proline substituted with another amino acid (e.g. , serine or alanine).
  • the above-described altered human lgG 1 hinge can additionally have a proline located carboxyl-terminal to the three cysteines of wild type human lgG 1 hinge region substituted by another amino acid residue (e.g. , serine, alanine).
  • the prolines of the core hinge region are not substituted.
  • a hinge, a carboxyl-terminus linker, or an amino-terminus linker polypeptide comprises or is a sequence that is at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a wild type
  • immunoglobulin hinge region such as a wild type human lgG 1 hinge, a wild type human lgG2 hinge, or a wild type human lgG4 hinge.
  • a hinge, a carboxyl-terminus linker, or an amino-terminus linker present in a ROR1 -binding polypeptide can be a hinge that is not based on or derived from an immunoglobulin hinge (i.e., not a wild-type immunoglobulin hinge or an altered immunoglobulin hinge).
  • hinges and carboxyl-terminus linkers include peptides of about five to about 150 amino acids derived from an interdomain region of a transmembrane protein or stalk region of a type II C-lectin, for instance, peptides of about eight to 25 amino acids and peptides of about seven to 18 amino acids.
  • interdomain or stalk region hinges, carboxyl-terminus linkers, and amino-terminus linkers have seven to 18 amino acids and can form an a-helical coiled coil structure.
  • interdomain or stalk region hinges, carboxyl- terminus linkers, or amino-terminus linkers contain 0, 1 , 2, 3, or 4 cysteines.
  • Exemplary interdomain or stalk region hinges, carboxyl-terminus linkers, and amino-terminus linkers are peptide fragments of the interdomain or stalk regions, such as ten to 150 amino acid fragments from the stalk regions of CD69, CD72, CD94, NKG2A and NKG2D.
  • hinge, carboxyl-terminus linker, and amino-terminal linker sequences have about 5 to 150 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, 40 to 50 amino acids, 50 to 60 amino acids, 5 to 60 amino acids, 5 to 40 amino acids, 8 to 20 amino acids, or 10 to 15 amino acids.
  • the hinge or linker can be primarily flexible, but can also provide more rigid characteristics or can contain primarily a-helical structure with minimal ⁇ -sheet structure.
  • the lengths or the sequences of the hinges and linkers can affect the binding affinities of the binding domains to which the hinges are directly or indirectly (via another region or domain, such as an heterodimerization domain) connected as well as one or more activities of the Fc region portions to which the hinges or linkers are directly or indirectly connected.
  • hinge, carboxyl-terminus linker, and amino-terminal linker sequences are stable in plasma and serum and are resistant to proteolytic cleavage.
  • the first lysine in the lgG 1 upper hinge region can be mutated to minimize proteolytic cleavage, for instance, the lysine can be substituted with methionine, threonine, alanine or glycine, or is deleted.
  • a ROR1 -binding polypeptide e.g. , an anti- ROR1 x anti-CD3 molecule
  • a ROR1 -binding polypeptide is capable of forming a heterodimer with a second polypeptide chain and comprises a hinge region (a) immediately amino-terminal to an immunoglobulin constant region (e.g. , amino-terminal to a CH2 domain wherein the immunglobulin constant region includes CH2 and CH3 domains, or amino-terminal to a CH3 domain wherein the immunoglobulin sub-regions includes CH3 and CH4 domains), (b) interposed between and connecting a binding domain (e.g.
  • immunoglobulin heterodimerization domain (c) interposed between and connecting a immunoglobulin heterodimerization domain and an immunoglobulin constant region (e.g. , wherein the immunoglobulin constant region includes CH2 and CH3 domains or CH3 and CH4 domains), (d) interposed between and connecting an immunoglobulin constant region and a binding domain, (e) at the amino-terminus of a polypeptide chain, or (f) at the carboxyl-terminus of a polypeptide chain.
  • immunoglobulin constant region e.g. , wherein the immunoglobulin constant region includes CH2 and CH3 domains or CH3 and CH4 domains
  • a polypeptide chain comprising a hinge region as described herein will be capable of associating with a different polypeptide chain to form a heterodimeric protein provided herein, and the heterodimer formed will contain a binding domain that retains its target specificity or its specific target binding affinity.
  • a hinge present in a polypeptide that forms a heterodimer with another polypeptide chain can be an immunoglobulin hinge, such as a wild-type immunoglobulin hinge region or an altered immunoglobulin hinge region thereof.
  • a hinge of one polypeptide chain of a heterodimeric protein is identical to a corresponding hinge of the other polypeptide chain of the heterodimer.
  • a hinge of one chain is different from that of the other chain (in their length or sequence).
  • a heterodimeric protein has a CD3- or TCR-binding domain in one chain and a ROR1 -binding domain in another chain. Having two different hinges in the two chains may allow the heterodimer to bind to the ROR1 first, and then to a CD3 or other TCR component second.
  • the heterodimer may recruit CD3 + T-cells to ROR1 -expressing cells (e.g. , ROR1 -expressing tumor or cancer cells), which in turn may damage or destroy the ROR1 -expressing cells.
  • a carboxyl-terminus linker or an amino-terminus linker is a flexible linker sequence comprising glycine-serine (e.g., Gly 4 Ser) repeats.
  • the linker comprises three Gly 4 Ser repeats (SEQ ID NO: 160) followed by a proline residue.
  • the proline residue is followed by an amino acid selected from the group consisting of glycine, arginine and serine.
  • a carboxyl-terminus linker or an amino-terminus linker comprises or consists of SEQ ID NO:265, SEQ ID NO: 301 , SEQ ID NO: 302 or SEQ ID NO: 303.
  • hinges and linkers (derived from H7 hinge, stalk region of a type II C-lectin, or interdomain region of a type I transmembrane protein)
  • a ROR1 -binding polypeptide or protein of the disclosure can comprise an "immunoglobulin dimerization domain" or “immunoglobulin heterodimenzation domain.”
  • an "immunoglobulin dimerization domain” or “immunoglobulin heterodimenzation domain,” as used herein, refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts or associates with a different immunoglobulin domain of another polypeptide chain, wherein the interaction of the different immunoglobulin heterodimenzation domains substantially contributes to or efficiently promotes heterodimenzation of the first and second polypeptide chains (i.e. , the formation of a dimer between two different polypeptide chains, which is also referred to as a "heterodimer” or “heterodimeric protein”).
  • the interactions between immunoglobulin heterodimenzation domains "substantially contributes to or efficiently promotes" the heterodimenzation of first and second polypeptide chains if there is a statistically significant reduction in the dimerization between the first and second polypeptide chains in the absence of the immunoglobulin heterodimenzation domain of the first polypeptide chain and/or the immunoglobulin heterodimenzation domain of the second polypeptide chain.
  • the first and second polypeptide chains when the first and second polypeptide chains are co-expressed, at least 60%, at least about 60% to about 70%, at least about 70% to about 80% , at least 80% to about 90% , 91 %, 92%, 93% , 94%, 95%, 96%, 97% , 98%, or 99% of the first and second polypetpide chains form heterodimers with each other.
  • immunoglobulin heterodimenzation domains include an immunoglobulin CH 1 domain, an immunoglobulin CL1 domain (e.g. , CK or CA isotypes), or derivatives thereof, including wild-type immunoglobulin CH 1 and CL domains and altered (or mutated) immunoglobulin CH 1 and CL domains, such as provided herein.
  • immunoglobulin CH 1 domain an immunoglobulin CH 1 domain
  • immunoglobulin CL1 domain e.g. CK or CA isotypes
  • derivatives thereof including wild-type immunoglobulin CH 1 and CL domains and altered (or mutated) immunoglobulin CH 1 and CL domains, such as provided herein.
  • Dimerization/heterodimerization domains can be used where it is desired to form heterodimers from two non-identical polypeptide chains, where one or both polypeptide chains comprise a binding domain.
  • one polypeptide chain member of certain heterodimers described herein does not contain a binding domain.
  • a heterodimeric protein of the present disclosure comprises an immunoglobulin heterodimerization domain in each polypeptide chain. The immunoglobulin
  • heterodimerization domains in the polypeptide chains of a heterodimer are different from each other and thus can be differentially modified to facilitate heterodimerization of both chains and to minimize homodimerization of either chain.
  • heterodimerization domains provided herein allow for efficient heterodimerization between different polypeptides and facilitate purification of the resulting heterodimeric protein.
  • immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different single chain polypeptides include immunoglobulin CH1 and CL domains, for instance, human CH1 and CL domains.
  • an immunoglobulin heterodimerization domain is a wild-type CH 1 domain, such as a wild type lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH1 domain.
  • an immunoglobulin heterodimerization domain is a wild-type human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH1 domain as set forth in SEQ ID NOS: 1 14, 186-192 and 194, respectively, of PCT Publication No. WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
  • an immunoglobulin heterodimerization domain is a wild-type human lgG 1 CH1 domain as set forth in SEQ ID NO: 1 14 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
  • an immunoglobulin heterodimerization domain is an altered immunoglobulin CH1 domain, such as an altered lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 IgD, IgE, or IgM CH1 domain.
  • an immunoglobulin heterodimerization domain is an altered human lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH1 domain.
  • a cysteine residue of a wild-type CH1 domain e.g.
  • a human CH1 involved in forming a disulfide bond with a wild type immunoglobulin CL domain (e.g. , a human CL) is deleted or substituted in the altered immunoglobulin CH1 domain such that a disulfide bond is not formed between the altered CH1 domain and the wild-type CL domain.
  • a wild type immunoglobulin CL domain e.g. , a human CL
  • an immunoglobulin heterodimerization domain is a wild- type CL domain, such as a wild type CK domain or a wild type CA domain.
  • an immunoglobulin heterodimerization domain is a wild type human CK or human CA domain as set forth in SEQ ID NOS: 1 12 and 1 13, respectively, of
  • an immunoglobulin heterodimerization domain is an altered immunoglobulin CL domain, such as an altered CK or CA domain, for instance, an altered human CK or human CA domain.
  • a cysteine residue of a wild-type CL domain (e.g. , a human CL) involved in forming a disulfide bond with a wild type immunoglobulin CH1 domain (e.g. , a human CH 1) is deleted or substituted in the altered immunoglobulin CL domain.
  • Such altered CL domains can further comprise an amino acid deletion at their amino-termini.
  • An exemplary CK domain is set forth in SEQ ID NO: 141 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein), in which the first arginine and the last cysteine of the wild type human Ck domain are both deleted.
  • only the last cysteine of the wild type human Ck domain is deleted in the altered Ck domain because the first arginine deleted from the wild type human Ck domain can be provided by a linker that has an arginine at its carboxyl-terminus and links the amino-terminus of the altered Ck domain with another domain (e.g. , an immunoglobulin sug-region, such as a sub- region comprising immunoglobulin CH2 and CH3 domains).
  • An exemplary CA domain is set forth in SEQ ID NO: 140 of WO201 1/090762 (US 2013/0129723) (said sequence
  • an immunoglobulin heterodimerization domain is an altered CK domain that contains one or more amino acid substitutions, as compared to a wild type CK domain, at positions that may be involved in forming the interchain-hydrogen bond network at a CK-CK interface.
  • an immunoglobulin heterodimerization domain is an altered human CK domain having one or more amino acids at positions N29, N30, Q52, V55, T56, S68 or T70 that are substituted with a different amino acid.
  • an immunoglobulin heterodimerization domain is an altered human CK domain having one, two, three or four amino acid substitutions at positions N29, N30, V55, or T70.
  • the amino acid used as a substitute at the above-noted positions can be an alanine, or an amino acid residue with a bulk side chain moiety such as arginine, tryptophan, tyrosine, glutamate, glutamine, or lysine.
  • Additional amino acid residues that can be used to substitute amino acid residues of the wild type human Ck sequence at the above noted positions include aspartate, methionine, serine and phenyalanine.
  • Exemplary altered human CK domains are set forth in SEQ ID NOS: 142-178 of WO201 1/090762 (US 2013/0129723) (said sequences
  • Altered human CK domains are those that facilitate heterodimerization with a CH1 domain, but minimize homodimerization with another CK domain.
  • Representative altered human CK domains are set forth in SEQ ID NOS: 160 (N29W V55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176 (N29Y N30Y V55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E), 838 (N30D V55A T70E), 839 (N30M V55A T70E), 840 (N30S V55A T70E), and 841 (N29W V55
  • both the immunoglobulin heterodimerization domains (i.e. , immunoglobulin CH1 and CL domains) of a polypeptide heterodimer have mutations so that the resulting immunoglobulin heterodimerization domains form salt bridges (i.e. , ionic interactions) between the amino acid residues at the mutated sites.
  • the immunoglobulin heterodimerization domains of a polypeptide heterodimer can be a mutated CH 1 domain in combination with a mutated Ck domain.
  • valine at position 68 (V68) of the wild type human CH1 domain is substituted by an amino acid residue having a negative charge (e.g. , aspartate or glutamate), whereas leucine at position 29 (L29) of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted is substituted by an amino acid residue having a positive charge (e.g. , lysine, arginine or histidine).
  • V68 of the wild type CH1 can be substituted by an amino acid residue having a positive charge
  • L29 of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted can be substituted by an amino acid residue having a negative charge
  • Exemplary mutated CH1 sequences in which V68 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:844 and 845 of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
  • Exemplary mutated Ck sequences in which L29 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:842 and 843 of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
  • Positions other than V68 of human CH1 domain and L29 of human Ck domain can be substituted with amino acids having opposite charges to produce ionic interactions between the amino acids in addition or alternative to the mutations in V68 of CH1 domain and L29 of Ck domain.
  • Such positions can be identified by any suitable method, including random mutagenesis, analysis of the crystal structure of the CH 1 -Ck pair to identify amino acid residues at the CH 1 -Ck interface, and further identifying suitable positions among the amino acid residues at the CH 1 -Ck interface using a set of criteria (e.g. , propensity to engage in ionic interactions, proximity to a potential partner residue, etc.).
  • polypeptide heterodimers of the present disclosure contain only one pair of immunoglobulin heterodimenzation domains.
  • a first chain of a polypeptide heterodimer can comprise a CH 1 domain as an immunoglobulin
  • a second chain can comprise a CL domain (e.g. , a CK or CA) as an immunoglobulin heterodimenzation domain.
  • a first chain can comprise a CL domain (e.g. , a CK or CA) as an immunoglobulin heterodimenzation domain
  • a second chain can comprise a CH 1 domain as an immunoglobulin heterodimenzation domain.
  • the immunoglobulin heterodimenzation domains of the first and second chains are capable of associating to form a heterodimeric protein of this disclosure.
  • heterodimeric proteins of the present disclosure can have two pairs of immunoglobulin heterodimenzation domains.
  • a first chain of a heterodimer can comprise two CH 1 domains, while a second chain can have two CL domains that associate with the two CH 1 domains in the first chain.
  • a first chain can comprise two CL domains, while a second chain can have two CH 1 domains that associate with the two CL domains in the first chain.
  • a first polypeptide chain comprises a CH 1 domain and a CL domain
  • a second polypeptide chain comprises a CL domain and a CH 1 domain that associate with the CH 1 domain and the CL domain, respectively, of the first polypeptide chain.
  • heterodimeric protein comprises only one
  • the immunoglobulin heterodimenzation domain of each chain can be located amino-terminal to the immunoglobulin constant region of that chain.
  • the immunoglobulin heterodimenzation domain in each chain can be located carboxyl-terminal to the
  • both immunoglobulin heterodimenzation domains in each chain can be located amino-terminal to the immunoglobulin constant region of that chain.
  • both immunoglobulin heterodimenzation domains in each chain can be located carboxyl-terminal to the immunoglobulin constant region of that chain.
  • one immunoglobulin heterodimenzation domain in each chain can be located amino-terminal to the immunoglobulin constant region of that chain, while the other immunoglobulin heterodimerization domain of each chain can be located carboxyl-terminal to the
  • the immunoglobulin constant region is interposed between the two immunoglobulin
  • ROR1 -binding polypeptides of the present disclosure comprise an immunoglobulin constant region (also referred to as a constant region) in a polypeptide chain.
  • an immunoglobulin constant region slows clearance of the homodimeric and heterodimeric proteins formed from two ROR1 -binding polypeptide chains from circulation after administration to a subject.
  • an immunoglobulin constant region further enables relatively easy modulation of dimeric polypeptide effector functions (e.g.
  • an immunoglobulin constant region of one or both of the polypeptide chains of the polypeptide homodimers and heterodimers of the present disclosure will be capable of mediating one or more of these effector functions
  • one or more of these effector functions are reduced or absent in an immunoglobulin constant region of one or both of the polypeptide chains of the polypeptide homodimers and heterodimers of the present disclosure, as compared to a corresponding wild-type immunoglobulin constant region.
  • ROR1 -binding polypeptides designed to elicit RTCC such as, e.g. , via the inclusion of a CD3-binding domain (for example ROR248, ROR246, ROR252, ROR250, or ROR243), an CD3-binding domain (for example ROR248, ROR246, ROR252, ROR250, or ROR243), an CD3-binding domain (for example ROR248, ROR246, ROR252, ROR250, or ROR243), an CD3-binding domain (for example ROR248, ROR246, ROR252, ROR250, or ROR243), an CD3-binding domain (for example ROR248, ROR246, ROR252, ROR250, or ROR243), an CD3-binding domain (for example ROR248, ROR246, ROR252, ROR250, or ROR243), an CD3-binding domain (for example ROR248, ROR246, ROR252, ROR250, or ROR243), an
  • immunoglobulin constant region preferably has reduced or no effector function relative to a corresponding wild-type immunoglobulin constant region.
  • An immunoglobulin constant region present in ROR1 -binding polypeptides of the present disclosure can comprise or is derived from part or all of: a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof.
  • an immunoglobulin constant region can comprise a CH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3 domains, a CH4 domain, two CH4 domains, and a CH2 domain and part of a CH3 domain.
  • a ROR1 - binding polypeptide or protein does not comprise a CH1 domain.
  • a CH2 domain that can form an immunoglobulin constant region of a ROR1 -binding polypeptide of the present disclosure can be a wild type immunoglobulin CH2 domain or an altered immunoglobulin CH2 domain thereof from certain immunoglobulin classes or subclasses (e.g. , lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, or IgD) and from various species (including human, mouse, rat, and other mammals).
  • a CH2 domain of an anti-ROR1 x anti-CD3 molecule is a wild type human immunoglobulin CH2 domain, such as wild type CH2 domains of human lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, or IgD, as set forth in SEQ ID NOS: 1 15, 199-201 and 195-197, respectively, of PCT Publication WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
  • the CH2 domain is a wild type human lgG1 CH2 domain as set forth in SEQ ID NO: 1 15 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
  • a CH2 domain of an anti-ROR1 x anti-CD3 molecule is an altered immunoglobulin CH2 region (e.g. , an altered human lgG 1 CH2 domain) that comprises an amino acid substitution at the asparagine of position 297 (e.g. , asparagine to alanine).
  • an amino acid substitution reduces or eliminates glycosylation at this site and abrogates efficient Fc binding to FcvR and C1 q.
  • the sequence of an altered human lgG1 CH2 domain with an Asn to Ala substitution at position 297 is set forth in SEQ ID NO:324 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
  • a CH2 domain of an anti-ROR1 x anti-CD3 molecule is an altered immunoglobulin CH2 region (e.g. , an altered human lgG 1 CH2 domain) that comprises at least one substitution or deletion at positions 234 to 238.
  • an immunoglobulin CH2 region can comprise a substitution at position 234, 235, 236, 237 or 238, positions 234 and 235, positions 234 and 236, positions 234 and 237, positions 234 and 238, positions 234-236, positions 234, 235 and 237, positions 234, 236 and 238, positions 234, 235, 237, and 238, positions 236-238, or any other combination of two, three, four, or five amino acids at positions 234-238.
  • an altered CH2 region can comprise one or more (e.g. , two, three, four or five) amino acid deletions at positions 234-238, for instance, at one of position 236 or position 237 while the other position is substituted.
  • the above-noted mutation(s) decrease or eliminate the antibody- dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide homodimer or heterodimer that comprises the altered CH2 domain.
  • the amino acid residues at one or more of positions 234-238 has been replaced with one or more alanine residues.
  • a CH2 domain of an anti-ROR1 x anti-CD3 molecule is an altered immunoglobulin CH2 region (e.g. , an altered human lgG 1 CH2 domain) that comprises one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331 .
  • an immunoglobulin CH2 region can comprise a substitution at position 253, 310, 318, 320, 322, or 331 , positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of two, three, four, five or six amino acids at positions 253, 310, 318, 320, 322, and 331.
  • the above-noted mutation(s) decrease or eliminate the complement-dependent cytotoxicity (CDC) of a polypeptide homodimer or heterodimer that comprises the altered CH2 domain.
  • an altered CH2 region of an anti-ROR1 x anti-CD3 molecule can further comprise one or more (e.g. , two, three, four, or five) additional substitutions at positions 234-238.
  • an immunoglobulin CH2 region can comprise a substitution at positions 234 and 297, positions 234, 235, and 297, positions 234, 236 and 297, positions 234-236 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any combination of two, three, four, or five amino acids at positions 234-238 in addition to position 297.
  • an altered CH2 region can comprise one or more (e.g. , two, three, four or five) amino acid deletions at positions 234-238, such as at position 236 or position 237.
  • the additional mutation(s) decreases or eliminates the antibody- dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide homodimer or heterodimer that comprises the altered CH2 domain.
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • the amino acid residues at one or more of positions 234-238 have been replaced with one or more alanine residues.
  • only one of the amino acid residues at positions 234-238 has been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g. , alanine or serine).
  • a mutated CH2 region of an anti-ROR1 x anti-CD3 molecule in addition to one or more (e.g. , 2, 3, 4, or 5) amino acid substitutions at positions 234-238, a mutated CH2 region of an anti-ROR1 x anti-CD3 molecule (e.g. , an altered human lgG 1 CH2 domain) in a fusion protein of the present disclosure can contain one or more (e.g. , 2, 3, 4, 5, or 6) additional amino acid substitutions (e.g. , substituted with alanine) at one or more positions involved in complement fixation (e.g. , at positions I253, H310, E318, K320, K322, or P331).
  • additional amino acid substitutions e.g. , substituted with alanine
  • mutated immunoglobulin CH2 regions include human lgG 1 , lgG2, lgG4 and mouse lgG2a CH2 regions with alanine substitutions at positions 234, 235, 237 (if present), 318, 320 and 322.
  • An exemplary mutated immunoglobulin CH2 region is mouse IGHG2c CH2 region with alanine
  • an altered CH2 region of an anti-ROR1 x anti-CD3 molecule can further comprise one or more (e.g. , two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331 .
  • an immunoglobulin CH2 region can comprise a (1) substitution at position 297, (2) one or more substitutions or deletions or a combination thereof at positions 234-238, and one or more (e.g.
  • amino acid substitutions at positions 1253, H310, E318, K320, K322, and P331 such as one, two, three substitutions at positions E318, K320 and K322.
  • the amino acids at the above-noted positions can be substituted by alanine or serine.
  • an immunoglobulin CH2 region polypeptide of an anti- ROR1 x anti-CD3 molecule comprises: (i) an amino acid substitution at the asparagines of position 297 and one amino acid substitution at position 234, 235, 236 or 237; (ii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at two of positions 234-237; (iii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at three of positions 234-237; (iv) an amino acid substitution at the asparagine of position 297, amino acid substitutions at positions 234, 235 and 237, and an amino acid deletion at position 236; (v) amino acid substitutions at three of positions 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three of positions 234-237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320 and 322.
  • Exemplary altered immunoglobulin CH2 regions with amino acid substitutions at the asparagine of position 297 include: human lgG1 CH2 region with alanine substitutions at L234, L235, G237 and N297 and a deletion at G236 (SEQ ID NO:325 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), human lgG2 CH2 region with alanine substitutions at V234, G236, and N297 (SEQ ID NO:326 of
  • an altered CH2 region of an anti-ROR1 x anti-CD3 molecule can contain one or more additional amino acid substitutions at one or more positions other than the above-noted positions.
  • Such amino acid substitutions can be conservative or non-conservative amino acid substitutions.
  • P233 can be changed to E233 in an altered lgG2 CH2 region (see, e.g. , SEQ ID NO:326 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein).
  • the altered CH2 region can contain one or more amino acid insertions, deletions, or both.
  • the insertion(s), deletion(s) or substitution(s) can be anywhere in an immunoglobulin CH2 region, such as at the N- or C-terminus of a wild type immunoglobulin CH2 region resulting from linking the CH2 region with another region (e.g. , a binding domain or an immunoglobulin
  • heterodimerization domain via a hinge.
  • an altered CH2 region in a polypeptide of the present disclosure comprises or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild type immunoglobulin CH2 region, such as the CH2 region of wild type human lgG1 , lgG2, or lgG4, or mouse lgG2a (e.g. , IGHG2C).
  • a wild type immunoglobulin CH2 region such as the CH2 region of wild type human lgG1 , lgG2, or lgG4, or mouse lgG2a (e.g. , IGHG2C).
  • An altered immunoglobulin CH2 region in a ROR1 -binding polypeptide of the present disclosure can be derived from a CH2 region of various immunoglobulin isotypes, such as lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, and IgD, from various species (including human, mouse, rat, and other mammals).
  • an altered immunoglobulin CH2 region in a fusion protein of the present disclosure can be derived from a CH2 region of human lgG 1 , lgG2 or lgG4, or mouse lgG2a (e.g. , IGHG2c), whose sequences are set forth in SEQ ID NOS: 1 15, 199, 201 , and 320 of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
  • an altered CH2 domain of an anti-ROR1 x anti-CD3 molecule is a human lgG1 CH2 domain with alanine substitutions at positions 235, 318, 320, and 322 (i.e. , a human lgG1 CH2 domain with L235A, E318A, K320A and K322A substitutions) (SEQ ID NO:595 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), and optionally an N297 mutation (e.g. , to alanine).
  • an altered CH2 domain is a human lgG 1 CH2 domain with alanine substitutions at positions 234, 235, 237, 318, 320 and 322 (i.e. , a human lgG 1 CH2 domain with L234A, L235A, G237A, E318A, K320A and K322A substitutions) (SEQ ID NO:596 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), and optionally an N297 mutation (e.g. , to alanine).
  • an immunoglobulin constant region of a ROR1 -binding protein or polypeptide comprises a human lgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A, according to the EU numbering system.
  • an altered CH2 domain of an anti-ROR1 x anti-CD3 molecule is an altered human lgG 1 CH2 domain with mutations known in the art that enhance immunological activities such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any combination thereof.
  • the CH3 domain that can form an immunoglobulin constant region of a ROR1 - binding polypeptide of the present disclosure can be a wild type immunoglobulin CH3 domain or an altered immunoglobulin CH3 domain thereof from certain immunoglobulin classes or subclasses (e.g. , lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM) of various species (including human, mouse, rat, and other mammals).
  • immunoglobulin classes or subclasses e.g. , lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM
  • a CH3 domain of an anti-ROR1 x anti-CD3 molecule is a wild type human immunoglobulin CH3 domain, such as wild type CH3 domains of human lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM as set forth in SEQ ID NOS: 1 16, 208- 210, 204-207, and 212, respectively of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
  • the CH3 domain is a wild type human lgG1 CH3 domain as set forth in SEQ ID NO: 1 16 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
  • a CH3 domain of an anti-ROR1 x anti-CD3 molecule is an altered human immunoglobulin CH3 domain, such as an altered CH3 domain based on or derived from a wild-type CH3 domain of human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM antibodies.
  • an altered CH3 domain can be a human lgG1 CH3 domain with one or two mutations at positions H433 and N434 (positions are numbered according to EU numbering). The mutations in such positions can be involved in complement fixation.
  • an altered CH3 domain of an anti-ROR1 x anti-CD3 molecule can be a human lgG 1 CH3 domain but with one or two amino acid substitutions at position F405 or Y407. The amino acids at such positions are involved in interacting with another CH3 domain.
  • an altered CH3 domain of an anti-ROR1 x anti-CD3 molecule can be an altered human lgG1 CH3 domain with its last lysine deleted. The sequence of this altered CH3 domain is set forth in SEQ ID NO:761 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
  • ROR1 -binding polypeptides e.g., an anti-ROR1 x anti-CD3 molecule
  • a polypeptide heterodimer comprise a CH3 pair that comprises so called "knobs-into-holes" mutations (see, Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et al. , Protein Engineering 9:617-21 , 1966). More specifically, mutations can be introduced into each of the two CH3 domains of each polypeptide chain so that the steric complementarity required for CH3/CH3 association obligates these two CH3 domains to pair with each other.
  • a CH3 domain in one single chain polypeptide of a polypeptide heterodimer can contain a T366W mutation (a "knob” mutation, which substitutes a small amino acid with a larger one), and a CH3 domain in the other single chain polypeptide of the polypeptide heterodimer can contain a Y407A mutation (a "hole” mutation, which substitutes a large amino acid with a smaller one).
  • Other exemplary knobs- into-holes mutations include (1) a T366Y mutation in one CH3 domain and a Y407T in the other CH3 domain, and (2) a T366W mutation in one CH3 domain and T366S, L368A and Y407V mutations in the other CH3 domain.
  • the CH4 domain that can form an immunoglobulin constant region of ROR1 -binding polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule) of the present disclosure can be a wild type immunoglobulin CH4 domain or an altered immunoglobulin CH4 domain thereof from IgE or IgM molecules.
  • the CH4 domain of an anti-ROR1 x anti-CD3 molecule is a wild type human immunoglobulin CH4 domain, such as wild type CH4 domains of human IgE and IgM molecules as set forth in SEQ ID NOS:213 and 214, respectively, of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
  • a CH4 domain of an anti-ROR1 x anti-CD3 molecule is an altered human immunoglobulin CH4 domain, such as an altered CH4 domain based on or derived from a CH4 domain of human IgE or IgM molecules, which have mutations that increase or decrease an immunological activity known to be associated with an IgE or IgM Fc region.
  • an immunoglobulin constant region of ROR1 -binding polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule) of the present disclosure comprises a combination of CH2, CH3 or CH4 domains (i.e. , more than one constant region domain selected from CH2, CH3 and CH4).
  • the immunoglobulin constant region can comprise CH2 and CH3 domains or CH3 and CH4 domains.
  • the immunoglobulin constant region can comprise two CH3 domains and no CH2 or CH4 domains (i.e. , only two or more CH3).
  • the multiple constant region domains that form an immunoglobulin constant region of an anti-ROR1 x anti-CD3 molecule can be based on or derived from the same immunoglobulin molecule, or the same class or subclass
  • the immunoglobulin constant region is an IgG CH2CH3 (e.g. , lgG 1 CH2CH3, lgG2 CH2CH3, and lgG4 CH2CH3) and can be a human (e.g. , human lgG 1 , lgG2, and lgG4) CH2CH3.
  • the immunoglobulin constant region of an anti-ROR1 x anti-CD3 molecule comprises (1) wild type human lgG1 CH2 and CH3 domains, (2) human lgG 1 CH2 with N297A substitution (i.e. , CH2(N297A)) and wild type human lgG 1 CH3, or (3) human lgG1 CH2(N297A) and an altered human lgG 1 CH3 with the last lysine deleted.
  • the multiple constant region domains of an anti-ROR1 x anti-CD3 molecule can be based on or derived from different immunoglobulin molecules, or different classes or subclasses immunoglobulin molecules.
  • an immunoglobulin constant region comprises both human IgM CH3 domain and human lgG 1 CH3 domain.
  • the multiple constant region domains that form an immunoglobulin constant region of an anti-ROR1 x anti-CD3 molecule can be directly linked together or can be linked to each other via one or more (e.g. , about 2-10) amino acids.
  • Exemplary immunoglobulin constant regions that can be used in an anti-ROR1 x anti-CD3 molecule are set forth in SEQ ID NOS:305-309, 321 , 323, 341 , 342, and 762 of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
  • the immunoglobulin constant regions of both ROR1 -binding polypeptides of a polypeptide homodimer or heterodimer of an anti-ROR1 x anti-CD3 molecule are identical to each other.
  • the immunoglobulin constant region of one polypeptide chain of a heterodimeric protein is different from the immunoglobulin constant region of the other polypeptide chain of the heterodimer.
  • one immunoglobulin constant region of a heterodimeric protein can contain a CH3 domain with a "knob" mutation, whereas the other immunoglobulin constant region of the heterodimeric protein can contain a CH3 domain with a "hole" mutation.
  • the disclosure relates to ROR1 -binding proteins and polypeptides that may comprise any of the sequences shown in Table 3. Sequences for various cloned sequences and components are also presented in Table 3 and summarized in Table 12. Amino acid sequences given for polypeptide constructs do not include the human or rabbit
  • VL region gccaagatcacctgcaccctgtcctccgcccacaagaccgacaccatcga kitctlssahktdtidwyq (SEQ ID NO:39) ctggtaccagcagctgcagggcgaggcccccggtacctgatgcaggtgc qlqgeaprylmqvqsd
  • R12 VL (LI) gtgaagctgacctgcaccctgtcctccgcccacaagaccgacaccatcga Itctlssahktdtidwyq (SEQ ID NO:55) ctggtaccagcagcagcccgagaagggcccccggtacctgatgcaggtg qqpekgprylmqvqsd
  • ROR036 caggtgcagctggtggagtccggcggcggctggtgaagcccggcggct qvqlvesggglvkpggsl SEQ ID NO:90 ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:91)

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Abstract

The present disclosure relates to protein molecules that specifically bind to receptor tyrosine kinase-like orphan receptor 1 (ROR1), which may have at least one humanized ROR1-binding domain. Such molecules are useful for the treatment of cancer. The protein molecule binding to ROR1 may have a second binding domain that binds to another target. In one embodiment, multispecific polypeptide molecules bind both ROR1-expressing cells and the T-cell receptor complex on T-cells to induce target-dependent T-cell cytotoxicity, activation, and proliferation. The disclosure also provides pharmaceutical compositions comprising the ROR1-binding poypeptide molecules, nucleic acid molecules encoding these polypeptides and methods of making these molecules.

Description

RECEPTOR TYROSINE KINASE-LIKE ORPHAN RECEPTOR 1 BINDING PROTEINS AND RELATED COMPOSITIONS AND METHODS
[001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 62/091 ,307, filed on December 12, 2014 and of U.S. Provisional Patent Application No.
62/221 , 194, filed on September 21 , 2015. The contents of both these applications are herein incorporated by reference in their entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: EMER-037_01 WO_SeqList_ST25.txt, date recorded: December 1 1 , 2015, file size 595, 175 bytes).
FIELD OF THE DISCLOSURE
[003] The present disclosure relates to molecules that specifically bind to receptor tyrosine kinase-like orphan receptor 1 (ROR1), which may have at least one humanized ROR1 - binding domain. These molecules are useful for the characterization or treatment of disorders characterized by expression of ROR1 , such as cancer. A protein therapeutic binding to ROR1 may be a monospecific protein therapeutic or a multispecific protein therapeutic. A multspecific protein therapeutic may bind both ROR1 -expressing cells and the T-cell receptor complex on T-cells to induce target-dependent T-cell cytotoxicity, activation and proliferation.
BACKGROUND OF THE DISCLOSURE
[004] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a glycosylated type I membrane protein normally expressed in early embryonic development and attenuated during fetal development. Normal non-embryonic and non-fetal tissues do not express R OR1 at detectable levels, except for hematogones, which are unusual CD5 precursor B cells. However, ROR1 surface expression has been reported to be upregulated in many hematologic and solid cancers. For example, ROR1 has been found to be expressed in chronic lymphocytic leukemia (CLL) and mantle cell leukemia (MCL) (Baskar et al., Clin. Cancer Res., 14: 396-404 (2008); Hudecek et al., Blood, 1 16: 4532-4541 (2010)).
Additionally, ROR1 acts as a receptor for Wnt ligands and can activate non-canonical Wnt signaling, which can provide a survival advantage to cancer cells.
[005] Currently there are no FDA approved therapeutics that specifically target or bind ROR1 . There is a need for new therapeutics to treat malignancies in which ROR1 is expressed (for example, pancreatic cancer, triple negative breast cancer, and ovarian cancer).
SUMMARY OF THE DISCLOSURE
[006] In certain embodiments, the disclosure encompasses a receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding domain that specifically binds to human ROR1 , wherein the ROR1 -binding polypeptide comprises (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12 or a sequence that differs from SEQ ID NO: 12 by at least one amino acid substitution; (b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14 or a sequence that differs from SEQ ID NO: 14 by at least one amino acid substitution; (c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268, or a sequence that differs from SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268 by at least one amino acid substitution; (d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:266 or a sequence that differs from SEQ ID NO:6 or SEQ ID NO:266 by at least one amino acid substitution; (e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:8 or a sequence that differs from SEQ ID NO:8 by at least one amino acid substitution; and (f) the HCDR3 has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO:267 or a sequence that differs from SEQ ID NO: 10 or SEQ ID NO:267 by at least one amino acid substitution. In some embodiments, the disclosure relates to a ROR1 - binding domain that binds an epitope near the junction of the Frizzled and Kringle domains of ROR1 (e.g., human ROR1). In certain aspects, this epitope is a discontinuous and/or conformational epitope. In some embodiments, a ROR1 -binding domain binds residues in stretches 259-273 (LCQTEYIFARSNPMI (residues 259-273 of SEQ ID NO: 128)) and 389- 403 (PACDSKDSKEKNKME (residues 389-403 of SEQ ID NO: 128)) of the ROR1 ectodomain.
[007] In certain embodiments, the ROR1 -binding domain of a ROR1 -binding polypeptide comprises (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 ,
HCDR2, and HCDR3, wherein (a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO:47 or a sequence that differs from SEQ ID NO:47 by at least one amino acid substitution; (b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO:49 or a sequence that differs from SEQ ID NO:49 by at least one amino acid substitution; (c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO:51 , or a sequence that differs from SEQ ID NO:51 by at least one amino acid substitution; (d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:41 or a sequence that differs from SEQ ID NO:41 by at least one amino acid substitution; (e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:43 or a sequence that differs from SEQ ID NO:43 by at least one amino acid substitution; and (f) the HCDR3 has an amino acid sequence set forth in SEQ ID NO:45 or a sequence that differs from SEQ ID NO:45 by at least one amino acid substitution.
[008] The disclosure relates to a ROR1 -binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12; (b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14; (c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268; (d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:266; (e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:8; and (f) the HCDR3 has an amino acid sequence set forth in SEQ ID NO:267.
[009] The at least one amino acid substitution may be a conservative or a non-conservative amino acid substitution. In some embodiments, an LCDR1 , LCDR2, LCDR3, HCDR1 , HCDR2, and/or HCDR3 differs from a recited sequence by 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, a CDR of the present disclosure contains about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g. , conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared to the CDR sequence of a known monoclonal antibody.
[0010] In some embodiments, the disclosure encompasses a ROR1 -binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; (ii) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252; (iii) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:333 or SEQ ID NO:337; or (iv) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:335 or SEQ ID NO:339. In some embodiments, a ROR1 -binding domain comprises (i) an immunoglobulin light chain variable region comprising an amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; (ii) an immunoglobulin heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252; (iii) the immunoglobulin light chain comprises an amino acid sequence set forth in SEQ ID NO:333 or SEQ ID NO:337; or (iv) the immunoglobulin light chain comprises an amino acid sequence set forth in SEQ ID NO:335 or SEQ ID NO:339.
[0011] In some embodiments, the disclosure relates to a ROR1 -binding domain wherein (i) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:23 and the
immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27; (ii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27; (iii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:59; (iv) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:61 ; (v) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:59; (vi) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in
SEQ ID NO:61 , (vii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:210; (viii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:210; (ix) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:212; (x) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:212; (xi) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the
immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232; (xii) the
immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232; (xiii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:244; (xiv) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:250 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:252; (xv) the immunoglobulin light chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:333 and the immunoglobulin heavy chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:335; or (xvi) the immunoglobulin light chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:337 and the immunoglobulin heavy chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:339.
[0012] In further embodiments, the disclosure encompasses a ROR1 -binding domain wherein (i) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27; (ii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27; (iii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59; (iv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 ; (v) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59; (vi) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 , (vii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:210; (viii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:210; (ix) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:212; (x) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:212; (xi) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the
immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232; (xii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232; (xiii) the
immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:244; (xiv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:250 and the
immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:252; (xv) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:333 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:335; or (xvi) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:337 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:339.
[0013] In certain aspects, at least one of the light chain variable region or the heavy chain variable region of a ROR1 -binding domain is humanized. In some embodiments, a ROR1- binding domain is a single chain variable fragment (scFv). The light chain variable region of said scFv may be carboxy-terminal or amino-terminal to the heavy chain variable region of said scFv. In some variations, a ROR1 -binding domain specifically binds to human ROR1 and comprises SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69. In some embodiments, the disclosure encompasses a ROR1 - binding polypeptide comprising a ROR1 -binding domain as described herein and an immunoglobulin constant region. A ROR1 -binding polypeptide may specifically bind to human ROR1 and comprise SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91 .
[0014] In some embodiments, a ROR1 -binding domain binds to human ROR1 with a dissociation constant that is lower than the dissociation constant of a ROR1 -binding domain comprising SEQ ID NO:73. Some ROR1 -binding domains have a reduced isoelectric point compared to the isoelectric point of a ROR1 -binding domain comprising an immunoglobulin light chain variable region of SEQ ID NO:220 and an immunoglobulin heavy chain variable region of SEQ ID NO:232. In some embodiments, a ROR1 -binding domain having a lower dissociation constant and/or a reduced isolectric point comprises one or more CDR amino acid sequences set forth in Table 3.
[0015] In certain embodiments, a ROR1 -binding domain (or a ROR1 -binding polypeptide comprising this domain) is conjugated to a drug or a toxin. In one aspect, an anti-ROR1 x anti-CD3 molecule as described herein is conjugated to a drug or a toxin. In some embodiments, ROR248, ROR246, ROR252, ROR250, or ROR243 is conjugated to a drug or a toxin.
[0016] The disclosure encompasses an isolated nucleic acid molecule encoding a ROR1 - binding domain described herein or a portion of said ROR1 -binding domain. In non-limiting examples, this isolated nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID
NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:209, SEQ ID NO:21 1 , SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221 , SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231 , SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241 , SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251 , SEQ ID NO:253, or SEQ ID NO:255. [0017] The disclosure further encompasses a ROR1 -binding polypeptide comprising: (i) a ROR1 -binding domain and (ii) a second binding domain. In some embodiments, a ROR1 - binding polypeptide comprises, in order from amino-terminus to carboxyl-terminus or in order from carboxyl-terminus to amino-terminus, (i) a ROR1 -binding domain, (ii) a hinge region and (iii) an immunoglobulin constant region. In some embodiments, a ROR1 -binding polypeptide comprises, in order from amino-terminus to carboxyl-terminus, (i) a ROR1 - binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl- terminus linker, and (v) a second binding domain. In other embodiments, a ROR1 -binding polypeptide comprises, in order from carboxyl-terminus to amino-terminus, (i) a ROR1 - binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) an amino- terminus linker, and (v) a second binding domain. In certain variations, the first and/or the second binding domain is an scFv. Non-limiting examples of carboxyl-terminus and amino- terminus linkers include flexible linkers comprising glycine-serine (e.g., (Gly4Ser)) repeats and linkers derived from (i) a stalk region of a type II C lectin or (ii) an immunoglobulin hinge region. In certain aspects, a carboxyl-terminus linker (or an amino-terminus linker) comprises or consists of SEQ ID NO:265, SEQ ID NO:301 , SEQ ID NO:302, or SEQ ID NO:303. In some aspects, the disclosure relates to a ROR1 -binding polypeptide (e.g., multispecific), wherein (i) the ROR1 -binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3; and (ii) the second binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3. In some embodiments, a ROR1 -binding polypeptide comprises, in order from amino-terminus to carboxyl-terminus or in order from carboxyl- terminus to amino-terminus, (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) a CD3-binding domain; wherein said ROR1 -binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 12; (b) the LCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 14; (c) the LCDR3 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID
NO:268; (d) the HCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:266; (e) the HCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:8; and (f) the HCDR3 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:267; and wherein said CD3-binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 10, 1 1 1 and 1 12, respectively; or (b') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 272, 273 and 274, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 269, 270 and 271 , respectively; (c') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 278, 279 and 280, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 275, 276 and 277, respectively; (d') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 284, 285 and 286, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 281 , 282 and 283, respectively; (e') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 290, 291 and 292, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 287, 288 and 289, respectively; or (Γ) the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 296, 297 and 298, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 293, 294 and 295, respectively.
[0018] In some embodiments, a ROR1 -binding polypeptide comprises an immunoglobulin constant region that comprises immunoglobulin CH2 and CH3 domains of lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 or IgD. In one embodiment, an immunoglobulin constant region comprises a human lgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A, according to the EU numbering system.
[0019] In certain aspects, a ROR1 -binding polypeptide of the disclosure (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) does not exhibit or exhibits minimal antibody- dependent cell-mediated cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDC) activity. In some embodiments, ROR248, ROR246, ROR252, ROR250, or ROR243 does not exhibit or exhibits minimal ADCC activity or CDC activity.
[0020] In certain variations, a ROR1 -binding polypeptide comprises a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex, including TCR alpha/beta chains. The ROR1 - binding polypeptide may induce redirected T-cell cytotoxicity (RTCC). In some embodiments, a ROR1 -binding polypeptide that also binds a T-cell, CD3, CD3z or a TCR complex may induce T-cell activation, T-cell proliferation and/or T-cell-dependent lysis of ROR1 - expressing cells (e.g., tumor or cancer cells). In certain embodiments, a ROR1 -binding polypeptide that also binds a T-cell, CD3, CD3z or a TCR complex does not induce or induces a minimally detectable cytokine release from said T cell.
[0021] In some embodiments, the second binding domain of a ROR1 -binding polypeptide described herein competes for binding to CD3z with a monoclonal antibody selected from CRIS-7, HuM291 and I2C. In certain embodiments, the second binding domain specifically binds CD3 and comprises an immunoglobulin light chain variable region and an
immunoglobulin heavy chain variable region; wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:260; or at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:261 ; and wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:259.
[0022] In some aspects, the second binding domain of a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) described herein is a humanized binding domain. In certain embodiments, the second binding domain of a ROR1 -binding polypeptide (e.g., an anti- ROR1 x anti-CD3 molecule) described herein is a single-chain variable fragment (scFv).
[0023] In some embodiments, the second binding domain of a ROR1 -binding polypeptide described herein comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region derived from a monoclonal antibody selected from CRIS-7, HuM291 and I2C. In certain embodiments, the second binding domain of a ROR1 -binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 10, 1 1 1 and 1 12, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 272, 273 and 274, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 269, 270 and 271 , respectively. Alternatively, the second binding domain of a ROR1 -binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 278, 279 and 280, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 275, 276 and 277, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 284, 285 and 286, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 281 , 282 and 283, respectively. In another embodiment, the second binding domain of a ROR1 - binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 290, 291 and
292, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 287, 288 and 289, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 296, 297 and 298, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs:
293, 294 and 295, respectively. In some aspects, the second binding domains comprising the CDR sequences recited in this paragraph are humanized.
[0024] In some embodiments, a ROR1 -binding domain competes for binding to human ROR1 with a single chain variable fragment (scFv) having the amino acid sequence set forth in SEQ ID NO: 18, SEQ I D NO:21 or SEQ ID NO:53.
[0025] The ROR1 -binding domain of a ROR1 -binding polypeptide (e.g. , an anti-ROR1 x anti- CD3 molecule) described herein may be a humanized binding domain.
[0026] In some embodiments, the ROR1 -binding domain of a ROR1 -binding polypeptide (e.g. , an anti-ROR1 x anti-CD3 molecule) described herein is a single chain variable fragment (scFv). In some embodiments, a ROR1 -binding domain that is an scFv comprises SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69.
[0027] In certain aspects, the disclosure encompasses a ROR1 -binding polypeptide, wherein the light chain variable region of the ROR1 -binding domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:39, SEQ I D NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; and the heavy chain variable region of the ROR1 -binding domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:2; SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ I D NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252. [0028] The disclosure relates to a ROR1 -binding polypeptide, wherein the light chain variable region of the ROR1 -binding domain comprises the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; and the heavy chain variable region of the ROR1 -binding domain comprises an amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252. The ROR1 -binding domain may be an scFv and may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO:21 , SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 or SEQ ID NO:69.
[0029] In some embodiments, a ROR1 -binding polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:71 , SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91 , SEQ ID NO:214, SEQ ID NO:218, or SEQ ID NO:222. In other embodiments, a ROR1 -binding polypeptide may comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID
NO:299, or SEQ ID NO:300. In additional embodiments, a ROR1 -binding polypeptide may comprise an amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321 , SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, or SEQ ID NO:331 .
[0030] In some embodiments, a ROR1 -binding polypeptide comprises in order from amino- terminus to carboxyl-terminus (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker, and (v) a second binding domain, wherein said ROR1 -binding polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321 , SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, or SEQ ID NO:331 .
[0031] In certain embodiments, a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) comprises a ROR1 -binding domain that is an scFv comprising an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; and wherein said light chain variable region and said heavy chain variable region are joined by an amino acid sequence comprising (Gly4Ser)n, wherein n=1 -5 (SEQ ID NO: 127).
[0032] In some embodiments, a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) comprises a second binding domain that is an scFv comprising an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; and wherein said light chain variable region and said heavy chain variable region are joined by an amino acid sequence comprising (Gly4Ser)n, wherein n=1 -5 (SEQ ID NO: 127).
[0033] The disclosure relates to a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) that is a dimer of two identical polypeptides, wherein each polypeptide may be a ROR1 -binding polypeptide comprising the sequences disclosed herein. In some embodiments, the disclosure encompasses a dimer of two identical polypeptides, wherein each polypeptide in the dimer is ROR248, ROR246, ROR252, ROR250, or ROR243.
[0034] A ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) may further comprise an immunoglobulin heterodimerization domain. The immunoglobulin heterodimerization domain may optionally comprise an immunoglobulin CH1 domain or an immunoglobulin CL domain.
[0035] In some embodiments, a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) is a heterodimeric ROR1 -binding protein comprising (i) a first polypeptide chain comprising, in order from amino-terminus to carboxyl-terminus or from carboxyl-terminus to amino-terminus, (a) a ROR1 -binding domain that specifically binds human ROR1 , (b) a first hinge region, (c) a first immunoglobulin constant region, and (d) a first immunoglobulin heterodimerization domain; and (ii) a second polypeptide chain comprising, in order from amino-terminus to carboxyl-terminus or from carboxyl-terminus to amino-terminus, (a') a second hinge region, (b') a second immunoglobulin constant region, and (c') a second immunoglobulin heterodimerization domain that is different from the first immunoglobulin heterodimerization domain of the first single chain polypeptide, wherein the first and second immunoglobulin heterodimerization domains associate with each other to form a heterodimer. The first immunoglobulin heterodimerization domain may comprise an immunoglobulin CH1 domain and the second immunoglobulin heterodimerization domain may comprise an immunoglobulin CL domain, or the first immunoglobulin heterodimerization domain may comprise an immunoglobulin CL domain and the second immunoglobulin heterodimerization domain may comprise an immunoglobulin CH1 domain. At least one of the first and second immunoglobulin constant regions may comprise immunoglobulin CH2 and CH3 domains of lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD or any combination thereof; an immunoglobulin CH3 domain of lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM or any combination thereof; or immunoglobulin CH3 and CH4 domains of IgE, IgM or a combination thereof. The ROR1 -binding polypeptide heterodimer may comprise a second polypeptide chain that further comprises a second binding domain. The second binding domain may be amino-terminal to the second hinge region. A heterodimer may comprise ROR248, ROR246, ROR252, ROR250, or ROR243.
[0036] In some embodiments, a ROR1 -binding polypeptide is a bispecific single chain antibody molecule comprising a ROR1 binding domain and a CD3 binding domain, wherein the binding domains are arranged in the order VH ROR1 -VL ROR1 -VH CD3-VL CD3 or VL ROR1 -VH ROR1 -VH CD3-VL CD3 or VH CD3-VL CD3-VH ROR1 -VL ROR1 or VH CD3-VL CD3-VL ROR1-VH ROR1 or VH ROR1 -VL ROR1 -VL CD3-VH CD3 or VL ROR1 -VH ROR1 - VL CD3-VH CD3 or VL CD3-VH CD3-VH ROR1 -VL ROR1 or VL CD3-VH CD3-VL ROR1 - VH ROR1 . In some embodiments, these VH and VL regions are linked, such as in an scFv. In some embodiments, the scFvs are joined by a linker with or without an immunoglobulin CH1 CH2 region. In some embodiments, the two scFvs are joined as described in, for example, US20130295121 , US 20130129730, WO 2010/037836, WO 2004/106381 or WO 201 1/121 1 10, each of which is incorporated herein by reference in its entirety.
[0037] The disclosure encompasses an isolated nucleic acid molecule encoding a ROR1 - binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) described herein or a portion of said ROR1 -binding polypeptide. The isolated nucleic acid molecule may comprise a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:209, SEQ ID NO:21 1 , SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221 , SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231 , SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241 , SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251 , SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, or SEQ ID NO:338.
[0038] The disclosure relates to an expression vector comprising a nucleic acid segment encoding a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) described herein, wherein the nucleic acid segment is operatively linked to regulatory sequences suitable for expression of the nucleic acid segment in a host cell. The nucleic acid segment of the expression vector may comprise a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:209, SEQ ID NO:21 1 , SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221 , SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231 , SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241 , SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251 , SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, or SEQ ID NO:338.
[0039] The disclosure encompasses an expression vector comprising first and second expression units, wherein the first and second expression units respectively comprise first and second nucleic acid segments encoding the first and second polypeptide chains of a heterodimeric ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule) described herein, and wherein the first and second nucleic acid segments are operably linked to regulatory sequences suitable for expression of the nucleic acid segments in a host cell.
[0040] The disclosure includes a recombinant host cell comprising an expression vector described herein.
[0041] The disclosure relates to a method for producing a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243), the method comprising culturing a recombinant host cell comprising an expression vector described herein under conditions whereby the nucleic acid segment of the vector is expressed, thereby producing the ROR1-binding polypeptide. The method may further comprise recovering the ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein).
[0042] The disclosure encompasses a method for producing a heterodimeric ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule), the method comprising culturing a recombinant host cell comprising first and second expression units, wherein the first and second expression units respectively comprise first and second nucleic acid segments encoding the first and second polypeptide chains of a heterodimeric ROR1 -binding protein, wherein the first and second nucleic acid segments are operably linked to regulatory sequences suitable for expression of the nucleic acid segments in a host cell, and wherein said culturing is under conditions whereby the first and second nucleic acid segments are expressed and the encoded polypeptide chains are produced as the heterodimeric ROR1 - binding protein. The method may further comprise recovering the heterodimeric ROR1 - binding protein (e.g., an anti-ROR1 x anti-CD3 molecule).
[0043] In some embodiments, the disclosure relates to a pharmaceutical composition comprising a ROR1-binding polypeptide or ROR1 -binding protein described herein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243), and a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, a pharmaceutical composition comprises a ROR1-binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) that is a homodimer or a heterodimer. A "homodimer" may be a dimer formed from two identical polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243). A pharmaceutical composition may be formulated in a dosage form selected from the group consisting of: an oral unit dosage form, an intravenous unit dosage form, an intranasal unit dosage form, a suppository unit dosage form, an intradermal unit dosage form, an intramuscular unit dosage form, an intraperitoneal unit dosage form, a
subcutaneous unit dosage form, an epidural unit dosage form, a sublingual unit dosage form, and an intracerebral unit dosage form. The pharmaceutical composition formulated as an oral unit dosage form may be selected from the group consisting of: tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained-release formulations, aerosols, and sprays.
[0044] The disclosure also relates to a method for inducing redirected T-cell cytotoxicity (RTCC) against a cell expressing ROR1 , the method comprising: contacting said ROR1 - expressing cell with a ROR1-binding polypeptide or ROR1 -binding protein (e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein, wherein the second binding domain specifically binds a T-cell, CD3, CD3£ or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; and wherein said contacting is under conditions whereby RTCC against the ROR1 - expressing cell is induced. In some embodiments, the disclosure relates to a method for inducing redirected T-cell cytotoxicity (RTCC) against a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 - expressing cell with a ROR1-binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; wherein said contacting is under conditions whereby RTCC against the ROR1 -expressing cell is induced.
[0045] The disclosure encompasses a method for inducing T-cell dependent lysis of a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide or ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein, wherein the second binding domain specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; and wherein said contacting is under conditions whereby T-cell dependent lysis of the ROR1 -expressing cell is induced. In some embodiments, the disclosure relates to a method for inducing T-cell dependent lysis of a cell expressing receptor tyrosine kinase- like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; wherein said contacting is under conditions whereby T-cell dependent lysis of the ROR1 -expressing cell is induced.
[0046] The disclosure encompasses a method for treating a disorder (e.g., cancer) in a subject, wherein said disorder is characterized by expression of ROR1 , the method comprising administering to the subject a therapeutically effective amount of a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein. In certain embodiments, the disclosure relates to a method for treating a disorder in a subject, wherein said disorder is characterized by expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising administering to the subject a therapeutically effective amount of a ROR1 -binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR. The disclosure also relates to a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein for the manufacture of a medicament for treatment of a disorder (e.g., cancer) in a subject, wherein said disorder is characterized by expression of ROR1 . In some embodiments, the disclosure relates to a receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 for the manufacture of a medicament for treatment of a disorder in a subject, wherein said disorder is characterized by expression of ROR1 . The disclosure includes a ROR1 -binding polypeptide or ROR1 -binding protein (e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein for use in treating a disorder (e.g., cancer) in a subject, wherein said disorder is characterized by expression of ROR1 . In some embodiments, the disclosure relates to a receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 for use in treating a disorder in a subject, wherein said disorder is characterized by expression of ROR1 . The cancer treated by the ROR1 -binding polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) described herein may be breast cancer (e.g., triple negative breast cancer (TNBC)), pancreatic cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), acute
lymphoblastic leukemia (ALL), melanoma, adrenal cancer, bladder cancer or prostate cancer.
[0047] These and other embodiments and/or other aspects of the disclosure will become evident upon reference to the following detailed description of the disclosure and the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] Figure 1 (top panel) is a graph showing the results of a flow cytometry study measuring the binding of four humanized R12 molecules (ROR033, ROR034, ROR035 and ROR036), the chimeric parental R12 molecule ROR016 and the chimeric R1 1 molecule ROR015 to the ROR1 (+) cell line Kasumi-2. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0049] Figure 2 (top panel) is a graph showing the results of a flow cytometry study measuring the binding of two chimeric R1 1 molecules (ROR015, ROR063) to the ROR1 (+) cell line MDA-MB-231 . Phosphate buffered saline (PBS) was used as a control. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph. [0050] Figure 3 (top panel) is a graph showing the results of a flow cytometry study measuring the binding of the hemi-humanized R1 1 molecule ROR091 , the chimeric molecule ROR063 and the fully humanized molecule ROR101 to the ROR1 (+) cell line MDA- MB-231 . Phosphate buffered saline (PBS) was used as a control. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0051] Figure 4A is a graph showing the results of a flow cytometry study measuring the binding of the parental R1 1 antibody ROR066 and the chimeric R1 1 molecule with the scFv in the VL-VH orientation (ROR063) to the ROR1 (+) cell line MDA-MB-231 . Mean
fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
[0052] Figure 4B is a graph showing the results of a flow cytometry study measuring the binding of the parental R12 antibody ROR069 and the chimeric R12 molecule ROR016 to the ROR1 (+) cell line MDA-MB-231 . Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
[0053] Figure 5 is a graph showing the results of a flow cytometry study measuring the binding of the chimeric bispecific R12 molecule ROR021 , the humanized bispecific R12 molecule ROR050, the chimeric bispecific R1 1 molecule ROR070 and the humanized bispecific R1 1 molecule ROR084 to the ROR1 (+) cell line Kasumi-2. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis.
[0054] Figure 6 (top panel) is a graph showing the results of a chromium-51 release assay measuring the effectiveness of the humanized bispecific R12 molecule ROR050 and the parental chimeric bispecific R12 molecule ROR021 at inducing target-dependent T-cell cytotoxicity in 4 hours against MDA-MB-231 cells. Percent specific lysis relative to a total lysis control is shown on the y-axis. Concentration (pM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0055] Figure 7 (top panel) is a graph showing the results of a chromium-51 release assay measuring the effectiveness of the the chimeric bispecific R1 1 molecule ROR070 and the chimeric bispecific R1 1 molecule ROR020 at inducing target-dependent T-cell cytotoxicity in 4 hours against MDA-MB-231 cells. Percent specific lysis relative to a total lysis control is shown on the y-axis. Concentration (pM) of the ROR1 -binding molecules is shown on the x- axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0056] Figure 8 is a graph showing the results of a flow cytometry study measuring the effectiveness of the bispecific R1 1 molecules ROR070 and ROR084 and the bispecific R12 molecules ROR021 and ROR050 at inducing target-dependent T-cell cytotoxicity in 18 hours against Kasumi-2 cells. Percentage of live target cells remaining after 18 hours is shown on the y-axis. Concentration (pM) of the ROR1 -binding molecules is shown on the x-axis.
[0057] Figure 9A is a graph showing the results of a flow cytometry study measuring the effectiveness of the bispecific R1 1 molecules ROR070 and ROR084 and the bispecific R12 molecules ROR021 and ROR050 at inducing CD4+ (CD8") T-cell proliferation in the presence of Kasumi-2 cells. Percentage of dividing (proliferating) cells is shown on the y- axis. Concentration (pM) of the ROR1 -binding molecules is shown on the x-axis.
[0058] Figure 9B is a graph showing the results of a flow cytometry study measuring the effectiveness of the bispecific R1 1 molecules ROR070 and ROR084 and the bispecific R12 molecules ROR021 and ROR050 at inducing CD8+ T-cell proliferation in the presence of Kasumi-2 cells. Percentage of dividing (proliferating) cells is shown on the y-axis.
Concentration (pM) of the ROR1 -binding molecules is shown on the x-axis.
[0059] Figure 10 (top panel) is a graph showing the results of a flow cytometry study measuring the binding of the chimeric molecule ROR063 and the fully humanized molecules ROR101 , ROR1 1 1 , and ROR1 12 to the ROR1 (+) cell line Kasumi-2. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0060] Figure 11 (top panel) is a graph showing the results of a flow cytometry study measuring the binding of the hemi-humanized molecule ROR091 and the fully humanized molecules ROR1 19, ROR1 1 1 , and ROR1 12 to the ROR1 (+) cell line Kasumi-2. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis.
Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0061] Figure 12 is a graph showing the results of a study measuring the serum stability in human serum of anti-ROR1 x anti-CD3 bispecific molecules ROR133 and ROR193 over the number of days specified on the x-axis.
[0062] Figure 13 is a graph showing the results of a study measuring the ability of the ROR1 -directed bispecific molecules ROR182 and ROR192 to inhibit subcutaneous tumor outgrowth of MDA-MB-231 tumors co-implanted with human T cells in NOD/SCID mice. The mean tumor volume (mm3) is shown on the y-axis. Mice were treated with 3 of anti- ROR1 molecules on day 0, 4 and 8 post tumor challenge.
[0063] Figure 14 is a graph showing the results of a study measuring the effect of treatment with ROR1 -directed bispecific molecules ROR1 82 and ROR192 on body weight of
NOD/SCID mice co-implanted with MDA-MB-231 tumors and human T cells. Percentage of maximum body weight is shown on the y-axis. Mice were treated with 3 μg of anti-ROR1 molecules on day 0, 4 and 8 post tumor challenge.
[0064] Figure 15A is a graph (top panel) showing the results of a study measuring the ability of codon-optimized anti-ROR1 molecules ROR238, ROR242, ROR243, and ROR244 to induce redirected T-cell cytotoxicity (RTCC) of PC-3 cells which were stably transfected with GFP. Percentage specific mortality is shown on the y-axis. Concentration (pM) of the anti- ROR1 molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0065] Figure 15B is a graph (top panel) showing the results of a study measuring the ability of codon-optimized anti-ROR1 molecules ROR241 , ROR250, ROR251 , ROR252, and ROR253 to induce redirected T-cell cytotoxicity (RTCC) of PC-3 cells which were stably transfected with GFP. Percentage specific mortality is shown on the y-axis. Concentration (pM) of the anti-ROR1 molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0066] Figure 16A is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR241 , ROR250, ROR251 , ROR252, and ROR253) and the parental anti-ROR1 molecule ROR185 to the ROR1 (+) cell line Kasumi-2. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0067] Figure 16B is a graph (top panel) showing the results of a flow cytometry study measuring the measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR241 , ROR250, ROR251 , ROR252, and ROR253) and the parental anti-ROR1 molecule ROR185 to the CD3(+) cell line Jurkat-60. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 - binding molecules is shown on the x-axis. The table (bottom panel) shows the EC5o values obtained from the data in the graph.
[0068] Figure 17A is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR241 , ROR238, ROR239, ROR242, ROR243, and ROR244) and the parental anti-ROR1 molecules ROR185 and ROR192 to the ROR1 (+) cell line Kasumi-2. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0069] Figure 17B is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR241 , ROR238, ROR239, ROR242, ROR243, and ROR244) and the parental anti-ROR1 molecules ROR185 and ROR192 to the CD3(+) cell line Jurkat-60. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0070] Figure 18A is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR239, ROR246, ROR247, ROR248, and ROR249) and the parental anti-ROR1 molecule ROR192 to the ROR1 (+) cell line Kasumi-2. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
[0071] Figure 18B is a graph (top panel) showing the results of a flow cytometry study measuring the binding of codon optimized bispecific anti-ROR1 molecules (ROR239, ROR246, ROR247, ROR248, and ROR249) and the parental anti-ROR1 molecule ROR192 to the CD3(+) cell line Jurkat-60. Mean fluorescence intensity (MFI) of bound molecules on live cells is shown on the y-axis. Concentration (nM) of the ROR1 -binding molecules is shown on the x-axis. The table (bottom panel) shows the EC50 values obtained from the data in the graph.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0072] The disclosure provides binding domains that specifically bind to receptor tyrosine kinase-like orphan receptor 1 (ROR1) and binding molecules (e.g. polypeptides and proteins) that specifically bind to ROR1 . These binding molecules may bind specifically to ROR1 and to another target. In some embodiments, a ROR1 -binding molecule described herein binds to ROR1 more effectively than a parent (non-humanized) antibody used to construct the humanized molecule. Administration of a therapeutically effective amount of a ROR1 -binding polypeptide or protein to a patient in need thereof is useful for treatment of certain disorders associated with the expression of ROR1 , including certain cancers. In one embodiment, a ROR1 -binding polypeptide or protein binds both a target cell expressing ROR1 and a T-cell, thereby "cross-linking" the target cell expressing ROR1 and the T-cell. The binding of both domains to their targets elicits potent target-dependent redirected T-cell cytotoxicity (RTCC) (e.g., induces target-dependent T-cell cytotoxicity, T-cell activation and/or T-cell proliferation). The ROR1 -binding therapeutics of the disclosure offer various advantages in treating patients, for example, effective binding to ROR1 , efficient induction of RTCC activity, reduced levels of cytokine release and/or a lower risk of adverse events (e.g., toxicity). In certain aspects, ROR1 -binding proteins bind to ROR1 more effectively in certain formats (e.g., scFv compared to parent antibody) and/or certain orientations (e.g., VL-VH compared to VH-VL), leading to higher potency and improved utility in treating disorders associated with expression of ROR1 . In some embodiments, a target cell expresses ROR1 at a higher level than a non-target cell (e.g., normal cell or non-cancerous cell in the same subject, organ, or tissue) expresses ROR1 . In other embodiments, a target cell expresses ROR1 while a non-target cell (e.g., normal cell or non-cancerous cell in the same subject, organ, or tissue) does not express ROR1 .
[0073] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define a term that contradicts that term's definition in the application, the definition that appears in this application controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.
[0074] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components unless otherwise indicated. The use of the alternative (e.g. , "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used synonymously. In addition, it should be understood that the
polypeptides comprising the various combinations of the components (e.g. , domains or regions) and substituents described herein, are disclosed by the present application to the same extent as if each polypeptide was set forth individually. Thus, selection of particular components of individual polypeptides is within the scope of the present disclosure.
[0075] As used herein, the term "binding domain" or "binding region" refers to the domain, region, portion, or site of a protein, polypeptide, oligopeptide, or peptide or antibody or binding domain derived from an antibody that possesses the ability to specifically recognize and bind to a target molecule, such as an antigen, ligand, receptor, substrate, or inhibitor (e.g. , ROR1 , CD3). Exemplary binding domains include single-chain antibody variable regions (e.g. , domain antibodies, sFv, scFv, scFab), receptor ectodomains, and ligands (e.g. , cytokines, chemokines). In certain embodiments, the binding domain comprises or consists of an antigen binding site (e.g. , comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g. , human FRs optionally comprising one or more amino acid substitutions). A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, including Western blot, ELISA, phage display library screening, and BIACORE® interaction analysis. As used herein, a ROR1 -binding polypeptide can have a "first binding domain" and, optionally, a "second binding domain." In certain embodiments, the "first binding domain" is a ROR1 -binding domain and the format is an antibody or antibody-like protein or domain. In certain embodiments comprising both the first and second binding domains, the second binding domain is a T-cell binding domain such as a scFv derived from a mouse monoclonal antibody (e.g. , CRIS-7) or phage display (e.g., I2C) that binds to a T-cell surface antigen (e.g. , CD3). In other embodiments, the second binding domain is a second ROR1 -binding domain. In yet other embodiments, the second binding domain is a binding domain other than a ROR1 -binding domain or a T-cell binding domain.
[0076] A binding domain or protein "specifically binds" a target if it binds the target with an affinity or Ka (i.e. , an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10s M"1 , while not significantly binding other components present in a test sample. Binding domains can be classified as "high affinity" binding domains and "low affinity" binding domains. "High affinity" binding domains refer to those binding domains with a Ka of at least 107 M"1 , at least 108 M"1 , at least 109 M"1 , at least 1010 M"1 , at least 1011 M"1 , at least 1012 M"1 , or at least 1013 M"1. "Low affinity" binding domains refer to those binding domains with a Ka of up to 107 M"1 , up to 106 M"1 , up to 10s M" Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g. , 10"5 M to 10"13 M). Affinities of binding domain polypeptides and single chain polypeptides according to the present disclosure can be readily determined using conventional techniques (see, e.g. , Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283, 173, 5,468,614, or the equivalent).
[0077] "CD3" is known in the art as a multi-protein complex of six chains (see, e.g. , Abbas and Lichtman, 2003; Janeway et al. , p. 172 and 178, 1999), which are subunits of the T-cell receptor complex. In mammals, the CD3 subunits of the T-cell receptor complex are a CD3Y chain, a CD35 chain, two CD3z chains, and a homodimer of ΟΏ3 chains. The CD3Y, CD35, and CD3z chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3v, CD35, and CD3z chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T-cell receptor chains. The intracellular tails of the CD3v, CD35, and CD3z chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each ΟΏ3 chain has three. It is believed the ITAMs are important for the signaling capacity of a TCR complex. CD3 as used in the present disclosure can be from various animal species, including human, monkey, mouse, rat, or other mammals.
[0078] As used herein, a "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary
conservative substitutions are well-known in the art (see, e.g. , WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71 -77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8). In certain embodiments, a conservative substitution includes a leucine to serine substitution.
[0079] As used herein, the term "derivative" refers to a modification of one or more amino acid residues of a peptide by chemical or biological means, either with or without an enzyme, e.g. , by glycosylation, alkylation, acylation, ester formation, or amide formation.
[0080] As used herein, a polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. In certain embodiments, the polypeptide or amino acid sequence which is derived from a particular sequence (sometimes referred to as the "starting" or "parent" or "parental" sequence) has an amino acid sequence that is essentially identical to the starting sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or at least 50-150 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence. For example, a binding domain can be derived from an antibody, e.g., a Fab, F(ab')2, Fab', scFv, single domain antibody (sdAb), etc. In some embodiments, a ROR1 -binding domain sequence is derived from a ROR1 -binding antibody or protein by means of a computer algorithm or in silico.
[0081] Polypeptides derived from another polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) can have one or more mutations relative to the starting polypeptide, e.g. , one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. The polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variations necessarily have less than 100% sequence identity or similarity with the starting polypeptide. In one embodiment, the variant will have an amino acid sequence from about 60% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide. In another embodiment, the variant will have an amino acid sequence from about 75% to less thant 100%, from about 80% to less than 100%, from about 85% to less than 100%, from about 90% to less than 100%, from about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.
[0082] As used herein, unless otherwise provided, a position of an amino acid residue in a variable region of an immunoglobulin molecule is numbered according to the IMGT criteria (Brochet, X, et al, Nucl. Acids Res. (2008) 36, W503-508), and a position of an amino acid residue in a constant region of an immunoglobulin molecule is numbered according to EU nomenclature (Ward et al. , 1995 Therap. Immunol. 2:77-94). The Kabat numbering convention (Kabat, Sequences of Proteins of Immunological Interest, 5th ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991)) is an alternative system used to refer to a position of an amino acid residue in a variable region of an immunoglobulin molecule and is sometimes used to refer to a position of an amino acid residue in a variable region of an immunoglobulin molecule herein.
[0083] As used herein, the term "dimer" refers to a biological entity that consists of two subunits associated with each other via one or more forms of intramolecular forces, including covalent bonds (e.g. , disulfide bonds) and other interactions (e.g. , electrostatic interactions, salt bridges, hydrogen bonding, and hydrophobic interactions), and is stable under appropriate conditions (e.g. , under physiological conditions, in an aqueous solution suitable for expressing, purifying, and/or storing recombinant proteins, or under conditions for non- denaturing and/or non-reducing electrophoresis). A "heterodimer" or "heterodimeric protein," as used herein, refers to a dimer formed from two different polypeptides. A heterodimer may comprise an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243. A heterodimer does not include an antibody formed from four polypeptides (i.e. , two light chains and two heavy chains). A "homodimer" or "homodimeric protein," as used herein, refers to a dimer formed from two identical polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243).
[0084] In some embodiments, a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein) comprises, in order from amino-terminus to carboxyl-terminus or in order from carboxyl-terminus to amino-terminus, (i) the ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) a second binding domain. As used herein and depending on context, a "hinge region" or a "hinge" refers to a polypeptide region between a binding domain (e.g., a ROR1 -binding domain) and an immunoglobulin constant region. As used herein and depending on context, a "linker" may refer to (1) a polypeptide region between VH and VL regions in a single-chain Fv (scFv) or (2) a polypeptide region between an immunoglobulin constant region and a second binding domain in a ROR1 -binding polypeptide comprising two binding domains (e.g., an anti-ROR1 x anti-CD3 molecule as described herein). A polypeptide region between an immunoglobulin constant region and a second binding domain in a ROR1 -binding polypeptide comprising two binding domains may also be referred to as a "carboxyl-terminus linker" or an "amino-terminus linker." Non-limiting examples of carboxyl-terminus and amino-terminus linkers include flexible linkers comprising glycine-serine repeats, and linkers derived from (a) an interdomain region of a
transmembrane protein (e.g. , a type I transmembrane protein); (b) a stalk region of a type II C-lectin; or (c) an immunoglobulin hinge. Non-limiting examples of hinges and linkers are provided in Tables 1 and 2. In some embodiments, a "linker" provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. In certain embodiments, a linker is comprised of five to about 35 amino acids, for instance, about 15 to about 25 amino acids.
[0085] A "wild-type immunoglobulin hinge region" refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, a wild type immunoglobulin hinge region sequence is human, and can comprise a human IgG hinge region.
[0086] An "altered wild-type immunoglobulin hinge region" or "altered immunoglobulin hinge region" refers to (a) a wild type immunoglobulin hinge region with up to 30% amino acid changes (e.g. , up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (b) a portion of a wild type immunoglobulin hinge region that has a length of about 5 amino acids (e.g. , about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) up to about 120 amino acids (for instance, having a length of about 10 to about 40 amino acids or about 15 to about 30 amino acids or about 15 to about 20 amino acids or about 20 to about 25 amino acids), has up to about 30% amino acid changes (e.g. , up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1 % amino acid substitutions or deletions or a combination thereof), and has an IgG core hinge region as disclosed in WO 201 1/090762 (US
2013/0129723) and WO 201 1/090754 (US 2013/0095097).
[0087] As used herein, the term "humanized" refers to a process of making an antibody or immunoglobulin binding proteins and polypeptides derived from a non-human species (e.g. , mouse or rat) less immunogenic to humans, while still retaining antigen-binding properties of the original antibody, using genetic engineering techniques. In some embodiments, the binding domain(s) of an antibody or immunoglobulin binding proteins and polypeptides (e.g. , light and heavy chain variable regions, Fab, scFv) are humanized. Non-human binding domains can be humanized using techniques known as CDR grafting (Jones et al. , Nature 321 :522 (1986)) and variants thereof, including "reshaping" (Verhoeyen, et al. , 1988 Science 239: 1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al. , Bio/Technol 1991 9:266-271), "hyperchimerization" (Queen, et al. , 1989 Proc Natl Acad Sci USA 86: 10029-10033; Co, et a/. , 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et a/. , 1992 J Immunol 148: 1 149-1 154), and "veneering" (Mark, et al. , "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291 - 312). If derived from a non-human source, other regions of the antibody or immunoglobulin binding proteins and polypeptides, such as the hinge region and constant region domains, can also be humanized.
[0088] An "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain", as used herein, refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts or associates with a different immunoglobulin domain of a second polypeptide chain, wherein the interaction of the different immunoglobulin heterodimerization domains substantially contributes to or efficiently promotes heterodimerization of the first and second polypeptide chains (i.e. , the formation of a dimer between two different polypeptide chains, which is also referred to as a "heterodimer"). The interactions between
immunoglobulin heterodimerization domains "substantially contributes to or efficiently promotes" the heterodimerization of first and second polypeptide chains if there is a statistically significant reduction in the dimerization between the first and second polypeptide chains in the absence of the immunoglobulin heterodimerization domain of the first polypeptide chain and/or the immunoglobulin heterodimerization domain of the second polypeptide chain. In certain embodiments, when the first and second polypeptide chains are co-expressed, at least 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least 80% to about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second polypetpide chains form heterodimers with each other.
Representative immunoglobulin heterodimerization domains include an immunoglobulin CH1 domain, an immunoglobulin CL domain (e.g. , CK or CA isotypes), or derivatives thereof, including wild type immunoglobulin CH1 and CL domains and altered (or mutated) immunoglobulin CH1 and CL domains, as provided therein.
[0089] An "immunoglobulin constant region" or "constant region" is a term defined herein to refer to a peptide or polypeptide sequence that corresponds to or is derived from part or all of one or more constant region domains. In certain embodiments, the immunoglobulin constant region corresponds to or is derived from part or all of one or more constant region domains, but not all constant region domains of a source antibody. In certain embodiments, the constant region comprises IgG CH2 and CH3 domains, e.g., lgG 1 CH2 and CH3 domains. In certain embodiments, the constant region does not comprise a CH1 domain. In certain embodiments, the constant region domains making up the constant region are human. In some embodiments (for example, in certain variations of a ROR1 -binding polypeptide or protein comprising a second binding domain that specifically binds CD3 or another T-cell surface antigen; for example, ROR248, ROR246, ROR252, ROR250, or ROR243), the constant region domains of a fusion protein of this disclosure lack or have minimal effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation and complement-dependent cytotoxicity (CDC), while retaining the ability to bind some Fc receptors (such as FcRn, the neonatal Fc receptor) and retaining a relatively long half life in vivo. In other variations, a fusion protein of this disclosure includes constant domains that retain such effector function of one or both of ADCC and CDC. In certain embodiments, a binding domain of this disclosure is fused to a human lgG1 constant region, wherein the lgG 1 constant region has one or more of the following amino acids mutated: leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamate at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (numbering according to EU). For example, any one or more of these amino acids can be changed to alanine. In a further embodiment, an lgG 1 Fc domain has each of L234, L235, G237, E318, K320, and K322 (according to EU numbering) mutated to an alanine (i.e. , L234A, L235A, G237A, E318A, K320A, and K322A, respectively), and optionally an N297A mutation as well (i.e. , essentially eliminating glycosylation of the CH2 domain).
[0090] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from the portion of a source antibody that is responsible for binding to antibody receptors on cells and the C1 q component of complement. Fc stands for "fragment crystalline," the fragment of an antibody that will readily form a protein crystal. Distinct protein fragments, which were originally described by proteolytic digestion, can define the overall general structure of an immunoglobulin protein. As originally defined in the literature, the Fc fragment consists of the disulfide-linked heavy chain hinge regions, CH2, and CH3 domains. However, more recently the term has been applied to a single chain consisting of CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide-linked dimer with a second such chain. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31 : 169-217, 1994. As used herein, the term Fc includes variants of naturally occuring sequences.
[0091] In some embodiments, a CD3-binding domain comprises a protein scaffold as generally disclosed in, for example, in US Patent Application Publication Nos.
2003/0133939, 2003/01 18592, and 2005/0136049. A CD3-binding domain may comprise, in order from amino-terminus to carboxyl-terminus: a first binding domain, a hinge region, and an immunoglobulin constant region. In other embodiments, a CD3-binding domain comprises a protein scaffold as generally disclosed in, for example, in US Patent Application Publication No. 2009/0148447. A CD3-binding domain may comprise, in order from amino- terminus to carboxyl-terminus: an immunoglobulin constant region, a hinge region and a first binding domain.
[0092] CD3-binding domains disclosed herein may incorporate a multi-specific binding protein scaffold. Multi-specific binding proteins and polypeptides using scaffolds are disclosed, for instance, in PCT Application Publication No. WO 2007/146968, U.S. Patent Application Publication No. 2006/0051844, PCT Application Publication No. WO
2010/040105, PCT Application Publication No. WO 2010/003108, U.S. Patent No. 7, 166,707 and U.S. Patent No. 8,409,577, which are each incorporated herein by reference in their entirety. A CD3-binding domain may comprise two binding domains (the domains can be designed to specifically bind the same or different targets), a hinge region, a linker (e.g., a carboxyl-terminus or an amino-terminus linker), and an immunoglobulin constant region. A CD3-binding protein may be a homodimeric protein comprising two identical, disulfide- bonded polypeptides. For example, a CD3-binding protein may comprise ROR248, ROR246, ROR252, ROR250, or ROR243.
[0093] As used herein, the "stalk region" of a type II C-lectin refers to the portion of the extracellular domain of the type II C-lectin that is located between the C-type lectin-like domain (CTLD; e.g. , similar to CTLD of natural killer cell receptors) and the transmembrane domain. For example, in the human CD94 molecule (GenBank Accession No. AAC50291 .1 , PRI November 30, 1995), the extracellular domain corresponds to amino acid residues 34- 179, whereas the CTLD corresponds to amino acid residues 61 -176. Accordingly, the stalk region of the human CD94 molecule includes amino acid residues 34-60, which is found between the membrane and the CTLD (see Boyington et al. , Immunity 10:75, 1999; for descriptions of other stalk regions, see also Beavil et al. , Proc. Nat'l. Acad. Sci. USA 89:753, 1992; and Figdor et al. , Nature Rev. Immunol. 2:77, 2002). These type II C-lectins can also have from six to 10 junction amino acids between the stalk region and the transmembrane region or the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1 , PRI June 15, 2010) has a transmembrane domain ranging from amino acids 71 -93 and an extracellular domain ranging from amino acids 94- 233. The CTLD is comprised of amino acids 1 19-231 , and the stalk region comprises amino acids 99-1 16, which is flanked by junctions of five and two amino acids. Other type II C- lectins, as well as their extracellular ligand-bind domains, interdomain or stalk regions, and CTLDs are known in the art (see, e.g. , GenBank Accession Nos. NPJD01993.2;
AAH07037.1 , PRI July 15, 2006; NP_001773.1 , PRI June 20, 1010; AAL65234.1 , PRI January 17, 2002, and CAA04925.1 , PRI November 14, 2006, for the sequences of human CD23, CD69, CD72, NKG2A and NKG2D and their descriptions, respectively).
[0094] As used herein, the "interdomain region" of a transmembrane protein (e.g. , a type I transmembrane protein) refers to a portion of the extracellular domain of the transmembrane protein that is located between two adjacent domains. Examples of interdomain regions include regions linking adjacent Ig domains of immunoglobulin superfamily members (e.g. , an immunoglobulin hinge region from IgG, IgA, IgD, or IgE; the region linking the IgV and lgC2 domains of CD2; or the region linking the IgV and IgC domains of CD80 or CD86). Another example of an interdomain region is the region linking the non-lg and lgC2 domain of CD22, a type I sialic acid-binding Ig-like lectin.
[0095] A polypeptide region "derived from" a stalk region of a type II C-lectin, or "derived from" a transmembrane protein interdomain region (e.g. , an immunoglobulin hinge region), refers to an about five to about 150 amino acid sequence, an about 5 to about 100 amino acid sequence, an about 5 to about 50 amino acid sequence, an about 5 to about 40 amino acid sequence, an about 5 to about 30 amino acid sequence, an about 5 to about 25 amino acid sequence, an about 5 to about 20 amino acid sequence, an about 10 to about 25 amino acid sequence, an about 10 to about 20 amino acid sequence, about 8 to about 20 amino acid sequence, about 9 to about 20 amino acid sequence, about 10 to about 20 amino acid sequence, about 1 1 to about 20 amino acid sequence, about 12 to about 20 amino acid sequence, about 13 to about 20 amino acid sequence, about 14 to about 20 amino acid sequence, about 15 to about 20 amino acid sequence, or an about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid sequence, wherein all or at least a portion of which includes (i) a wild-type stalk region or interdomain region sequence; (ii) a fragment of the wild-type stalk region or interdomain region sequence; (iii) a polypeptide having at least 80%, 85%, 90%, or 95% amino acid sequence identity with either (i) or (ii); or (iv) either (i) or (ii) in which one, two, three, four, or five amino acids have a deletion, insertion, substitution, or any combination thereof, for instance, the one or more changes are substitutions or the one or more mutations include only one deletion. In some embodiments, a derivative of a stalk region is more resistant to proteolytic cleavage as compared to the wild-type stalk region sequence, such as those derived from about eight to about 20 amino acids of NKG2A, NKG2D, CD23, CD64, CD72, or CD94.
[0096] As used herein, the term "junction amino acids" or "junction amino acid residues" refers to one or more (e.g. , about 2-10) amino acid residues between two adjacent regions or domains of a polypeptide, such as between a hinge and an adjacent immunoglobulin constant region or between a hinge and an adjacent binding domain or between a peptide linker and an adjacent immunoglobulin variable domain or an adjacent immunoglobulin constant region. Junction amino acids can result from the construct design of a polypeptide (e.g. , amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a polypeptide).
[0097] As used herein, the phrase a "linker between CH3 and CH1 or CL" refers to one or more (e.g. , about 2-12, about 2-10, about 4-10, about 5-10, about 6-10, about 7-10, about 8- 10, about 9-10, about 8-12, about 9-12, or about 10-12) amino acid residues between the C- terminus of a CH3 domain (e.g. , a wild type CH3 or a mutated CH3) and the N-terminus of a CH1 domain or CL domain (e.g. , Ck).
[0098] As used herein, the term "patient in need" refers to a patient at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a ROR1 -binding protein or polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243) or a composition thereof provided herein.
[0099] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions 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 are considered to be "pharmaceutically acceptable."
[00100] As used herein, the term "promoter" refers to a region of DNA involved in binding RNA polymerase to initiate transcription.
[00101] As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon
substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer ef al. (1991) Nucleic Acid Res. 19:5081 ; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91 -98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" are intended to include DNA molecules (e.g. , cDNA or genomic DNA), RNA molecules (e.g. , mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
[00102] The term "expression" refers to the biosynthesis of a product encoded by a nucleic acid. For example, in the case of nucleic acid segment encoding a polypeptide of interest, expression involves transcription of the nucleic acid segment into mRNA and the translation of mRNA into one or more polypeptides.
[00103] The terms "expression unit" and "expression cassette" are used interchangeably herein and denote a nucleic acid segment encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell. An expression unit typically comprises a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in operable configuration. In addition to a transcriptional promoter and terminator, an expression unit can further include other nucleic acid segments such as, e.g. , an enhancer or a polyadenylation signal.
[00104] The term "expression vector," as used herein, refers to a nucleic acid molecule, linear or circular, comprising one or more expression units. In addition to one or more expression units, an expression vector can also include additional nucleic acid segments such as, for example, one or more origins of replication or one or more selectable markers. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both.
[00105] As used herein, the term "sequence identity" refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparator sequence, the sequences are said to be "identical" at that position. The percentage "sequence identity" is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of "sequence identity." Percentage of "sequence identity" is determined by comparing two optimally aligned sequences over a comparison window. The comparison window for nucleic acid sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids in length. The comparison windon for polypeptide sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length. In order to optimally align sequences for comparison, the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant. An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of "identical" positions between the reference and comparator sequences. Percentage "sequence identity" between two sequences can be determined using the version of the program "BLAST 2 Sequences" which was available from the National Center for Biotechnology Information as of September 1 , 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing "BLAST 2 Sequences," parameters that were default parameters as of September 1 , 2004, can be used for word size (3), open gap penalty (1 1), extension gap penalty (1), gap dropoff (50), expect value (10) and any other required parameter including but not limited to matrix option. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or "substantial sequence identity" if the two sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to each other.
[00106] As used herein, a "polypeptide" or "polypeptide chain" is a single, linear and contiguous arrangement of covalently linked amino acids. It does not include two polypeptide chains that link together in a non-linear fashion, such as via an interchain disulfide bond (e.g. , a half immunoglobulin molecule in which a light chain links with a heavy chain via a disulfide bond). Polypeptides can have or form one or more intrachain disulfide bonds. With regard to polypeptides as described herein, reference to amino acid residues corresponding to those specified by SEQ ID NO includes post-translational modifications of such residues.
[00107] As used herein, "ROR1 -binding protein" may be used interchangeably with "ROR1 - binding polypeptide." Such molecules specifically bind to receptor tyrosine kinase-like orphan receptor 1 (ROR1) (e.g. , human ROR1), also known as neurotrophic tyrosine kinase, receptor-related 1 or NTRKR1 . ROR1 is a type I membrane protein, with an extracellular domain comprising an Ig-like domain, a Frizzled (cysteine-rich) domain and a Kringle domain. The ROR1 -binding proteins of the disclosure bind to the extracellular domain of ROR1 . The term "ROR1 " may refer to any isoform of ROR1. Exemplary human ROR1 nucleotide and amino acid sequences are provided in SEQ ID NO: 127 and SEQ ID NO: 128, respectively. In some embodiments, a ROR1 -binding protein is an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243. In certain embodiments, a ROR1 -binding protein is a humanized or a chimeric antibody. In various embodiments, a ROR1 -binding protein is a construct that induces redirected T-cell cytotoxicity. For example, a ROR1 -binding protein may comprise a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex. In certain embodiments, a ROR1 - binding protein is an anti-ROR1 x anti-CD3 molecule in the format of an scFv-Fc-scFv molecule, an scFv-scFv molecule, or a diabody. In some embodiments, a ROR1 -binding protein comprises from amino-terminus to carboxyl-terminus (or from carboxyl-terminus to amino-terminus), (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker, and (v) a second binding domain (e.g, a CD3-binding domain). In certain aspects, a ROR1 -binding protein is a homodimer or a heterodimer.
[00108] A "protein" is a macromolecule comprising one or more polypeptide chains. A protein can also comprise non-peptidic components, such as carbohydrate groups.
Carbohydrates and other non-peptidic substituents can be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless. A protein may be an antibody or an antigen-binding fragment of an antibody. In some embodiments, a protein may also be an scFv-Fc-scFv molecule, scFv-scFv dimer, or a diabody.
[00109] The terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl-terminus of the reference sequence, but is not necessarily at the carboxyl-terminus of the complete polypeptide.
[00110] "T-cell receptor" (TCR) is a molecule found on the surface of T-cells that, along with CD3, is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It consists of a disulfide-linked heterodimer of the highly variable a and β chains in most T-cells. In other T-cells, an alternative receptor made up of variable Y and δ chains is expressed. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin variable domain, one
immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see Abbas and Lichtman, Cellular and Molecular Immunology (5th Ed.), Editor: Saunders, Philadelphia, 2003; Janeway et al. , Immunobiology: The Immune System in Health and Disease, 4th Ed., Current Biology Publications, p148, 149, and 172, 1999). TCR as used in the present disclosure can be from various animal species, including human, mouse, rat, or other mammals.
[00111] "TCR complex," as used herein, refers to a complex formed by the association of CD3 chains with other TCR chains. For example, a TCR complex can be composed of a CD3v chain, a CD35 chain, two CD3z chains, a homodimer of ΟΏ3 chains, a TCRa chain, and a TCRp chain. Alternatively, a TCR complex can be composed of a CD3v chain, a CD35 chain, two CD3z chains, a homodimer of ΟΏ3 chains, a TCRv chain, and a TCR5 chain.
[00112] "A component of a TCR complex," as used herein, refers to a TCR chain (i.e. , TCRa, TCRp, TCRv or TCR5), a CD3 chain (i.e. , CD3v, CD35, CD3z or CD3Q, or a complex formed by two or more TCR chains or CD3 chains (e.g. , a complex of TCRa and TCRp, a complex of TCRv and TCR5, a complex of CD3z and CD35, a complex of CD3v and CD3£, or a sub-TCR complex of TCRa, TCRp, CD3v, CD35, and two CD3z chains).
[00113] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC," as used herein, refer to a cell-mediated process in which nonspecific cytotoxic cells that express FcyRs (e.g., monocytic cells such as Natural Killer (NK) cells and macrophages) recognize bound antibody (or other protein capable of binding FcyRs) on a target cell and subsequently cause lysis of the target cell. In principle, any effector cell with an activating FcyR can be triggered to mediate ADCC. The primary cells for mediating ADCC are NK cells, which express only FcyRIII, whereas monocytes, depending on their state of activation, localization, or differentiation, can express FcyRI, FcyRII, and FcyRIII. For a review of FcyR expression on hematopoietic cells, see, e.g., Ravetch et al., 1991 , Annu. Rev. Immunol., 9:457-92. [00114] The term "having ADCC activity," as used herein in reference to a polypeptide or protein, means that the polypeptide or protein (for example, one comprising an
immunoglobulin hinge region and an immunoglobulin constant region having CH2 and CH3 domains, such as derived from IgG (e.g., lgG 1)), is capable of mediating antibody- dependent cell-mediated cytotoxicity (ADCC) through binding of a cytolytic Fc receptor (e.g., FcyRIII) on a cytolytic immune effector cell expressing the Fc receptor (e.g., an NK cell). In some embodiments, a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243) may have null effector function (e.g., null ADCC activity) as the result of mutations.
[00115] "Complement-dependent cytotoxicity" and "CDC," as used herein, refer to a process in which components in normal serum ("complement"), together with an antibody or other C1 q-complement-binding protein bound to a target antigen, exhibit lysis of a target cell expressing the target antigen. Complement consists of a group of serum proteins that act in concert and in an orderly sequence to exert their effect.
[00116] The terms "classical complement pathway" and "classical complement system," as used herein, are synonymous and refer to a particular pathway for the activation of complement. The classical pathway requires antigen-antibody complexes for initiation and involves the activation, in an orderly fashion, of nine major protein components designated C1 through C9. For several steps in the activation process, the product is an enzyme that catalyzes the subsequent step. This cascade provides amplification and activation of large amounts of complement by a relatively small initial signal.
[00117] The term "having CDC activity," as used herein in reference to a polypeptide or protein, means that the polypeptide or protein (for example, one comprising an
immunoglobulin hinge region and an immunoglobulin constant region having CH2 and CH3 domains, such as derived from IgG (e.g., lgG 1)) is capable of mediating complement- dependent cytotoxicity (CDC) through binding of C1 q complement protein and activation of the classical complement system. In some embodiments, a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243) may have null effector function (e.g., null CDC activity) as the result of mutations.
[00118] "Redirected T-cell cytotoxicity" and "RTCC," as used herein, refer to a T-cell- mediated process in which a cytotoxic T-cell is recruited to a target cell using a multi-specific protein that is capable of specifically binding both the cytotoxic T-cell and the target cell, and whereby a target-dependent cytotoxic T-cell response is elicited against the target cell. Polypeptides and proteins comprising anti-ROR1 and anti-CD3 binding domains (for example, ROR248, ROR246, ROR252, ROR250, or ROR243), as disclosed herein, are, in some embodiments, capable of RTCC. In certain embodiments, a ROR1 -binding protein that induces RTCC is an anti-ROR1 x anti-CD3 molecule in the format of an scFv-Fc-scFv molecule, an scFv-scFv molecule, or a diabody. In some embodiments, a ROR1 -binding protein that induces RTCC comprises from amino-terminus to carboxyl-terminus (or from carboxyl-terminus to amino-terminus), (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker, and (v) a second binding domain (e.g, a CD3-binding domain). In certain aspects, a ROR1 -binding protein that induces RTCC is a homodimer or a heterodimer.
[00119] As used herein, the term "treatment," "treating," or "ameliorating" refers to either a therapeutic treatment or prophylactic/preventative treatment. A treatment is therapeutic if at least one symptom of disease in an individual receiving treatment improves or a treatment can delay worsening of a progressive disease in an individual, or prevent onset of additional associated diseases.
[00120] As used herein, the term "therapeutically effective amount (or dose)" or "effective amount (or dose)" of a specific binding molecule or compound (e.g., an anti-ROR1 x anti- CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243) refers to that amount of the compound sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner or a statistically significant improvement in organ function. When referring to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously (in the same formuation or concurrently in separate formulations).
[00121] As used herein, the term "transformation," "transfection," and "transduction" refer to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell. As used herein, the term "genetic transformation" refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell. The transferred nucleic acid can be introduced into a cell via an expression vector.
[00122] As used herein, the term "variant" or "variants" refers to a nucleic acid or polypeptide differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide. For instance, a variant may exhibit at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity compared to the active portion or full length reference nucleic acid or polypeptide.
[00123] The terms "light chain variable region" (also referred to as "light chain variable domain" or "VL" or VL) and "heavy chain variable region" (also referred to as "heavy chain variable domain" or "VH" or VH) refer to the variable binding region from an antibody light and heavy chain, respectively. The variable binding regions are made up of discrete, well- defined sub-regions known as "complementarity determining regions" (CDRs) and
"framework regions" (FRs). In one embodiment, the FRs are humanized. The term "CL" refers to an "immunoglobulin light chain constant region" or a "light chain constant region," i.e., a constant region from an antibody light chain. The term "CH" refers to an
"immunoglobulin heavy chain constant region" or a "heavy chain constant region," which is further divisible, depending on the antibody isotype into CH1 , CH2, and CH3 (IgA, IgD, IgG), or CH1 , CH2, CH3, and CH4 domains (IgE, IgM). A "Fab" (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CH1 domain of the heavy chain linked to the light chain via an inter-chain disulfide bond.
[00124] The present disclosure describes binding domains that specifically bind ROR1 (e.g., human ROR1), as well as polypeptides and proteins comprising these binding domains. In some embodiments, the ROR1 -binding proteins and polypeptides comprise a second binding domain, which may bind to ROR1 or to a different target. The polypeptides and proteins comprising binding domains of this disclosure can further comprise immunoglobulin constant regions, linker peptides, hinge regions, immunoglobulin
dimerization/heterodimerization domains, junctional amino acids, tags, etc. These components of the disclosed polypeptides and proteins are described in further detail below.
[00125] Additionally, the ROR1 -binding polypeptides and proteins disclosed herein can be in the form of an antibody or a fusion protein of any of a variety of different formats (e.g. , the fusion protein can be in the form of a ROR1 -binding bispecific or multispecific molecule). Non-limiting examples of bispecific molecules include a scFv-Fc-scFv molecule. Some bispecific molecules comprise or consist of an anti-ROR1 scFv linked to a second binding domain scFv and do not include other sequences such as an immunoglobulin constant region. In other embodiments, a ROR1 -binding protein is a diabody.
[00126] A ROR1 -binding protein in accordance with the present disclosure generally includes at least one ROR1 -binding polypeptide chain comprising (a) a ROR1 -binding domain as set forth herein. In certain variations, the ROR1 -binding polypeptide further includes (b) a hinge region carboxyl-terminal to the ROR1 -binding domain, and (c) an immunoglobulin constant region. In further variations, the ROR1 -binding polypeptide further includes (d) a carboxyl-terminus linker carboxyl-terminal to the immunoglobulin constant region, and (e) a second binding domain carboxyl-terminal to the carboxyl-terminus linker.
[00127] In yet other variations, a ROR1 -binding polypeptide comprises (b) a hinge region amino-terminal to the ROR1 -binding domain, and (c) an immunoglobulin sub-region amino- terminal to the hinge region.
[00128] In some embodiments, ROR1 -binding polypeptides are capable of
homodimerization, typically through disulfide bonding, via the immunoglobulin constant region and/or hinge region (e.g. , via an immunoglobulin constant region comprising IgG CH2 and CH3 domains and an IgG hinge region). Thus, in certain embodiments of the present disclosure, two identical single chain ROR1 -binding polypeptides (e.g., an anti-ROR1 x anti- CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243) homodimerize to form a dimeric ROR1 -binding protein.
[00129] In other embodiments, a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) includes a heterodimerization domain that is capable of heterodimerization with a different heterodimerization domain in a second, non-identical polypeptide chain. In certain variations, the second polypeptide chain for heterodimerization includes a second binding domain. Accordingly, in certain embodiments of the present disclosure, two non-identical polypeptide chains, one comprising the ROR1 -binding domain and the second optionally comprising a second binding domain, dimerize to form a heterodimeric ROR1 -binding protein. In some embodiments, a heterodimer comprises ROR248, ROR246, ROR252, ROR250, or ROR243. Examples of types of heterodimers include those described in International Appl. Publ. No. WO 201 1/090754 (US 2013/0095097) and in US
2013/0129723.
[00130] In some embodiments, a ROR1 -binding domain, protein or polypeptide is conjugated to a drug or a toxic moiety. In one aspect, an anti-ROR1 x anti-CD3 molecule as described herein is conjugated to a drug or a toxic moiety. In some embodiments, ROR248, ROR246, ROR252, ROR250, or ROR243 is conjugated to a drug or a toxic moiety.
[00131] ROR1 -binding polypeptides, proteins, and their various components used in the therapeutics of the present disclosure are further described below.
[00132] As indicated above, the disclosure relates to binding domains that specifically bind ROR1 . In some variations, a ROR1 -binding domain is capable of competing for binding to ROR1 with an antibody having VL and VH regions having amino acid sequences as shown in SEQ ID NO:4 and SEQ ID NO:2, respectively (e.g. , R1 1), or with a single-chain Fv (scFv) having an amino acid sequence as shown in SEQ ID NO: 18, SEQ ID NO:21 , SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33 or SEQ ID NO:35. In other variations, a ROR1 -binding domain is capable of competing for binding to ROR1 with an antibody having VL and VH regions having amino acid sequences as shown in SEQ ID NO:39 and SEQ ID NO:37, respectively (e.g. , R12), or with a single-chain Fv (scFv) having an amino acid sequence as shown in SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 or SEQ ID NO:69. Monoclonal rabbit anti-ROR1 antibodies R1 1 and R12 are described in, for example, U.S. Patent Application Publication No. 2013/0251642 and Yang et al., PLoS ONE 6(6): e21018 (201 1).
[00133] In some embodiments, a ROR1 -binding domain binds an epitope near the junction of the Frizzled and Kringle domains of ROR1 (e.g., human ROR1). In certain aspects, this epitope is a discontinuous and/or conformational epitope. In some embodiments, a ROR1 - binding domain binds residues in stretches 259-273 (LCQTEYIFARSNPMI (residues 259- 273 of SEQ ID NO: 128)) and 389-403 (PACDSKDSKEKNKME (residues 389-403 of SEQ ID NO: 128)) of the ROR1 ectodomain. For example, a ROR1 -binding domain may bind at least one, at least two, at least three, at least four, at least five, at least six, or at least seven residues in these stretches of the ROR1 ectodomain.
[00134] A ROR1-binding domain may comprise sequences shown in Table 3. In some instances, a ROR1 -binding domain comprises at least one sequence shown in Table 3 and binds an epitope near the junction of the Frizzled and Kringle domains of ROR1 (e.g., human ROR1). In some cases, the sequences shown in the disclosure, including in Table 3, contain amino acid substitutions relative to a parent sequence. For example, ROR1 19 has the amino acid sequence of the L6H15 construct with the asparagine residue at position 94 substituted with alanine. In certain embodiments, a ROR1 -binding domain comprises (i) an immunoglobulin light chain variable region (VL) comprising CDRs LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region (VH) comprising CDRs HCDR1 , HCDR2, and HCDR3. Suitable ROR1 -binding domains include those having VL and VH regions derived from rabbit antibody R1 1 or R12. In some such embodiments, (i) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12 or a sequence that differs from SEQ ID NO: 12 by at least one amino acid substitution; (ii) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14 or a sequence that differs from SEQ ID NO: 14 by at least one amino acid substitution; (iii) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268, or a sequence that differs from SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268 by at least one amino acid substitution; (iv) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:266 or a sequence that differs from SEQ ID NO:6 or SEQ ID NO:266 by at least one amino acid substitution; (v) the
HCDR2 has an amino acid sequence set forth in SEQ ID NO:8 or a sequence that differs from SEQ ID NO:8 by at least one amino acid substitution; and (vi) the HCDR3 has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO:267 or a sequence that differs from SEQ ID NO: 10 or SEQ ID NO:267 by at least one amino acid substitution. The amino acid substitution described above may be a conservative or a non-conservative amino acid substitution. In other such embodiments, (i) the LCDR1 has an amino acid sequence set forth in SEQ ID NO:47 or a sequence that differs from SEQ ID NO:47 by at least one amino acid substitution; (ii) the LCDR2 has an amino acid sequence set forth in SEQ ID NO:49 or a sequence that differs from SEQ ID NO:49 by at least one amino acid substitution; (iii) the LCDR3 has an amino acid sequence set forth in SEQ ID NO:51 , or a sequence that differs from SEQ ID NO:51 by at least one amino acid substitution; (iv) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:41 or a sequence that differs from SEQ ID NO:41 by at least one amino acid substitution; (v) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:43 or a sequence that differs from SEQ ID NO:43 by at least one amino acid substitution; and (vi) the HCDR3 has an amino acid sequence set forth in SEQ ID NO:45 or a sequence that differs from SEQ ID NO:45 by at least one amino acid substitution. In certain cases, a ROR1 -binding domain comprises the CDR sequences listed in this paragraph and binds an epitope near the junction of the Frizzled and Kringle domains of ROR1 (e.g., human ROR1). In some embodiments, a ROR1 -binding domain comprises the CDR sequences listed in this paragraph and binds residues in stretches 259- 273 (LCQTEYI FARSN PM I (residues 259-273 of SEQ ID NO: 128)) and 389-403
(PACDSKDSKEKNKME (residues 389-403 of SEQ ID NO: 128)) of the ROR1 ectodomain.
[00135] In some embodiments, the disclosure relates to a ROR1 -binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12; (b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14; (c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268; (d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:266; (e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:8; and (f) the HCDR3 has an amino acid sequence set forth in SEQ ID NO:267.
[00136] In certain embodiments, a ROR1 -binding domain comprises humanized
immunoglobulin VL and/or VH regions. Techniques for humanizing immunoglobulin VL and VH regions are known in the art and are discussed, for example, in U.S. Patent Application Publication No. 2006/0153837. [00137] "Humanization" is expected to result in an antibody that is less immunogenic, with complete retention of the antigen-binding properties of the original molecule. In order to retain all of the antigen-binding properties of the original antibody, the structure of its antigen binding site should be reproduced in the "humanized" version. This can be achieved by grafting only the nonhuman CDRs onto human variable framework domains and constant regions, with or without retention of critical framework residues (Jones et al. , Nature 321 :522 (1986); Verhoeyen et al. , Science 239: 1539 (1988)) or by recombining the entire nonhuman variable domains (to preserve ligand-binding properties), but "cloaking" them with a humanlike surface through judicious replacement of exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol. 28:489 (1991)).
[00138] Essentially, humanization by CDR grafting involves recombining only the CDRs of a non-human antibody onto a human variable region framework and a human constant region. Theoretically, this should substantially reduce or eliminate immunogenicity (except if allotypic or idiotypic differences exist). However, it has been reported that some framework residues of the original antibody also may need to be preserved (Reichmann et al. , Nature, 332:323 (1988); Queen et al. , Proc. Natl. Acad. Sci. USA, 86: 10,029 (1989)).
[00139] The framework residues that need to be preserved are amenable to identification through computer modeling. Alternatively, critical framework residues can potentially be identified by comparing known antigen-binding site structures (Padlan, Molec. Immunol. , 31 (3): 169-217 (1994), incorporated herein by reference).
[00140] The residues that potentially affect antigen binding fall into several groups. The first group comprises residues that are contiguous with the antigen site surface, which could therefore make direct contact with antigens. These residues include the amino-terminal residues and those adjacent to the CDRs. The second group includes residues that could alter the structure or relative alignment of the CDRs, either by contacting the CDRs or another peptide chain in the antibody. The third group comprises amino acids with buried side chains that could influence the structural integrity of the variable domains. The residues in these groups are usually found in the same positions (Padlan, 1994, supra) although their positions as identified may differ depending on the numbering system (see Kabat et al. , "Sequences of proteins of immunological interest, 5th ed., Pub. No. 91 -3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md., 1991).
[00141] Knowledge about humanized antibodies in the art is applicable to the polypeptides according to the disclosure, even if these polypeptides are not antibodies.
[00142] Thus, in some embodiments, a ROR1 -binding domain of the disclosure is a humanized VL and/or VH region derived from the variable region sequences of rabbit monoclonal antibody R1 1 (VL is SEQ ID NO:4; VH is SEQ ID NO:2) or R12 (VL is SEQ ID NO:39; VH is SEQ ID NO:37). Monoclonal rabbit antibodies R1 1 and R12 are described in US 2013/0251642.
[00143] In related embodiments, a ROR1 -binding domain comprises or is a sequence that is at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) (e.g. , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250) or to a heavy chain variable region (VH) (e.g. , SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 ,
SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252), or both. In certain embodiments, a ROR1 -binding domain or polypeptide comprises or is a sequence that is at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence of a light chain (HC) (e.g. , SEQ ID NO:333 or SEQ ID NO:337) or to a heavy chain (HC) (e.g. , SEQ ID NO:335 or SEQ ID NO:339).
[00144] In some embodiments, the disclosure relates to ROR1 -binding domains wherein (i) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:23 and the
immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27; (ii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27; (iii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:59; (iv) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:61 ; (v) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:59; (vi) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:61 , (vii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:210; (viii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:210; (ix) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:212; (x) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:212; (xi) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the
immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232; (xii) the
immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232; (xiii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:244; or (xiv) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:250 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:252. In some embodiments, the disclosure relates to ROR1 -binding domains or polypeptides wherein (i) the immunoglobulin light chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:333 and the immunoglobulin heavy chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:335; or (ii) the immunoglobulin light chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:337 and the immunoglobulin heavy chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:339. [00145] The disclosure also relates to ROR1 -binding domains wherein (i) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27; (ii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27; (iii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59; (iv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the
immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 ; (v) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59; (vi) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 , (vii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:210; (viii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:210; (ix) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:212; (x) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:212; (xi) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the
immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232; (xii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232; (xiii) the
immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:244; or (xiv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:250 and the
immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:252. In certain embodiments, the disclosure also relates to ROR1 -binding domains and polypeptides wherein (i) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:333 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:335; or (ii) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:337 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:339.
[00146] In further embodiments, each CDR comprises no more than one, two, or three substitutions, insertions or deletions, as compared to that from a monoclonal antibody or fragment or derivative thereof that specifically binds to a target of interest (e.g. , ROR1).
[00147] A ROR1 -binding domain may specifically bind to human ROR1 and comprise SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69. A ROR1 -binding polypeptide may specifically bind to human ROR1 and comprise SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91 .
[00148] In certain embodiments, a binding domain VL and/or VH region of the present disclosure is derived from a VL and/or VH of a known monoclonal antibody (e.g. , R1 1 or R12) and optionally contains about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, about one or more (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g. , conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above- noted changes, when compared to the VL and/or VH sequence of a known monoclonal antibody. The insertion(s), deletion(s) or substitution(s) can be anywhere in the VL and/or VH region, including at the amino- or carboxyl-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes. In some embodiments, the binding domain containing the modified VL and/or VH region can still specifically bind its target with an affinity similar to the wild type binding domain.
[00149] In certain embodiments, a ROR1 -binding protein can comprise one or more additional binding domains (e.g. , second binding domain) that bind a target other than ROR1 . These other binding domains can comprise, for example, a particular cytokine or a molecule that targets the binding domain polypeptide to a particular cell type, a toxin, an additional cell receptor, an antibody, etc.
[00150] In certain embodiments, a ROR1 -binding molecule or protein can comprise a T-cell binding domain for recruitment of T-cells to target cells expressing ROR1 . In certain embodiments, a ROR1 -binding protein as described herein can comprise (i) a binding domain that specifically binds a TCR complex or a component thereof (e.g., TCRa, TCRp, CD3Y, CD35, and CD3£) and (ii) another binding domain that specifically binds to ROR1.
[00151] A ROR1-binding protein can utilize essentially any binding domain that binds a T- cell, e.g., an antibody derived binding domain. Exemplary anti-CD3 antibodies from which the CD3 binding domain can be derived include the CRIS-7 monoclonal antibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II., Springer Verlag, New York, (1986); VL and VH amino acid sequences respectively shown in SEQ ID NO: 131
(QWLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASGVPA RFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITR) and SEQ ID NO:132 (QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHVWKQRPGQGLEWIGYINP SSAYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNGFPYWGQ GTLVTVSA)); HuM291 (Chau et al. (2001) Transplantion 71 :941-950; VL and VH amino acid sequences respectively shown in SEQ ID NO: 133
(DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK) and SEQ ID NO:134 (QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHVWRQAPGQGLEWMGYINPRSGYTH YNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVS
S)); BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172:1691); OKT3 monoclonal antibody (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (also referred to as OKT3 AA-FL or OKT3 FL), a humanized, Fc variant with alanine substitutions at positions 234 and 235 (Herold et al. (2003) J. Clin. Invest. 1 1 :409); visilizumab (Carpenter et al. (2002) Blood 99:2712), G19-4 monoclonal antibody (Ledbetter et al., 1986, J. Immunol. 136:3945), 145- 2C1 1 monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140: 3766) and I2C monoclonal antibody (see, e.g., US 201 1/0293619 and US20120244162). For example, a CD3 binding domain may comprise a CD3 binding domain disclosed in U.S. Patent Application
Publication No. 20120244162, including a CD3 binding domain comprising a VL region selected from SEQ ID NO: 17, 21 , 35, 39, 53, 57, 71 , 75, 89, 83, 107, 1 1 1 , 125, 129, 143, 147, 161 , 165, 179 and 183 of US20120244162 and/or a VH region selected from SEQ ID NO:15, 19, 33, 37, 51 , 55, 69, 73, 87, 91. 105, 109, 123, 127, 141 , 145, 159, 163, 177 and 181 of US20120244162. In some embodiments, a CD3 binding domain comprises an amino acid sequence selected from SEQ ID NO: 23, 25, 41 , 43, 59, 61 , 77, 79, 95, 97, 1 13, 1 15, 131 , 133, 149, 151 , 167, 169, 185, and 187 of US20120244162. In some embodiments, a CD3 binding domain is one described in WO2004/106380, WO2005/040220A1 , US
2014/0099318 or derived from a CD3 binding domain thereof. An exemplary anti-TCR antibody is the BMA031 monoclonal antibody (Borst et al. (1990) Human Immunology 29: 175-188). The CD3 binding domain may be derived from any of the antibodies or sequences described in WO 2013/158856 (incorporated herein by reference in its entirety). In some embodiments, the CD3 binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 10, 1 1 1 and 1 12, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 272, 273 and 274, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 269, 270 and 271 , respectively. Alternatively, the second binding domain of a ROR1 -binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 278, 279 and 280, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 275, 276 and 277, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 284, 285 and 286, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 281 , 282 and 283, respectively. In another embodiment, the second binding domain of a RORI - binding polypeptide described herein may comprise: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 290, 291 and 292, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 287, 288 and 289, respectively; or (b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 296, 297 and 298, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 293, 294 and 295, respectively. The second binding domains comprising the CDR sequences recited in this paragraph may be humanized.
[00152] In some embodiments, a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) comprises, in order from amino-terminus to carboxyl-terminus (or in order from carboxyl-terminus to amino-terminus), (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) a CD3-binding domain; wherein said ROR1 -binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 12; (b) the LCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 14; (c) the LCDR3 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID
NO:268; (d) the HCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:266; (e) the HCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:8; and (f) the HCDR3 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:267; and wherein said CD3-binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 10, 1 1 1 and 1 12, respectively; or (b') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 272, 273 and 274, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 269, 270 and 271 , respectively; (c') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 278, 279 and 280, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 275, 276 and 277, respectively; (d') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 284, 285 and 286, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 281 , 282 and 283, respectively; (e') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 290, 291 and 292, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 287, 288 and 289, respectively; or (Γ) the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 296, 297 and 298, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 293, 294 and 295, respectively.
[00153] In some embodiments of a ROR1 -binding protein comprising a second binding domain that specifically binds CD3£, the second binding domain competes for binding to CD3£ with the CRIS-7, HuM291 or I2C monoclonal antibody. In certain variations, the CD3- binding domain comprises an immunoglobulin light chain variable region (VL) and an immunoglobulin heavy chain variable region (VH) derived from the CRIS-7, HuM291 or I2C monoclonal antibody (e.g. , the VL and VH of the second binding domain can be humanized variable regions comprising, respectively, the light chain CDRs and the heavy chain CDRs of the monoclonal antibody). A second binding domain may comprise the light chain variable region, the heavy chain variable region, or both, of the DRA222, TSC455, or TSC456 CD3- binding domains. The amino acid sequences of DRA222, TSC455, and TSC456 are provided in Table 3. TSC455 is also referred to herein as TSC394 F87Y. TSC455 is also referred to herein as TSC394 E86D F87Y or TSC394 DY. In some embodiments, the second binding domain specifically binds CD3 and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; wherein the
immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:260; or at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:261 ; and wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:259. In some embodiments, a ROR1 -binding polypeptide or protein further comprising a CD3-binding domain (e.g., ROR248, ROR246, ROR252, ROR250, or
ROR243) may have a low level of high molecular weight aggregates produced during recombinant expression of the polypeptide or protein. A ROR1-binding polypeptide or protein further comprising a CD3-binding domain (e.g., ROR248, ROR246, ROR252, ROR250, or ROR243) may exhibit longer stability in human serum, depending on the CD3- binding domain present in the polypeptide or protein.
[00154] In certain variations, the second binding domain of a ROR1 -binding polypeptide described herein is a CD3-binding domain and comprises one or more of the CD3-binding sequences (e.g., CDRs or variable regions) disclosed in US 2013/0129730, US
201 1/0293619, US 7,635,472, WO 2010/037836, WO 2004/106381 , or WO 201 1/121 1 10; each incorporated herein by reference in its entirety. In some embodiments, a CD3-binding domain comprises one or more of the following sequences: LCDR1 LCDR2 LCDR3
GSSTGAVTSGYYPN GTKFLAP ALWYSNRWV (SEQ ID NO:353) (SEQ ID NO:348) (SEQ ID NO:351)
RSSTGAVTSGYYPN ATDMRPS ALWYSNRVW (SEQ ID NO:353) (SEQ ID NO:349) (SEQ ID NO:352)
GSSTGAVTSGNYPN GTKFLAP VLWYSNRVW (SEQ ID NO:354) (SEQ ID NO:350) (SEQ ID NO:351)
[00155] In various embodiments, a CD3-binding domain comprises one or more of the following sequences:
Figure imgf000055_0001
[00156] In certain embodiments, a ROR1 -binding polypeptide used in the methods and compositions described herein is a bispecific single chain molecule comprising a ROR1 binding domain and a CD3 binding domain. In some embodiments, a ROR1 - and/or a CD3- binding domain is derived from an antibody and comprises a variable heavy chain (VH) and a variable light chain (VL). For example, an scFv comprises a VH and VL. These binding domains and variable chains may be arranged in any order that still retains some binding to the target(s). For example, the variable domains may be arranged in the order such as VH ROR1 -VL ROR1 -VH CD3-VL CD3; VL ROR1 -VH ROR1 -VH CD3-VL CD3; VH ROR1 -VL ROR1 -VL CD3-VH CD3; VL ROR1 -VH ROR1 -VL CD3-VH CD3; VH CD3-VL CD3-VH ROR1 -VL ROR1 ; VL CD3-VH CD3-VL ROR1-VH ROR1 ; VH CD3-VL CD3-VL ROR1 -VH ROR1 ; or VL CD3-VH CD3-VH ROR1 -VL ROR1 . The pairs of VH regions and VL regions in the binding domain binding to CD3 may be in the format of a single chain antibody (scFv). The VH and VL regions may be arranged in the order VH-VL or VL-VH. In some
embodiments, the scFv may bind to ROR1 more effectively than the antibody comprising the same VH and VL region sequences in the same orientation (see, e.g., Example 3). In certain embodiments, the scFv may bind more effectively to ROR1 in the VL-VH orientation than in the VH-VL orientation, or vice versa (see, e.g., Example 4). The VH-region may be positioned N-terminally to a linker sequence. The VL region may be positioned C-terminally to the linker sequence. The domain arrangement in the CD3 binding domain of the bispecific single chain molecule may be VH-VL, with said CD3 binding domain located C-terminally to the ROR1 binding domain. In some instances, a bispecific molecule may comprise an scFv binding to ROR1 linked to an scFv binding to CD3. These scFvs may be linked, for example, with a short peptide. In some embodiments, bispecific single chain molecules do not comprise a hinge region or a constant region (see, for example, US 2013/0295121 , US 2013/0129730, WO 2010/037836, WO 2004/106381 and WO 201 1/121 1 10; each incorporated herein by reference in its entirety).
[00157] In some embodiments, a binding domain is a single-chain Fv fragment (scFv) that comprises VH and VL regions specific for a target of interest. In certain embodiments, the VH and VL regions are human or humanized.
[00158] In certain embodiments, a ROR1 -binding domain comprises or is an scFv that is at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100% identical to an amino acid sequence of a scFv of SEQ ID NO: 18, 21 , 29, 31 , 33, 35, 53, 63, 65, 67 or 69.
[00159] In some variations, a binding domain is a single-chain Fv (scFv) comprising immunoglobulin VL and VH regions joined by a peptide linker. The use of peptide linkers for joining VL and VH regions is well-known in the art, and a large number of publications exist within this particular field. In some embodiments, a peptide linker is a 15mer consisting of three repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence ((Gly4Ser)3) (SEQ ID NO: 135). Other linkers have been used, and phage display technology, as well as selective infective phage technology, has been used to diversify and select appropriate linker sequences (Tang et a/. , J. Biol. Chem. 271 , 15682-15686, 1996; Hennecke et a/. , Protein Eng. 1 1 , 405-410, 1998). In certain embodiments, the VL and VH regions are joined by a peptide linker having an amino acid sequence comprising the formula (Gly4Ser)n, wherein n = 1 -5 (SEQ ID NO: 19). Other suitable linkers can be obtained by optimizing a simple linker (e.g. ,
(Gly4Ser)n) through random mutagenesis.
[00160] In some embodiments, a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) comprises, in order from amino-terminus to carboxyl-terminus (or in order from carboxyl-terminus to amino-terminus), (i) the ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker (or an amino-terminus linker), and (v) the second binding domain. As used herein in the context of a polypeptide construct comprising a first binding domain and a second binding domain, a "hinge region" or a "hinge" refers to a polypeptide region between the first binding domain and the Fc region. A "carboxyl-terminus linker" or "an amino-terminus linker" refers to a polypeptide region between the Fc region and the second binding domain. In some embodiments, a carboxyl- terminus (or an amino-terminus linker) linker comprises or consists of SEQ ID NO:265, SEQ ID NO:301 , SEQ ID NO:302, or SEQ ID NO:303. In certain embodiments, a hinge is a wild- type human immunoglobulin hinge region. In certain other embodiments, one or more amino acid residues can be added at the amino- or carboxyl-terminus of a wild type
immunoglobulin hinge region as part of a fusion protein construct design. For example, additional junction amino acid residues at the hinge amino-terminus can be "RT," "RSS," "TG," or "T," or at the hinge carboxyl-terminus can be "SG", or a hinge deletion can be combined with an addition, such as ΔΡ with "SG" added at the carboxyl-terminus.
[00161] In certain embodiments, a hinge, a carboxyl-terminus linker, or an amino-terminus linker is an altered immunoglobulin hinge in which one or more cysteine residues in a wild type immunoglobulin hinge region is substituted with one or more other amino acid residues (e.g. , serine or alanine).
[00162] Exemplary altered immunoglobulin hinges, carboxyl-terminus linkers, and amino- terminus linkers include an immunoglobulin human lgG1 hinge region having one, two or three cysteine residues found in a wild type human lgG1 hinge substituted by one, two or three different amino acid residues (e.g. , serine or alanine). An altered immunoglobulin hinge can additionally have a proline substituted with another amino acid (e.g. , serine or alanine). For example, the above-described altered human lgG 1 hinge can additionally have a proline located carboxyl-terminal to the three cysteines of wild type human lgG 1 hinge region substituted by another amino acid residue (e.g. , serine, alanine). In one embodiment, the prolines of the core hinge region are not substituted.
[00163] In certain embodiments, a hinge, a carboxyl-terminus linker, or an amino-terminus linker polypeptide comprises or is a sequence that is at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a wild type
immunoglobulin hinge region, such as a wild type human lgG 1 hinge, a wild type human lgG2 hinge, or a wild type human lgG4 hinge.
[00164] In further embodiments, a hinge, a carboxyl-terminus linker, or an amino-terminus linker present in a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) can be a hinge that is not based on or derived from an immunoglobulin hinge (i.e., not a wild-type immunoglobulin hinge or an altered immunoglobulin hinge). Examples for such hinges and carboxyl-terminus linkers include peptides of about five to about 150 amino acids derived from an interdomain region of a transmembrane protein or stalk region of a type II C-lectin, for instance, peptides of about eight to 25 amino acids and peptides of about seven to 18 amino acids.
[00165] In certain embodiments, interdomain or stalk region hinges, carboxyl-terminus linkers, and amino-terminus linkers have seven to 18 amino acids and can form an a-helical coiled coil structure. In certain embodiments, interdomain or stalk region hinges, carboxyl- terminus linkers, or amino-terminus linkers contain 0, 1 , 2, 3, or 4 cysteines. Exemplary interdomain or stalk region hinges, carboxyl-terminus linkers, and amino-terminus linkers are peptide fragments of the interdomain or stalk regions, such as ten to 150 amino acid fragments from the stalk regions of CD69, CD72, CD94, NKG2A and NKG2D.
[00166] In certain embodiments, hinge, carboxyl-terminus linker, and amino-terminal linker sequences have about 5 to 150 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, 40 to 50 amino acids, 50 to 60 amino acids, 5 to 60 amino acids, 5 to 40 amino acids, 8 to 20 amino acids, or 10 to 15 amino acids. The hinge or linker can be primarily flexible, but can also provide more rigid characteristics or can contain primarily a-helical structure with minimal β-sheet structure. The lengths or the sequences of the hinges and linkers can affect the binding affinities of the binding domains to which the hinges are directly or indirectly (via another region or domain, such as an heterodimerization domain) connected as well as one or more activities of the Fc region portions to which the hinges or linkers are directly or indirectly connected.
[00167] In certain embodiments, hinge, carboxyl-terminus linker, and amino-terminal linker sequences are stable in plasma and serum and are resistant to proteolytic cleavage. The first lysine in the lgG 1 upper hinge region can be mutated to minimize proteolytic cleavage, for instance, the lysine can be substituted with methionine, threonine, alanine or glycine, or is deleted.
[00168] In some embodiments of the disclosure, a ROR1 -binding polypeptide (e.g. , an anti- ROR1 x anti-CD3 molecule) is capable of forming a heterodimer with a second polypeptide chain and comprises a hinge region (a) immediately amino-terminal to an immunoglobulin constant region (e.g. , amino-terminal to a CH2 domain wherein the immunglobulin constant region includes CH2 and CH3 domains, or amino-terminal to a CH3 domain wherein the immunoglobulin sub-regions includes CH3 and CH4 domains), (b) interposed between and connecting a binding domain (e.g. , scFv) and a immunoglobulin heterodimerization domain, (c) interposed between and connecting a immunoglobulin heterodimerization domain and an immunoglobulin constant region (e.g. , wherein the immunoglobulin constant region includes CH2 and CH3 domains or CH3 and CH4 domains), (d) interposed between and connecting an immunoglobulin constant region and a binding domain, (e) at the amino-terminus of a polypeptide chain, or (f) at the carboxyl-terminus of a polypeptide chain. A polypeptide chain comprising a hinge region as described herein will be capable of associating with a different polypeptide chain to form a heterodimeric protein provided herein, and the heterodimer formed will contain a binding domain that retains its target specificity or its specific target binding affinity.
[00169] In certain embodiments, a hinge present in a polypeptide that forms a heterodimer with another polypeptide chain can be an immunoglobulin hinge, such as a wild-type immunoglobulin hinge region or an altered immunoglobulin hinge region thereof. In certain embodiments, a hinge of one polypeptide chain of a heterodimeric protein is identical to a corresponding hinge of the other polypeptide chain of the heterodimer. In certain other embodiments, a hinge of one chain is different from that of the other chain (in their length or sequence). The different hinges in the different chains allow different manipulation of the binding affinities of the binding domains to which the hinges are connected, so that the heterodimer is able to preferentially bind to the target of one binding domain over the target of the other binding domain. For example, in certain embodiments, a heterodimeric protein has a CD3- or TCR-binding domain in one chain and a ROR1 -binding domain in another chain. Having two different hinges in the two chains may allow the heterodimer to bind to the ROR1 first, and then to a CD3 or other TCR component second. Thus, the heterodimer may recruit CD3+ T-cells to ROR1 -expressing cells (e.g. , ROR1 -expressing tumor or cancer cells), which in turn may damage or destroy the ROR1 -expressing cells.
[00170] In certain embodiments, a carboxyl-terminus linker or an amino-terminus linker is a flexible linker sequence comprising glycine-serine (e.g., Gly4Ser) repeats. In certain embodiments, the linker comprises three Gly4Ser repeats (SEQ ID NO: 160) followed by a proline residue. In certain embodiments the proline residue is followed by an amino acid selected from the group consisting of glycine, arginine and serine. In some embodiments, a carboxyl-terminus linker or an amino-terminus linker comprises or consists of SEQ ID NO:265, SEQ ID NO: 301 , SEQ ID NO: 302 or SEQ ID NO: 303.
[00171] Some exemplary hinge, carboxyl-terminus linker, and amino-terminus linker sequences suitable for use in accordance with the present disclosure are shown in the Tables 1 and 2 below. Additional exemplary hinge and linker regions are set forth in SEQ ID NOs: 241 -244, 601 , 78, 763-791 , 228, 379-434, 618-749 of WO201 1/090762 (US
2013/0129723) (said sequences incorporated by reference herein). Table 1. Exemplary hinges and linkers
Figure imgf000060_0001
Hinae Reaion Amino Acid Seauence SEQ ID NO
H114 SGGGGSGGGGSGGGGSPS SEQ ID NO:302
H115 SGGGGSGGGGSGGGGSPSS SEQ ID NO:303
Table 2. Exemplary hinges and linkers (derived from H7 hinge, stalk region of a type II C-lectin, or interdomain region of a type I transmembrane protein)
Figure imgf000061_0001
Hinqe Amino Acid Sequence Molecule and/or SEQ ID NO:
Reqion hinqe from
which derived
H56 QMNSELSVLANS CD86 SEQ ID NO:200
H57 VSERPFPPNS CD22 SEQ ID NO:201
H58 KPFFTCGSADTCPNS CD72 SEQ ID NO:202
H59 KPFFTCGSADTCPNS CD72 SEQ ID NO:203
H60 QYNCPGQYTFSMPNS CD69 SEQ ID NO:204
H61 EPAFTPGPNIELQKDSDCPNS CD94 SEQ ID NO:205
H62 QRHNNSSLNTRTQKARHCPNS NKG2A SEQ ID NO:206
H63 NSLFNQEVQIPLTESYCPNS NKG2D SEQ ID NO:207
[00172] In certain embodiments, a ROR1 -binding polypeptide or protein of the disclosure (e.g., an anti-ROR1 x anti-CD3 molecule) can comprise an "immunoglobulin dimerization domain" or "immunoglobulin heterodimenzation domain."
[00173] An "immunoglobulin dimerization domain" or "immunoglobulin heterodimenzation domain," as used herein, refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts or associates with a different immunoglobulin domain of another polypeptide chain, wherein the interaction of the different immunoglobulin heterodimenzation domains substantially contributes to or efficiently promotes heterodimenzation of the first and second polypeptide chains (i.e. , the formation of a dimer between two different polypeptide chains, which is also referred to as a "heterodimer" or "heterodimeric protein"). The interactions between immunoglobulin heterodimenzation domains "substantially contributes to or efficiently promotes" the heterodimenzation of first and second polypeptide chains if there is a statistically significant reduction in the dimerization between the first and second polypeptide chains in the absence of the immunoglobulin heterodimenzation domain of the first polypeptide chain and/or the immunoglobulin heterodimenzation domain of the second polypeptide chain. In certain embodiments, when the first and second polypeptide chains are co-expressed, at least 60%, at least about 60% to about 70%, at least about 70% to about 80% , at least 80% to about 90% , 91 %, 92%, 93% , 94%, 95%, 96%, 97% , 98%, or 99% of the first and second polypetpide chains form heterodimers with each other.
Representative immunoglobulin heterodimenzation domains include an immunoglobulin CH 1 domain, an immunoglobulin CL1 domain (e.g. , CK or CA isotypes), or derivatives thereof, including wild-type immunoglobulin CH 1 and CL domains and altered (or mutated) immunoglobulin CH 1 and CL domains, such as provided herein.
[00174] Dimerization/heterodimerization domains can be used where it is desired to form heterodimers from two non-identical polypeptide chains, where one or both polypeptide chains comprise a binding domain. In certain embodiments, one polypeptide chain member of certain heterodimers described herein does not contain a binding domain. As indicated above, a heterodimeric protein of the present disclosure comprises an immunoglobulin heterodimerization domain in each polypeptide chain. The immunoglobulin
heterodimerization domains in the polypeptide chains of a heterodimer are different from each other and thus can be differentially modified to facilitate heterodimerization of both chains and to minimize homodimerization of either chain. Immunoglobulin
heterodimerization domains provided herein allow for efficient heterodimerization between different polypeptides and facilitate purification of the resulting heterodimeric protein.
[00175] As provided herein, immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different single chain polypeptides (e.g. , one short and one long) according to the present disclosure include immunoglobulin CH1 and CL domains, for instance, human CH1 and CL domains. In certain embodiments, an immunoglobulin heterodimerization domain is a wild-type CH 1 domain, such as a wild type lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH1 domain. In further embodiments, an immunoglobulin heterodimerization domain is a wild-type human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH1 domain as set forth in SEQ ID NOS: 1 14, 186-192 and 194, respectively, of PCT Publication No. WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein). In certain embodiments, an immunoglobulin heterodimerization domain is a wild-type human lgG 1 CH1 domain as set forth in SEQ ID NO: 1 14 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
[00176] In further embodiments, an immunoglobulin heterodimerization domain is an altered immunoglobulin CH1 domain, such as an altered lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 IgD, IgE, or IgM CH1 domain. In certain embodiments, an immunoglobulin heterodimerization domain is an altered human lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM CH1 domain. In still further embodiments, a cysteine residue of a wild-type CH1 domain (e.g. , a human CH1 ) involved in forming a disulfide bond with a wild type immunoglobulin CL domain (e.g. , a human CL) is deleted or substituted in the altered immunoglobulin CH1 domain such that a disulfide bond is not formed between the altered CH1 domain and the wild-type CL domain.
[00177] In certain embodiments, an immunoglobulin heterodimerization domain is a wild- type CL domain, such as a wild type CK domain or a wild type CA domain. In certain embodiments, an immunoglobulin heterodimerization domain is a wild type human CK or human CA domain as set forth in SEQ ID NOS: 1 12 and 1 13, respectively, of
WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein). In further embodiments, an immunoglobulin heterodimerization domain is an altered immunoglobulin CL domain, such as an altered CK or CA domain, for instance, an altered human CK or human CA domain.
[00178] In certain embodiments, a cysteine residue of a wild-type CL domain (e.g. , a human CL) involved in forming a disulfide bond with a wild type immunoglobulin CH1 domain (e.g. , a human CH 1) is deleted or substituted in the altered immunoglobulin CL domain. Such altered CL domains can further comprise an amino acid deletion at their amino-termini. An exemplary CK domain is set forth in SEQ ID NO: 141 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein), in which the first arginine and the last cysteine of the wild type human Ck domain are both deleted. In certain embodiments, only the last cysteine of the wild type human Ck domain is deleted in the altered Ck domain because the first arginine deleted from the wild type human Ck domain can be provided by a linker that has an arginine at its carboxyl-terminus and links the amino-terminus of the altered Ck domain with another domain (e.g. , an immunoglobulin sug-region, such as a sub- region comprising immunoglobulin CH2 and CH3 domains). An exemplary CA domain is set forth in SEQ ID NO: 140 of WO201 1/090762 (US 2013/0129723) (said sequence
incorporated by reference herein), in which the first arginine of a wild type human CA domain is deleted and the cysteine involved in forming a disulfide bond with a cysteine in a CH1 domain is substituted by a serine.
[00179] In further embodiments, an immunoglobulin heterodimerization domain is an altered CK domain that contains one or more amino acid substitutions, as compared to a wild type CK domain, at positions that may be involved in forming the interchain-hydrogen bond network at a CK-CK interface. For example, in certain embodiments, an immunoglobulin heterodimerization domain is an altered human CK domain having one or more amino acids at positions N29, N30, Q52, V55, T56, S68 or T70 that are substituted with a different amino acid. The numbering of the amino acids is based on their positions in the altered human CK sequence as set forth in SEQ ID NO: 141 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein). In certain embodiments, an immunoglobulin heterodimerization domain is an altered human CK domain having one, two, three or four amino acid substitutions at positions N29, N30, V55, or T70. The amino acid used as a substitute at the above-noted positions can be an alanine, or an amino acid residue with a bulk side chain moiety such as arginine, tryptophan, tyrosine, glutamate, glutamine, or lysine. Additional amino acid residues that can be used to substitute amino acid residues of the wild type human Ck sequence at the above noted positions (e.g. , N30) include aspartate, methionine, serine and phenyalanine. Exemplary altered human CK domains are set forth in SEQ ID NOS: 142-178 of WO201 1/090762 (US 2013/0129723) (said sequences
incorporated by reference herein). Altered human CK domains are those that facilitate heterodimerization with a CH1 domain, but minimize homodimerization with another CK domain. Representative altered human CK domains are set forth in SEQ ID NOS: 160 (N29W V55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176 (N29Y N30Y V55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E), 838 (N30D V55A T70E), 839 (N30M V55A T70E), 840 (N30S V55A T70E), and 841 (N30F V55A T70E) of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
[00180] In certain embodiments, in addition to or alternative to the mutations in Ck domains described herein, both the immunoglobulin heterodimerization domains (i.e. , immunoglobulin CH1 and CL domains) of a polypeptide heterodimer have mutations so that the resulting immunoglobulin heterodimerization domains form salt bridges (i.e. , ionic interactions) between the amino acid residues at the mutated sites. For example, the immunoglobulin heterodimerization domains of a polypeptide heterodimer can be a mutated CH 1 domain in combination with a mutated Ck domain. In the mutated CH1 domain, valine at position 68 (V68) of the wild type human CH1 domain is substituted by an amino acid residue having a negative charge (e.g. , aspartate or glutamate), whereas leucine at position 29 (L29) of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted is substituted by an amino acid residue having a positive charge (e.g. , lysine, arginine or histidine). The charge-charge interaction between the amino acid residue having a negative charge of the resulting mutated CH1 domain and the amino acid residue having a positive charge of the resulting mutated Ck domain forms a salt bridge, which stabilizes the heterodimeric interface between the mutated CH1 and Ck domains. Alternatively, V68 of the wild type CH1 can be substituted by an amino acid residue having a positive charge, whereas L29 of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted can be substituted by an amino acid residue having a negative charge. Exemplary mutated CH1 sequences in which V68 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:844 and 845 of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein). Exemplary mutated Ck sequences in which L29 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:842 and 843 of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
[00181] Positions other than V68 of human CH1 domain and L29 of human Ck domain can be substituted with amino acids having opposite charges to produce ionic interactions between the amino acids in addition or alternative to the mutations in V68 of CH1 domain and L29 of Ck domain. Such positions can be identified by any suitable method, including random mutagenesis, analysis of the crystal structure of the CH 1 -Ck pair to identify amino acid residues at the CH 1 -Ck interface, and further identifying suitable positions among the amino acid residues at the CH 1 -Ck interface using a set of criteria (e.g. , propensity to engage in ionic interactions, proximity to a potential partner residue, etc.).
[00182] In certain embodiments, polypeptide heterodimers of the present disclosure contain only one pair of immunoglobulin heterodimenzation domains. For example, a first chain of a polypeptide heterodimer can comprise a CH 1 domain as an immunoglobulin
heterodimenzation domain, while a second chain can comprise a CL domain (e.g. , a CK or CA) as an immunoglobulin heterodimenzation domain. Alternatively, a first chain can comprise a CL domain (e.g. , a CK or CA) as an immunoglobulin heterodimenzation domain, while a second chain can comprise a CH 1 domain as an immunoglobulin heterodimenzation domain. As set forth herein, the immunoglobulin heterodimenzation domains of the first and second chains are capable of associating to form a heterodimeric protein of this disclosure.
[00183] In certain other embodiments, heterodimeric proteins of the present disclosure can have two pairs of immunoglobulin heterodimenzation domains. For example, a first chain of a heterodimer can comprise two CH 1 domains, while a second chain can have two CL domains that associate with the two CH 1 domains in the first chain. Alternatively, a first chain can comprise two CL domains, while a second chain can have two CH 1 domains that associate with the two CL domains in the first chain. In certain embodiments, a first polypeptide chain comprises a CH 1 domain and a CL domain, while a second polypeptide chain comprises a CL domain and a CH 1 domain that associate with the CH 1 domain and the CL domain, respectively, of the first polypeptide chain.
[00184] In the embodiments where a heterodimeric protein comprises only one
heterodimenzation pair (i.e. , one immunoglobulin heterodimenzation domain in each chain), the immunoglobulin heterodimenzation domain of each chain can be located amino-terminal to the immunoglobulin constant region of that chain. Alternatively, the immunoglobulin heterodimenzation domain in each chain can be located carboxyl-terminal to the
immunoglobulin constant region of that chain.
[00185] In the embodiments where a heterodimeric protein comprises two
heterodimenzation pairs (i.e. , two immunoglobulin heterodimenzation domains in each chain), both immunoglobulin heterodimenzation domains in each chain can be located amino-terminal to the immunoglobulin constant region of that chain. Alternatively, both immunoglobulin heterodimenzation domains in each chain can be located carboxyl-terminal to the immunoglobulin constant region of that chain. In further embodiments, one immunoglobulin heterodimenzation domain in each chain can be located amino-terminal to the immunoglobulin constant region of that chain, while the other immunoglobulin heterodimerization domain of each chain can be located carboxyl-terminal to the
immunoglobulin constant region of that chain. In other words, in those embodiments, the immunoglobulin constant region is interposed between the two immunoglobulin
heterodimerization domains of each chain.
[00186] As indicated herein, in certain embodiments, ROR1 -binding polypeptides of the present disclosure (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) comprise an immunoglobulin constant region (also referred to as a constant region) in a polypeptide chain. The inclusion of an immunoglobulin constant region slows clearance of the homodimeric and heterodimeric proteins formed from two ROR1 -binding polypeptide chains from circulation after administration to a subject. By mutations or other alterations, an immunoglobulin constant region further enables relatively easy modulation of dimeric polypeptide effector functions (e.g. , ADCC, ADCP, CDC, complement fixation, and binding to Fc receptors), which can either be increased or decreased depending on the disease being treated, as known in the art and described herein. In certain embodiments, an immunoglobulin constant region of one or both of the polypeptide chains of the polypeptide homodimers and heterodimers of the present disclosure will be capable of mediating one or more of these effector functions In other embodiments, one or more of these effector functions are reduced or absent in an immunoglobulin constant region of one or both of the polypeptide chains of the polypeptide homodimers and heterodimers of the present disclosure, as compared to a corresponding wild-type immunoglobulin constant region. For example, for dimeric ROR1 -binding polypeptides designed to elicit RTCC, such as, e.g. , via the inclusion of a CD3-binding domain (for example ROR248, ROR246, ROR252, ROR250, or ROR243), an
immunoglobulin constant region preferably has reduced or no effector function relative to a corresponding wild-type immunoglobulin constant region.
[00187] An immunoglobulin constant region present in ROR1 -binding polypeptides of the present disclosure (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) can comprise or is derived from part or all of: a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof. For example, an immunoglobulin constant region can comprise a CH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3 domains, a CH4 domain, two CH4 domains, and a CH2 domain and part of a CH3 domain. In certain embodiments, a ROR1 - binding polypeptide or protein does not comprise a CH1 domain.
[00188] A CH2 domain that can form an immunoglobulin constant region of a ROR1 -binding polypeptide of the present disclosure (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) can be a wild type immunoglobulin CH2 domain or an altered immunoglobulin CH2 domain thereof from certain immunoglobulin classes or subclasses (e.g. , lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, or IgD) and from various species (including human, mouse, rat, and other mammals).
[00189] In certain embodiments, a CH2 domain of an anti-ROR1 x anti-CD3 molecule is a wild type human immunoglobulin CH2 domain, such as wild type CH2 domains of human lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, or IgD, as set forth in SEQ ID NOS: 1 15, 199-201 and 195-197, respectively, of PCT Publication WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein). In certain embodiments, the CH2 domain is a wild type human lgG1 CH2 domain as set forth in SEQ ID NO: 1 15 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
[00190] In certain embodiments, a CH2 domain of an anti-ROR1 x anti-CD3 molecule is an altered immunoglobulin CH2 region (e.g. , an altered human lgG 1 CH2 domain) that comprises an amino acid substitution at the asparagine of position 297 (e.g. , asparagine to alanine). Such an amino acid substitution reduces or eliminates glycosylation at this site and abrogates efficient Fc binding to FcvR and C1 q. The sequence of an altered human lgG1 CH2 domain with an Asn to Ala substitution at position 297 is set forth in SEQ ID NO:324 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
[00191] In certain embodiments, a CH2 domain of an anti-ROR1 x anti-CD3 molecule is an altered immunoglobulin CH2 region (e.g. , an altered human lgG 1 CH2 domain) that comprises at least one substitution or deletion at positions 234 to 238. For example, an immunoglobulin CH2 region can comprise a substitution at position 234, 235, 236, 237 or 238, positions 234 and 235, positions 234 and 236, positions 234 and 237, positions 234 and 238, positions 234-236, positions 234, 235 and 237, positions 234, 236 and 238, positions 234, 235, 237, and 238, positions 236-238, or any other combination of two, three, four, or five amino acids at positions 234-238. In addition or alternatively, an altered CH2 region can comprise one or more (e.g. , two, three, four or five) amino acid deletions at positions 234-238, for instance, at one of position 236 or position 237 while the other position is substituted. The above-noted mutation(s) decrease or eliminate the antibody- dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide homodimer or heterodimer that comprises the altered CH2 domain. In certain embodiments, the amino acid residues at one or more of positions 234-238 has been replaced with one or more alanine residues. In further embodiments, only one of the amino acid residues at positions 234-238 have been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g. , alanine or serine). [00192] In certain other embodiments, a CH2 domain of an anti-ROR1 x anti-CD3 molecule is an altered immunoglobulin CH2 region (e.g. , an altered human lgG 1 CH2 domain) that comprises one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331 . For example, an immunoglobulin CH2 region can comprise a substitution at position 253, 310, 318, 320, 322, or 331 , positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of two, three, four, five or six amino acids at positions 253, 310, 318, 320, 322, and 331. The above-noted mutation(s) decrease or eliminate the complement-dependent cytotoxicity (CDC) of a polypeptide homodimer or heterodimer that comprises the altered CH2 domain.
[00193] In certain other embodiments, in addition to the amino acid substitution at position 297, an altered CH2 region of an anti-ROR1 x anti-CD3 molecule (e.g. , an altered human lgG 1 CH2 domain) can further comprise one or more (e.g. , two, three, four, or five) additional substitutions at positions 234-238. For example, an immunoglobulin CH2 region can comprise a substitution at positions 234 and 297, positions 234, 235, and 297, positions 234, 236 and 297, positions 234-236 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any combination of two, three, four, or five amino acids at positions 234-238 in addition to position 297. In addition or alternatively, an altered CH2 region can comprise one or more (e.g. , two, three, four or five) amino acid deletions at positions 234-238, such as at position 236 or position 237. The additional mutation(s) decreases or eliminates the antibody- dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide homodimer or heterodimer that comprises the altered CH2 domain. In certain embodiments, the amino acid residues at one or more of positions 234-238 have been replaced with one or more alanine residues. In further embodiments, only one of the amino acid residues at positions 234-238 has been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g. , alanine or serine).
[00194] In certain embodiments, in addition to one or more (e.g. , 2, 3, 4, or 5) amino acid substitutions at positions 234-238, a mutated CH2 region of an anti-ROR1 x anti-CD3 molecule (e.g. , an altered human lgG 1 CH2 domain) in a fusion protein of the present disclosure can contain one or more (e.g. , 2, 3, 4, 5, or 6) additional amino acid substitutions (e.g. , substituted with alanine) at one or more positions involved in complement fixation (e.g. , at positions I253, H310, E318, K320, K322, or P331). Examples of mutated immunoglobulin CH2 regions include human lgG 1 , lgG2, lgG4 and mouse lgG2a CH2 regions with alanine substitutions at positions 234, 235, 237 (if present), 318, 320 and 322. An exemplary mutated immunoglobulin CH2 region is mouse IGHG2c CH2 region with alanine
substitutions at L234, L235, G237, E318, K320, and K322.
[00195] In still further embodiments, in addition to the amino acid substitution at position 297 and the additional deletion(s) or substitution(s) at positions 234-238, an altered CH2 region of an anti-ROR1 x anti-CD3 molecule (e.g. , an altered human lgG1 CH2 domain) can further comprise one or more (e.g. , two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331 . For example, an immunoglobulin CH2 region can comprise a (1) substitution at position 297, (2) one or more substitutions or deletions or a combination thereof at positions 234-238, and one or more (e.g. , 2, 3, 4, 5, or 6) amino acid substitutions at positions 1253, H310, E318, K320, K322, and P331 , such as one, two, three substitutions at positions E318, K320 and K322. The amino acids at the above-noted positions can be substituted by alanine or serine.
[00196] In certain embodiments, an immunoglobulin CH2 region polypeptide of an anti- ROR1 x anti-CD3 molecule comprises: (i) an amino acid substitution at the asparagines of position 297 and one amino acid substitution at position 234, 235, 236 or 237; (ii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at two of positions 234-237; (iii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at three of positions 234-237; (iv) an amino acid substitution at the asparagine of position 297, amino acid substitutions at positions 234, 235 and 237, and an amino acid deletion at position 236; (v) amino acid substitutions at three of positions 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three of positions 234-237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320 and 322.
[00197] Exemplary altered immunoglobulin CH2 regions with amino acid substitutions at the asparagine of position 297 include: human lgG1 CH2 region with alanine substitutions at L234, L235, G237 and N297 and a deletion at G236 (SEQ ID NO:325 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), human lgG2 CH2 region with alanine substitutions at V234, G236, and N297 (SEQ ID NO:326 of
WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), human lgG4 CH2 region with alanine substitutions at F234, L235, G237 and N297 and a deletion of G236 (SEQ ID NO:322 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), human lgG4 CH2 region with alanine substitutions at F234 and N297 (SEQ ID NO:343 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), human lgG4 CH2 region with alanine substitutions at L235 and N297 (SEQ ID NO:344 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), human lgG4 CH2 region with alanine substitutions at G236 and N297 (SEQ ID NO:345 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), and human lgG4 CH2 region with alanine substitutions at G237 and N297 (SEQ ID NO:346 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein). These CH2 regions can be used in a ROR1-binding polypeptide of the present disclosure (e.g., an anti-ROR1 x anti-CD3 molecule).
[00198] In certain embodiments, in addition to the amino acid substitutions described above, an altered CH2 region of an anti-ROR1 x anti-CD3 molecule (e.g. , an altered human lgG1 CH2 domain) can contain one or more additional amino acid substitutions at one or more positions other than the above-noted positions. Such amino acid substitutions can be conservative or non-conservative amino acid substitutions. For example, in certain embodiments, P233 can be changed to E233 in an altered lgG2 CH2 region (see, e.g. , SEQ ID NO:326 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein). In addition or alternatively, in certain embodiments, the altered CH2 region can contain one or more amino acid insertions, deletions, or both. The insertion(s), deletion(s) or substitution(s) can be anywhere in an immunoglobulin CH2 region, such as at the N- or C-terminus of a wild type immunoglobulin CH2 region resulting from linking the CH2 region with another region (e.g. , a binding domain or an immunoglobulin
heterodimerization domain) via a hinge.
[00199] In certain embodiments, an altered CH2 region in a polypeptide of the present disclosure (e.g, in an anti-ROR1 x anti-CD3 molecule) comprises or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild type immunoglobulin CH2 region, such as the CH2 region of wild type human lgG1 , lgG2, or lgG4, or mouse lgG2a (e.g. , IGHG2C).
[00200] An altered immunoglobulin CH2 region in a ROR1 -binding polypeptide of the present disclosure (e.g., an anti-ROR1 x anti-CD3 molecule) can be derived from a CH2 region of various immunoglobulin isotypes, such as lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, and IgD, from various species (including human, mouse, rat, and other mammals). In certain embodiments, an altered immunoglobulin CH2 region in a fusion protein of the present disclosure can be derived from a CH2 region of human lgG 1 , lgG2 or lgG4, or mouse lgG2a (e.g. , IGHG2c), whose sequences are set forth in SEQ ID NOS: 1 15, 199, 201 , and 320 of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
[00201] In certain embodiments, an altered CH2 domain of an anti-ROR1 x anti-CD3 molecule is a human lgG1 CH2 domain with alanine substitutions at positions 235, 318, 320, and 322 (i.e. , a human lgG1 CH2 domain with L235A, E318A, K320A and K322A substitutions) (SEQ ID NO:595 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), and optionally an N297 mutation (e.g. , to alanine). In certain other embodiments, an altered CH2 domain is a human lgG 1 CH2 domain with alanine substitutions at positions 234, 235, 237, 318, 320 and 322 (i.e. , a human lgG 1 CH2 domain with L234A, L235A, G237A, E318A, K320A and K322A substitutions) (SEQ ID NO:596 of WO201 1/090762 (US 2013/0129723), said sequence incorporated by reference herein), and optionally an N297 mutation (e.g. , to alanine).
[00202] In some embodiments, an immunoglobulin constant region of a ROR1 -binding protein or polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) comprises a human lgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A, according to the EU numbering system.
[00203] In certain embodiments, an altered CH2 domain of an anti-ROR1 x anti-CD3 molecule is an altered human lgG 1 CH2 domain with mutations known in the art that enhance immunological activities such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any combination thereof.
[00204] The CH3 domain that can form an immunoglobulin constant region of a ROR1 - binding polypeptide of the present disclosure (e.g., an anti-ROR1 x anti-CD3 molecule) can be a wild type immunoglobulin CH3 domain or an altered immunoglobulin CH3 domain thereof from certain immunoglobulin classes or subclasses (e.g. , lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM) of various species (including human, mouse, rat, and other mammals). In certain embodiments, a CH3 domain of an anti-ROR1 x anti-CD3 molecule is a wild type human immunoglobulin CH3 domain, such as wild type CH3 domains of human lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM as set forth in SEQ ID NOS: 1 16, 208- 210, 204-207, and 212, respectively of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein). In certain embodiments, the CH3 domain is a wild type human lgG1 CH3 domain as set forth in SEQ ID NO: 1 16 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein). In certain embodiments, a CH3 domain of an anti-ROR1 x anti-CD3 molecule is an altered human immunoglobulin CH3 domain, such as an altered CH3 domain based on or derived from a wild-type CH3 domain of human lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, or IgM antibodies. For example, an altered CH3 domain can be a human lgG1 CH3 domain with one or two mutations at positions H433 and N434 (positions are numbered according to EU numbering). The mutations in such positions can be involved in complement fixation. In certain other embodiments, an altered CH3 domain of an anti-ROR1 x anti-CD3 molecule can be a human lgG 1 CH3 domain but with one or two amino acid substitutions at position F405 or Y407. The amino acids at such positions are involved in interacting with another CH3 domain. In certain embodiments, an altered CH3 domain of an anti-ROR1 x anti-CD3 molecule can be an altered human lgG1 CH3 domain with its last lysine deleted. The sequence of this altered CH3 domain is set forth in SEQ ID NO:761 of WO201 1/090762 (US 2013/0129723) (said sequence incorporated by reference herein).
[00205] In certain embodiments, ROR1 -binding polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule) forming a polypeptide heterodimer comprise a CH3 pair that comprises so called "knobs-into-holes" mutations (see, Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et al. , Protein Engineering 9:617-21 , 1966). More specifically, mutations can be introduced into each of the two CH3 domains of each polypeptide chain so that the steric complementarity required for CH3/CH3 association obligates these two CH3 domains to pair with each other. For example, a CH3 domain in one single chain polypeptide of a polypeptide heterodimer can contain a T366W mutation (a "knob" mutation, which substitutes a small amino acid with a larger one), and a CH3 domain in the other single chain polypeptide of the polypeptide heterodimer can contain a Y407A mutation (a "hole" mutation, which substitutes a large amino acid with a smaller one). Other exemplary knobs- into-holes mutations include (1) a T366Y mutation in one CH3 domain and a Y407T in the other CH3 domain, and (2) a T366W mutation in one CH3 domain and T366S, L368A and Y407V mutations in the other CH3 domain.
[00206] The CH4 domain that can form an immunoglobulin constant region of ROR1 -binding polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule) of the present disclosure can be a wild type immunoglobulin CH4 domain or an altered immunoglobulin CH4 domain thereof from IgE or IgM molecules. In certain embodiments, the CH4 domain of an anti-ROR1 x anti-CD3 molecule is a wild type human immunoglobulin CH4 domain, such as wild type CH4 domains of human IgE and IgM molecules as set forth in SEQ ID NOS:213 and 214, respectively, of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein). In certain embodiments, a CH4 domain of an anti-ROR1 x anti-CD3 molecule is an altered human immunoglobulin CH4 domain, such as an altered CH4 domain based on or derived from a CH4 domain of human IgE or IgM molecules, which have mutations that increase or decrease an immunological activity known to be associated with an IgE or IgM Fc region.
[00207] In certain embodiments, an immunoglobulin constant region of ROR1 -binding polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule) of the present disclosure comprises a combination of CH2, CH3 or CH4 domains (i.e. , more than one constant region domain selected from CH2, CH3 and CH4). For example, the immunoglobulin constant region can comprise CH2 and CH3 domains or CH3 and CH4 domains. In certain other embodiments, the immunoglobulin constant region can comprise two CH3 domains and no CH2 or CH4 domains (i.e. , only two or more CH3). The multiple constant region domains that form an immunoglobulin constant region of an anti-ROR1 x anti-CD3 molecule can be based on or derived from the same immunoglobulin molecule, or the same class or subclass
immunoglobulin molecules. In certain embodiments, the immunoglobulin constant region is an IgG CH2CH3 (e.g. , lgG 1 CH2CH3, lgG2 CH2CH3, and lgG4 CH2CH3) and can be a human (e.g. , human lgG 1 , lgG2, and lgG4) CH2CH3. For example, in certain embodiments, the immunoglobulin constant region of an anti-ROR1 x anti-CD3 molecule comprises (1) wild type human lgG1 CH2 and CH3 domains, (2) human lgG 1 CH2 with N297A substitution (i.e. , CH2(N297A)) and wild type human lgG 1 CH3, or (3) human lgG1 CH2(N297A) and an altered human lgG 1 CH3 with the last lysine deleted.
[00208] Alternatively, the multiple constant region domains of an anti-ROR1 x anti-CD3 molecule can be based on or derived from different immunoglobulin molecules, or different classes or subclasses immunoglobulin molecules. For example, in certain embodiments, an immunoglobulin constant region comprises both human IgM CH3 domain and human lgG 1 CH3 domain. The multiple constant region domains that form an immunoglobulin constant region of an anti-ROR1 x anti-CD3 molecule can be directly linked together or can be linked to each other via one or more (e.g. , about 2-10) amino acids.
[00209] Exemplary immunoglobulin constant regions that can be used in an anti-ROR1 x anti-CD3 molecule are set forth in SEQ ID NOS:305-309, 321 , 323, 341 , 342, and 762 of WO201 1/090762 (US 2013/0129723) (said sequences incorporated by reference herein).
[00210] In certain embodiments, the immunoglobulin constant regions of both ROR1 -binding polypeptides of a polypeptide homodimer or heterodimer of an anti-ROR1 x anti-CD3 molecule are identical to each other. In certain other embodiments, the immunoglobulin constant region of one polypeptide chain of a heterodimeric protein is different from the immunoglobulin constant region of the other polypeptide chain of the heterodimer. For example, one immunoglobulin constant region of a heterodimeric protein can contain a CH3 domain with a "knob" mutation, whereas the other immunoglobulin constant region of the heterodimeric protein can contain a CH3 domain with a "hole" mutation.
[00211] The disclosure relates to ROR1 -binding proteins and polypeptides that may comprise any of the sequences shown in Table 3. Sequences for various cloned sequences and components are also presented in Table 3 and summarized in Table 12. Amino acid sequences given for polypeptide constructs do not include the human or rabbit
immunoglobulin leader sequences. CDR sequences and amino acid substitution positions shown are those defined using the IMGT criteria (Brochet, X, et al, Nucl. Acids Res. (2008) 36, W503-508). Table 3. Binding Polypeptide Sequences and Components
Figure imgf000075_0001
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Rll VH-VL cagtccgtgaaggagtccgagggcgacctggtgacccccgccggcaacc qsvkesegdlvtpagnlt SEQ ID N0:17 scFv tgaccctgacctgcaccgcctccggctccgacatcaacgactaccccatct Itctasgsdindypiswv (SEQ ID NO:18) cctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatc rqapgkglewigfinsgg
aactccggcggctccacctggtacgcctcctgggtgaagggccggttcac stwyaswvkgrftisrtst
catctcccggacctccaccaccgtggacctgaagatgacctccctgacca tvdlkmtslttddtatyfc
ccgacgacaccgccacctacttctgcgcccggggctactccacctactacg argystyygdfniwgpg
gcgacttcaacatctggggccccggcaccctggtgaccatctcctccggtg tlvtissggggsggggsgg
gaggcggttcaggcggaggtggatccggcggtggcggctccggtggcgg ggsggggselvmtqtps
cggatctgagctggtgatgacccagaccccctcctccacctccggcgccgt stsgavggtvtincqasq
gggcggcaccgtgaccatcaactgccaggcctcccagtccatcgactcca sidsnlawfqqkpgqpp
acctggcctggttccagcagaagcccggccagccccccaccctgctgatc tlliyrasnlasgvpsrfsg
taccgggcctccaacctggcctccggcgtgccctcccggttctccggctcc srsgteytltisgvqreda
cggtccggcaccgagtacaccctgaccatctccggcgtgcagcgggagg atyyclggvgnvsyrtsfg
acgccgccacctactactgcctgggcggcgtgggcaacgtgtcctaccgg ggtevvvkss
acctccttcggcggcggcaccgaggtggtggtgaagtcctcg
Rll VL-VH gagctggtgatgacccagaccccctcctccacctccggcgccgtgggcgg elvmtqtpsstsgavggt SEQ ID NO:20 scFv caccgtgaccatcaactgccaggcctcccagtccatcgactccaacctgg vtincqasqsidsnlawf (SEQ ID NO:21) cctggttccagcagaagcccggccagccccccaccctgctgatctaccgg qqkpgqpptlliyrasnl
gcctccaacctggcctccggcgtgccctcccggttctccggctcccggtcc asgvpsrfsgsrsgteytl
ggcaccgagtacaccctgaccatctccggcgtgcagcgggaggacgccg tisgvqredaatyyclgg
ccacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcct vgnvsyrtsfgggtevvv
tcggcggcggcaccgaggtggtggtgaagggtggaggcggttcaggcgg kggggsggggsggggsg
aggtggatccggcggtggcggctccggtggcggcggatctcagtccgtga gggsqsvkesegdlvtp
aggagtccgagggcgacctggtgacccccgccggcaacctgaccctgac agnltltctasgsdindyp
ctgcaccgcctccggctccgacatcaacgactaccccatctcctgggtgcg iswvrqapgkglewigfi
gcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcg nsggstwyaswvkgrft
gctccacctggtacgcctcctgggtgaagggccggttcaccatctcccgga isrtsttvdlkmtslttddt
cctccaccaccgtggacctgaagatgacctccctgaccaccgacgacacc atyfcargystyygdfni
gccacctacttctgcgcccggggctactccacctactacggcgacttcaac wgpgtlvtiss
atctggggccccggcaccctggtgaccatctcctcg
Humanized gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:22 Rll VL (L2) cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:23) ctggtaccagcagaagcccggcaagccccccaagctgctgatctaccgg yqqkpgkppklliyrasn
gcctccaacctggcctccggcgtgccctcccggttctccggctccggctcc lasgvpsrfsgsgsgtdftl ggcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggc tisslqpedvatyyclggv
cacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcctt gnvsyrtsfgggtkveik
cggcggcggcaccaaggtggagatcaag
Humanized gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:24 Rll VL (L6) cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:25) ctggttccagcagaagcccggcaagccccccaagctgctgatctaccggg fqqkpgkppklliyrasnl cctccaacctggcctccggcgtgccctcccggttctccggctccggctccg asgvpsrfsgsgsgtdftl
gcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggcc tisslqpedvatyyclggv
acctactactgcctgggcggcgtgggcaacgtgtcctaccggacctccttc gnvsyrtsfgggtkveik
ggcggcggcaccaaggtggagatcaag Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggt evqlvesggglvqpgrsl SEQ ID NO:26
Rll VH ccctgcggctgtcctgcaccgcctccggctccgacatcaacgactacccca rlsctasgsdindypisw (SEQ ID NO:27)
(H9) tctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttc vrqapgkglewigfinsg
atcaactccggcggctccacctggtacgcctcctgggtgaagggccggttc gstwyaswvkgrftisrd
accatctcccgggacgactccaagtccatcgcctacctgcagatgaactc dsksiaylqmnslktedt
cctgaagaccgaggacaccgccgtgtacttctgcacccggggctactcca avyfctrgystyygdfni
cctactacggcgacttcaacatctggggccagggcaccctggtgaccgtg wgqgtlvtvss
tcctcg
Hemi- gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac Diqmtqspsslsasvgd SEQ ID NO:28 humanized cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:29)
Rll VL-VH ctggtaccagcagaagcccggcaagccccccaagctgctgatctaccgg yqqkpgkppklliyrasn
scFv (L2H0) gcctccaacctggcctccggcgtgccctcccggttctccggctccggctcc lasgvpsrfsgsgsgtdftl
ggcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggc tisslqpedvatyyclggv
cacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcctt gnvsyrtsfgggtkveik
cggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgg ggggsggggsggggsgg
aggtggatccggcggtggcggctccggtggcggcggatctcagtccgtga ggsqsvkesegdlvtpa
aggagtccgagggcgacctggtgacccccgccggcaacctgaccctgac gnltltctasgsdindypi
ctgcaccgcctccggctccgacatcaacgactaccccatctcctgggtgcg swvrqapgkglewigfi
gcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcg nsggstwyaswvkgrft
gctccacctggtacgcctcctgggtgaagggccggttcaccatctcccgga isrtsttvdlkmtslttddt
cctccaccaccgtggacctgaagatgacctccctgaccaccgacgacacc atyfcargystyygdfni
gccacctacttctgcgcccggggctactccacctactacggcgacttcaac wgpgtlvtis
atctggggccccggcaccctggtgaccatctcctcg
Hemi- gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:30 humanized cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:31)
Rll VL-VH ctggttccagcagaagcccggcaagccccccaagctgctgatctaccggg fqqkpgkppklliyrasnl
scFv (L6H0) cctccaacctggcctccggcgtgccctcccggttctccggctccggctccg asgvpsrfsgsgsgtdftl
gcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggcc tisslqpedvatyyclggv
acctactactgcctgggcggcgtgggcaacgtgtcctaccggacctccttc gnvsyrtsfgggtkveik
ggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgga ggggsggggsggggsgg
ggtggatccggcggtggcggctccggtggcggcggatctcagtccgtgaa ggsqsvkesegdlvtpa
ggagtccgagggcgacctggtgacccccgccggcaacctgaccctgacct gnltltctasgsdindypi
gcaccgcctccggctccgacatcaacgactaccccatctcctgggtgcgg swvrqapgkglewigfi
caggcccccggcaagggcctggagtggatcggcttcatcaactccggcgg nsggstwyaswvkgrft
ctccacctggtacgcctcctgggtgaagggccggttcaccatctcccggac isrtsttvdlkmtslttddt
ctccaccaccgtggacctgaagatgacctccctgaccaccgacgacaccg atyfcargystyygdfni
ccacctacttctgcgcccggggctactccacctactacggcgacttcaaca wgpgtlvtiss
tctggggccccggcaccctggtgaccatctcctcg
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:32
Rll VL-VH cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:33) scFv (L2H9) ctggtaccagcagaagcccggcaagccccccaagctgctgatctaccgg yqqkpgkppklliyrasn
gcctccaacctggcctccggcgtgccctcccggttctccggctccggctcc lasgvpsrfsgsgsgtdftl ggcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggc tisslqpedvatyyclggv
cacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcctt gnvsyrtsfgggtkveik
cggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgg ggggsggggsggggsgg
aggtggatccggcggtggcggctccggtggcggcggatctgaggtgcag ggsevqlvesggglvqp
ctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggct grslrlsctasgsdindypi gtcctgcaccgcctccggctccgacatcaacgactaccccatctcctgggt swvrqapgkglewigfi
gcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccg nsggstwyaswvkgrft
gcggctccacctggtacgcctcctgggtgaagggccggttcaccatctccc isrddsksiaylqmnslkt
gggacgactccaagtccatcgcctacctgcagatgaactccctgaagacc edtavyfctrgystyygd
gaggacaccgccgtgtacttctgcacccggggctactccacctactacgg fniwgqgtlvtvss
cgacttcaacatctggggccagggcaccctggtgaccgtgtcctcg
Humanized gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac Diqmtqspsslsasvgd SEQ ID NO:34
Rll VL-VH cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:35) scFv (L6H9) ctggttccagcagaagcccggcaagccccccaagctgctgatctaccggg fqqkpgkppklliyrasnl
cctccaacctggcctccggcgtgccctcccggttctccggctccggctccg asgvpsrfsgsgsgtdftl
gcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggcc tisslqpedvatyyclggv
acctactactgcctgggcggcgtgggcaacgtgtcctaccggacctccttc gnvsyrtsfgggtkveik
ggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgga ggggsggggsggggsgg
ggtggatccggcggtggcggctccggtggcggcggatctgaggtgcagct ggsevqlvesggglvqp
ggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgt grslrlsctasgsdindypi cctgcaccgcctccggctccgacatcaacgactaccccatctcctgggtgc swvrqapgkglewigfi
ggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggc nsggstwyaswvkgrft
ggctccacctggtacgcctcctgggtgaagggccggttcaccatctcccgg isrddsksiaylqmnslkt
gacgactccaagtccatcgcctacctgcagatgaactccctgaagaccga edtavyfctrgystyygd
ggacaccgccgtgtacttctgcacccggggctactccacctactacggcg fniwgqgtlvtvss
acttcaacatctggggccagggcaccctggtgaccgtgtcctcg
Rabbit R12 caggagcagctggtggagtccggcggccggctggtgacccccggcggct qeqlvesggrlvtpggslt SEQ ID NO:36
VH region ccctgaccctgtcctgcaaggcctccggcttcgacttctccgcctactacat Isckasgfdfsayymsw (SEQ ID NO:37) gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc vrqapgkglewiatiyps
atctacccctcctccggcaagacctactacgccacctgggtgaacggccg sgktyyatwvngrftiss
gttcaccatctcctccgacaacgcccagaacaccgtggacctgcagatga dnaqntvdlqmnsltaa
actccctgaccgccgccgaccgggccacctacttctgcgcccgggactcct dratyfcardsyaddgal
acgccgacgacggcgccctgttcaacatctggggccccggcaccctggtg fniwgpgtlvtiss
accatctcctcc
Rabbit R12 gagctggtgctgacccagtccccctccgtgtccgccgccctgggctccccc elvltqspsvsaalgspa SEQ ID NO:38
VL region gccaagatcacctgcaccctgtcctccgcccacaagaccgacaccatcga kitctlssahktdtidwyq (SEQ ID NO:39) ctggtaccagcagctgcagggcgaggccccccggtacctgatgcaggtgc qlqgeaprylmqvqsd
agtccgacggctcctacaccaagcggcccggcgtgcccgaccggttctcc gsytkrpgvpdrfsgsss
ggctcctcctccggcgccgaccggtacctgatcatcccctccgtgcaggcc gadryliipsvqaddead
gacgacgaggccgactactactgcggcgccgactacatcggcggctacgt yycgadyiggyvfgggtq
gttcggcggcggcacccagctgaccgtgaccggctcctcg Itvtgss
R12 VH ggcttcgacttctccgcctactac GFDFSAYY SEQ ID NO:40
CDR1 (SEQ ID NO:41)
R12 VH atctacccctcctccggcaagacc IYPSSGKT SEQ ID NO:42
CDR2 (SEQ ID NO:43) Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
R12 VH gcccgggactcctacgccgacgacggcgccctgttcaacatc ARDSYADDGALFNI SEQ ID NO:44
CDR3 (SEQ ID NO:45)
R12 VL tccgcccacaagaccgacacc SAHKTDT SEQ ID NO:46
CDR1 (SEQ ID NO:47)
R12 VL gtgcagtccgacggctcctac VQSDGSY SEQ ID NO:48
CDR2 (SEQ ID NO:49)
R12 VL ggcgccgactacatcggcggctacgtg GADYIGGYV SEQ ID NO:50
CDR3 (SEQ ID NO:51)
R12 VH-VL caggagcagctggtggagtccggcggccggctggtgacccccggcggct qeqlvesggrlvtpggslt SEQ ID NO:52 scFv ccctgaccctgtcctgcaaggcctccggcttcgacttctccgcctactacat Isckasgfdfsayymsw (SEQ ID NO:53) gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc vrqapgkglewiatiyps
atctacccctcctccggcaagacctactacgccacctgggtgaacggccg sgktyyatwvngrftiss
gttcaccatctcctccgacaacgcccagaacaccgtggacctgcagatga dnaqntvdlqmnsltaa
actccctgaccgccgccgaccgggccacctacttctgcgcccgggactcct dratyfcardsyaddgal
acgccgacgacggcgccctgttcaacatctggggccccggcaccctggtg fniwgpgtlvtissggggs
accatctcctccggtggaggcggttcaggcggaggtggatccggcggtgg ggggsggggsggggsel
cggctccggtggcggcggatctgagctggtgctgacccagtccccctccgt vltqspsvsaalgspakit
gtccgccgccctgggctcccccgccaagatcacctgcaccctgtcctccgc ctlssahktdtidwyqql
ccacaagaccgacaccatcgactggtaccagcagctgcagggcgaggcc qgeaprylmqvqsdgs
ccccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggcc ytkrpgvpdrfsgsssga
cggcgtgcccgaccggttctccggctcctcctccggcgccgaccggtacct dryliipsvqaddeadyy
gatcatcccctccgtgcaggccgacgacgaggccgactactactgcggcg cgadyiggyvfgggtqlt
ccgactacatcggcggctacgtgttcggcggcggcacccagctgaccgtg vtgss
accggctcctcg
Humanized cagctggtgctgacccagtccccctccgcctccgcctccctgggcgcctcc qlvltqspsasaslgasvk SEQ ID NO:54
R12 VL (LI) gtgaagctgacctgcaccctgtcctccgcccacaagaccgacaccatcga Itctlssahktdtidwyq (SEQ ID NO:55) ctggtaccagcagcagcccgagaagggcccccggtacctgatgcaggtg qqpekgprylmqvqsd
cagtccgacggctcctacaccaagcggcccggcgtgcccgaccgcttctc gsytkrpgvpdrfsgsss
cggctcctcctccggcgccgagcggtacctgaccatctcctccctgcagtc gaeryltisslqsedead
cgaggacgaggccgactactactgcggcgccgactacatcggcggctac yycgadyiggyvfgggtq
gtgttcggcggcggcacccagctgaccgtgctgtcctcg Itvlss
Humanized cagcccgtgctgacccagtcctcctccgcctccgcctccctgggctcctccg qpvltqsssasaslgssvk SEQ ID NO:56
R12 VL (L2) tgaagctgacctgcaccctgtcctccgcccacaagaccgacaccatcgac Itctlssahktdtidwyq (SEQ ID NO:57) tggtaccagcagcagcccggcaaggccccccggtacctgatgcaggtgc qqpgkaprylmqvqsd
agtccgacggctcctacaccaagcggcccggcgtgcccgaccgcttctcc gsytkrpgvpdrfsgsss
ggctcctcctccggcgccgaccgctacctgaccatctccaacctgcagtcc gadryltisnlqsedead
gaggacgaggccgactactactgcggcgccgactacatcggcggctacg yycgadyiggyvfgggtq
tgttcggcggcggcacccagctgaccgtgctgtcctcg Itvlss
Humanized gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggct evqlvesggglvqpggsl SEQ ID NO:58
R12 VH ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:59)
(HI) gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc wvrqapgkglewiatiy
atctacccctcctccggcaagacctactacgccacctgggtgaagggccg pssgktyyatwvkgrfti
gttcaccatctcccgggacaactccaagaacaccctgtacctgcagatga srdnskntlylqmnslra
actccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactcct edtavyfcardsyaddg
acgccgacgacggcgccctgttcaacatctggggccagggcaccctggtg alfniwgqgtlvtvss
accgtgtcctcc Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized caggtgcagctggtggagtccggcggcggcctggtgaagcccggcggct qvqlvesggglvkpggsl SEQ ID NO:60 R12 VH ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:61) (H2) gtcctggattcggcaggcccccggcaagggcctggagtggatcgccacca wirqapgkglewiatiyp
tctacccctcctccggcaagacctactacgccacctgggtgaagggccgg ssgktyyatwvkgrftisr
ttcaccatctcccgggacaacgccaagaactccctgtacctgcagatgaa dnaknslylqmnslrae
ctccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactccta dtavyfcardsyaddgal cgccgacgacggcgccctgttcaacatctggggccagggcaccctggtga fniwgqgtlvtvss
ccgtgtcctcc
Humanized gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggct evqlvesggglvqpggsl SEQ ID NO:62 R12 VH-VL ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:63) scFv (H1L1) gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc wvrqapgkglewiatiy
atctacccctcctccggcaagacctactacgccacctgggtgaagggccg pssgktyyatwvkgrfti
gttcaccatctcccgggacaactccaagaacaccctgtacctgcagatga srdnskntlylqmnslra
actccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactcct edtavyfcardsyaddg
acgccgacgacggcgccctgttcaacatctggggccagggcaccctggtg alfniwgqgtlvtvssgg
accgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtgg ggsggggsggggsgggg
cggctccggtggcggcggatctcagctggtgctgacccagtccccctccgc sqlvltqspsasaslgasv
ctccgcctccctgggcgcctccgtgaagctgacctgcaccctgtcctccgc kltctlssahktdtidwyq
ccacaagaccgacaccatcgactggtaccagcagcagcccgagaaggg qqpekgprylmqvqsd
cccccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggc gsytkrpgvpdrfsgsss
ccggcgtgcccgaccgcttctccggctcctcctccggcgccgagcggtacc gaeryltisslqsedead
tgaccatctcctccctgcagtccgaggacgaggccgactactactgcggc yycgadyiggyvfgggtq
gccgactacatcggcggctacgtgttcggcggcggcacccagctgaccgt Itvlss
gctgtcctcg
Humanized gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggct evqlvesggglvqpggsl SEQ ID NO:64 R12 VH-VL ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:65) scFv (H1L2) gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc wvrqapgkglewiatiy
atctacccctcctccggcaagacctactacgccacctgggtgaagggccg pssgktyyatwvkgrfti
gttcaccatctcccgggacaactccaagaacaccctgtacctgcagatga srdnskntlylqmnslra
actccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactcct edtavyfcardsyaddg
acgccgacgacggcgccctgttcaacatctggggccagggcaccctggtg alfniwgqgtlvtvssgg
accgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtgg ggsggggsggggsgggg
cggctccggtggcggcggatctcagcccgtgctgacccagtcctcctccgc sqpvltqsssasaslgssv
ctccgcctccctgggctcctccgtgaagctgacctgcaccctgtcctccgcc kltctlssahktdtidwyq
cacaagaccgacaccatcgactggtaccagcagcagcccggcaaggccc qqpgkaprylmqvqsd
cccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggccc gsytkrpgvpdrfsgsss
ggcgtgcccgaccgcttctccggctcctcctccggcgccgaccgctacctg gadryltisnlqsedead
accatctccaacctgcagtccgaggacgaggccgactactactgcggcgc yycgadyiggyvfgggtq
cgactacatcggcggctacgtgttcggcggcggcacccagctgaccgtgc Itvlss
tgtcctcg
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized caggtgcagctggtggagtccggcggcggcctggtgaagcccggcggct qvqlvesggglvkpggsl SEQ ID NO:66 R12 VH-VL ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:67) scFv (H2L1) gtcctggattcggcaggcccccggcaagggcctggagtggatcgccacca wirqapgkglewiatiyp
tctacccctcctccggcaagacctactacgccacctgggtgaagggccgg ssgktyyatwvkgrftisr
ttcaccatctcccgggacaacgccaagaactccctgtacctgcagatgaa dnaknslylqmnslrae
ctccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactccta dtavyfcardsyaddgal cgccgacgacggcgccctgttcaacatctggggccagggcaccctggtga fniwgqgtlvtvssgggg
ccgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtggc sggggsggggsggggsq
ggctccggtggcggcggatctcagctggtgctgacccagtccccctccgcc Ivltqspsasaslgasvklt
tccgcctccctgggcgcctccgtgaagctgacctgcaccctgtcctccgcc ctlssahktdtidwyqq
cacaagaccgacaccatcgactggtaccagcagcagcccgagaagggc qpekgprylmqvqsdg
ccccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggcc sytkrpgvpdrfsgsssg
cggcgtgcccgaccgcttctccggctcctcctccggcgccgagcggtacct aeryltisslqsedeady
gaccatctcctccctgcagtccgaggacgaggccgactactactgcggcg ycgadyiggyvfgggtql
ccgactacatcggcggctacgtgttcggcggcggcacccagctgaccgtg tvlss
ctgtcctcg
Humanized caggtgcagctggtggagtccggcggcggcctggtgaagcccggcggct qvqlvesggglvkpggsl SEQ ID NO:68 R12 VH-VL ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:69) scFv (H2L2) gtcctggattcggcaggcccccggcaagggcctggagtggatcgccacca wirqapgkglewiatiyp
tctacccctcctccggcaagacctactacgccacctgggtgaagggccgg ssgktyyatwvkgrftisr
ttcaccatctcccgggacaacgccaagaactccctgtacctgcagatgaa dnaknslylqmnslrae
ctccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactccta dtavyfcardsyaddgal cgccgacgacggcgccctgttcaacatctggggccagggcaccctggtga fniwgqgtlvtvssgggg
ccgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtggc sggggsggggsggggsq
ggctccggtggcggcggatctcagcccgtgctgacccagtcctcctccgcc pvltqsssasaslgssvkl
tccgcctccctgggctcctccgtgaagctgacctgcaccctgtcctccgccc tctlssahktdtidwyqq
acaagaccgacaccatcgactggtaccagcagcagcccggcaaggcccc qpgkaprylmqvqsdg
ccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggcccg sytkrpgvpdrfsgsssg
gcgtgcccgaccgcttctccggctcctcctccggcgccgaccgctacctga adryltisnlqsedeady
ccatctccaacctgcagtccgaggacgaggccgactactactgcggcgcc ycgadyiggyvfgggtql
gactacatcggcggctacgtgttcggcggcggcacccagctgaccgtgct tvlss
gtcctcg
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR015 cagtccgtgaaggagtccgagggcgacctggtgacccccgccggcaacc qsvkesegdlvtpagnlt SEQ ID NO:70 tgaccctgacctgcaccgcctccggctccgacatcaacgactaccccatct Itctasgsdindypiswv (SEQ ID NO:71)
Chimeric
cctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatc rqapgkglewigfinsgg
Rll VH-VL
aactccggcggctccacctggtacgcctcctgggtgaagggccggttcac stwyaswvkgrftisrtst
scFv-Fc
catctcccggacctccaccaccgtggacctgaagatgacctccctgacca tvdlkmtslttddtatyfc
ccgacgacaccgccacctacttctgcgcccggggctactccacctactacg argystyygdfniwgpg
gcgacttcaacatctggggccccggcaccctggtgaccatctcctccggtg tlvtissggggsggggsgg
gaggcggttcaggcggaggtggatccggcggtggcggctccggtggcgg ggsggggselvmtqtps
cggatctgagctggtgatgacccagaccccctcctccacctccggcgccgt stsgavggtvtincqasq
gggcggcaccgtgaccatcaactgccaggcctcccagtccatcgactcca sidsnlawfqqkpgqpp
acctggcctggttccagcagaagcccggccagccccccaccctgctgatc tlliyrasnlasgvpsrfsg
taccgggcctccaacctggcctccggcgtgccctcccggttctccggctcc srsgteytltisgvqreda
cggtccggcaccgagtacaccctgaccatctccggcgtgcagcgggagg atyyclggvgnvsyrtsfg
acgccgccacctactactgcctgggcggcgtgggcaacgtgtcctaccgg ggtevvvksssepkssd
acctccttcggcggcggcaccgaggtggtggtgaagtcctcgagtgagcc kthtcppcpapeaaga
caaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaag psvflfppkpkdtlmisrt
ccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccc pevtcvvvdvshedpe
tcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcc vkfnwyvdgvevhnak
acgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggt tkpreeqynstyrvvsvl
gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgta tvlhqdwlngkayacav
ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggca snkalpapiektiskakg
aggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgag qprepqvytlppsrdelt
aaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtac knqvsltclvkgfypsdia
accctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac vewesngqpennyktt
ctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggaga ppvldsdgsfflyskltvd
gcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgga ksrwqqgnvfscsvmh
ctccgacggctccttcttcctctacagcaagctcaccgtggacaagagcag ealhnhytqkslslspgk
gtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgca
caaccactacacgcagaagagcctctccctgtctccgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR063 gagctggtgatgacccagaccccctcctccacctccggcgccgtgggcgg elvmtqtpsstsgavggt SEQ ID NO:72 caccgtgaccatcaactgccaggcctcccagtccatcgactccaacctgg vtincqasqsidsnlawf (SEQ ID NO:73)
Chimeric
cctggttccagcagaagcccggccagccccccaccctgctgatctaccgg qqkpgqpptlliyrasnl
Rll VL-VH
gcctccaacctggcctccggcgtgccctcccggttctccggctcccggtcc asgvpsrfsgsrsgteytl
scFv-Fc
ggcaccgagtacaccctgaccatctccggcgtgcagcgggaggacgccg tisgvqredaatyyclgg
ccacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcct vgnvsyrtsfgggtevvv
tcggcggcggcaccgaggtggtggtgaagggtggaggcggttcaggcgg kggggsggggsggggsg
aggtggatccggcggtggcggctccggtggcggcggatctcagtccgtga gggsqsvkesegdlvtp
aggagtccgagggcgacctggtgacccccgccggcaacctgaccctgac agnltltctasgsdindyp
ctgcaccgcctccggctccgacatcaacgactaccccatctcctgggtgcg iswvrqapgkglewigfi
gcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcg nsggstwyaswvkgrft
gctccacctggtacgcctcctgggtgaagggccggttcaccatctcccgga isrtsttvdlkmtslttddt
cctccaccaccgtggacctgaagatgacctccctgaccaccgacgacacc atyfcargystyygdfni
gccacctacttctgcgcccggggctactccacctactacggcgacttcaac wgpgtlvtisssepkssd
atctggggccccggcaccctggtgaccatctcctcgagtgagcccaaatct kthtcppcpapeaaga
tctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgg psvflfppkpkdtlmisrt
gtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga pevtcvvvdvshedpe
tctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa vkfnwyvdgvevhnak
gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa tkpreeqynstyrvvsvl
tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt tvlhqdwlngkayacav
ggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggcgt snkalpapiektiskakg
acgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaaac qprepqvytlppsrdelt
catctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg knqvsltclvkgfypsdia
cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct vewesngqpennyktt
ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaat ppvldsdgsfflyskltvd
gggcagccggagaacaactacaagaccacgcctcccgtgctggactccg ksrwqqgnvfscsvmh
acggctccttcttcctctacagcaagctcaccgtggacaagagcaggtgg ealhnhytqkslslspgk
cagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaa
ccactacacgcagaagagcctctccctgtctccgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR016 caggagcagctggtggagtccggcggccggctggtgacccccggcggct qeqlvesggrlvtpggslt SEQ ID NO:74 ccctgaccctgtcctgcaaggcctccggcttcgacttctccgcctactacat Isckasgfdfsayymsw (SEQ ID NO:75)
Chimeric
gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc vrqapgkglewiatiyps
R12 VH-VL
atctacccctcctccggcaagacctactacgccacctgggtgaacggccg sgktyyatwvngrftiss
scFv-Fc
gttcaccatctcctccgacaacgcccagaacaccgtggacctgcagatga dnaqntvdlqmnsltaa
actccctgaccgccgccgaccgggccacctacttctgcgcccgggactcct dratyfcardsyaddgal
acgccgacgacggcgccctgttcaacatctggggccccggcaccctggtg fniwgpgtlvtissggggs
accatctcctccggtggaggcggttcaggcggaggtggatccggcggtgg ggggsggggsggggsel
cggctccggtggcggcggatctgagctggtgctgacccagtccccctccgt vltqspsvsaalgspakit
gtccgccgccctgggctcccccgccaagatcacctgcaccctgtcctccgc ctlssahktdtidwyqql
ccacaagaccgacaccatcgactggtaccagcagctgcagggcgaggcc qgeaprylmqvqsdgs
ccccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggcc ytkrpgvpdrfsgsssga
cggcgtgcccgaccggttctccggctcctcctccggcgccgaccggtacct dryliipsvqaddeadyy
gatcatcccctccgtgcaggccgacgacgaggccgactactactgcggcg cgadyiggyvfgggtqlt
ccgactacatcggcggctacgtgttcggcggcggcacccagctgaccgtg vtgsssepkssdkthtcp
accggctcctcgagtgagcccaaatcttctgacaaaactcacacatgccc pcpapeaagapsvflfp
accgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccc pkpkdtlmisrtpevtcv
cccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacat vvdvshedpevkfnwy
gcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactg vdgvevhnaktkpree
gtacgtggacggcgtggaggtgcataatgccaagacaaagccgcggga qynstyrvvsvltvlhqd
ggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgc wlngkayacavsnkalp
accaggactggctgaatggcaaggcgtacgcgtgcgcggtctccaacaa apiektiskakgqprep
agccctcccagcccccatcgagaaaaccatctccaaagccaaagggcag qvytlppsrdeltknqvs
ccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac Itclvkgfypsdiavewe
caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcg sngqpennykttppvld
acatcgccgtggagtgggagagcaatgggcagccggagaacaactaca sdgsfflyskltvdksrwq
agaccacgcctcccgtgctggactccgacggctccttcttcctctacagca qgnvfscsvmhealhn
agctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatg hytqkslslspgk
ctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctct
ccctgtctccgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR073 gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:76 cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:77)
Hemi- ctggtaccagcagaagcccggcaagccccccaagctgctgatctaccgg yqqkpgkppklliyrasn
humanized
gcctccaacctggcctccggcgtgccctcccggttctccggctccggctcc lasgvpsrfsgsgsgtdftl Rll VL-VH
ggcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggc tisslqpedvatyyclggv
scFv-Fc
cacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcctt gnvsyrtsfgggtkveik
(L2H0)
cggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgg ggggsggggsggggsgg
aggtggatccggcggtggcggctccggtggcggcggatctcagtccgtga ggsqsvkesegdlvtpa
aggagtccgagggcgacctggtgacccccgccggcaacctgaccctgac gnltltctasgsdindypi
ctgcaccgcctccggctccgacatcaacgactaccccatctcctgggtgcg swvrqapgkglewigfi
gcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcg nsggstwyaswvkgrft
gctccacctggtacgcctcctgggtgaagggccggttcaccatctcccgga isrtsttvdlkmtslttddt
cctccaccaccgtggacctgaagatgacctccctgaccaccgacgacacc atyfcargystyygdfni
gccacctacttctgcgcccggggctactccacctactacggcgacttcaac wgpgtlvtisssepkssd
atctggggccccggcaccctggtgaccatctcctcgagtgagcccaaatct kthtcppcpapeaaga
tctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgg psvflfppkpkdtlmisrt
gtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga pevtcvvvdvshedpe
tctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa vkfnwyvdgvevhnak
gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa tkpreeqynstyrvvsvl
tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt tvlhqdwlngkayacav
ggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggcgt snkalpapiektiskakg
acgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaaac qprepqvytlppsrdelt
catctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg knqvsltclvkgfypsdia
cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct vewesngqpennyktt
ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaat ppvldsdgsfflyskltvd
gggcagccggagaacaactacaagaccacgcctcccgtgctggactccg ksrwqqgnvfscsvmh
acggctccttcttcctctacagcaagctcaccgtggacaagagcaggtgg ealhnhytqkslslspgk
cagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaa
ccactacacgcagaagagcctctccctgtctccgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR091 gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:78
Hemi- cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:79) humanized ctggttccagcagaagcccggcaagccccccaagctgctgatctaccggg fqqkpgkppklliyrasnl
Rll VL-VH cctccaacctggcctccggcgtgccctcccggttctccggctccggctccg asgvpsrfsgsgsgtdftl
scFv-Fc gcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggcc tisslqpedvatyyclggv
(L6HO) acctactactgcctgggcggcgtgggcaacgtgtcctaccggacctccttc gnvsyrtsfgggtkveik
ggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgga ggggsggggsggggsgg
ggtggatccggcggtggcggctccggtggcggcggatctcagtccgtgaa ggsqsvkesegdlvtpa
ggagtccgagggcgacctggtgacccccgccggcaacctgaccctgacct gnltltctasgsdindypi
gcaccgcctccggctccgacatcaacgactaccccatctcctgggtgcgg swvrqapgkglewigfi
caggcccccggcaagggcctggagtggatcggcttcatcaactccggcgg nsggstwyaswvkgrft
ctccacctggtacgcctcctgggtgaagggccggttcaccatctcccggac isrtsttvdlkmtslttddt
ctccaccaccgtggacctgaagatgacctccctgaccaccgacgacaccg atyfcargystyygdfni
ccacctacttctgcgcccggggctactccacctactacggcgacttcaaca wgpgtlvtisssepkssd
tctggggccccggcaccctggtgaccatctcctcgagtgagcccaaatctt kthtcppcpapeaaga
ctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggg psvflfppkpkdtlmisrt
tgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgat pevtcvvvdvshedpe
ctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaag vkfnwyvdgvevhnak
accctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataat tkpreeqynstyrvvsvl
gccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtg tvlhqdwlngkayacav
gtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggcgta snkalpapiektiskakg
cgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaaacc qprepqvytlppsrdelt
atctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgc knqvsltclvkgfypsdia
ccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctg vewesngqpennyktt
gtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatg ppvldsdgsfflyskltvd
ggcagccggagaacaactacaagaccacgcctcccgtgctggactccga ksrwqqgnvfscsvmh
cggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggc ealhnhytqkslslspgk
agcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaac
cactacacgcagaagagcctctccctgtctccgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR081 gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:80 cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:81)
Humanized
ctggtaccagcagaagcccggcaagccccccaagctgctgatctaccgg yqqkpgkppklliyrasn
Rll VL-VH
gcctccaacctggcctccggcgtgccctcccggttctccggctccggctcc lasgvpsrfsgsgsgtdftl scFv-Fc
ggcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggc tisslqpedvatyyclggv
(L2H9)
cacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcctt gnvsyrtsfgggtkveik
cggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgg ggggsggggsggggsgg
aggtggatccggcggtggcggctccggtggcggcggatctgaggtgcag ggsevqlvesggglvqp
ctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggct grslrlsctasgsdindypi gtcctgcaccgcctccggctccgacatcaacgactaccccatctcctgggt swvrqapgkglewigfi
gcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccg nsggstwyaswvkgrft
gcggctccacctggtacgcctcctgggtgaagggccggttcaccatctccc isrddsksiaylqmnslkt
gggacgactccaagtccatcgcctacctgcagatgaactccctgaagacc edtavyfctrgystyygd
gaggacaccgccgtgtacttctgcacccggggctactccacctactacgg fniwgqgtlvtvsssepk
cgacttcaacatctggggccagggcaccctggtgaccgtgtcctcgagtg ssdkthtcppcpapeaa
agcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacct gapsvflfppkpkdtlmi
gaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga srtpevtcvvvdvshed
caccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgt pevkfnwyvdgvevhn
gagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg aktkpreeqynstyrvvs
gaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc vltvlhqdwlngkayac
acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaat avsnkalpapiektiska
ggcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccat kgqprepqvytlppsrd
cgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggt eltknqvsltclvkgfyps
gtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcc diavewesngqpenny
tgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgg kttppvldsdgsfflysklt
gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtg vdksrwqqgnvfscsv
ctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag mhealhnhytqkslsls
agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggc Pgk
tctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaat
ga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR101 gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:82 cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID NO:83)
Humanized
ctggttccagcagaagcccggcaagccccccaagctgctgatctaccggg fqqkpgkppklliyrasnl Rll VL-VH
cctccaacctggcctccggcgtgccctcccggttctccggctccggctccg asgvpsrfsgsgsgtdftl
scFv-Fc
gcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggcc tisslqpedvatyyclggv
(L6H9)
acctactactgcctgggcggcgtgggcaacgtgtcctaccggacctccttc gnvsyrtsfgggtkveik
ggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgga ggggsggggsggggsgg
ggtggatccggcggtggcggctccggtggcggcggatctgaggtgcagct ggsevqlvesggglvqp
ggtggagtccggcggcggcctggtgcagcccggccggtccctgcggctgt grslrlsctasgsdindypi cctgcaccgcctccggctccgacatcaacgactaccccatctcctgggtgc swvrqapgkglewigfi
ggcaggcccccggcaagggcctggagtggatcggcttcatcaactccggc nsggstwyaswvkgrft
ggctccacctggtacgcctcctgggtgaagggccggttcaccatctcccgg isrddsksiaylqmnslkt
gacgactccaagtccatcgcctacctgcagatgaactccctgaagaccga edtavyfctrgystyygd
ggacaccgccgtgtacttctgcacccggggctactccacctactacggcg fniwgqgtlvtvsssepk
acttcaacatctggggccagggcaccctggtgaccgtgtcctcgagtgag ssdkthtcppcpapeaa
cccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctga gapsvflfppkpkdtlmi
agccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggaca srtpevtcvvvdvshed
ccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtga pevkfnwyvdgvevhn
gccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga aktkpreeqynstyrvvs
ggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcac vltvlhqdwlngkayac
gtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgg avsnkalpapiektiska
caaggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcg kgqprepqvytlppsrd
agaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgt eltknqvsltclvkgfyps
acaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctg diavewesngqpenny
acctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtggga kttppvldsdgsfflysklt
gagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg vdksrwqqgnvfscsv
gactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc mhealhnhytqkslsls
aggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctct Pgk
gcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR033 gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggct evqlvesggglvqpggsl SEQ ID NO:84 ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:85)
Humanized
gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc wvrqapgkglewiatiy
R12 VH-VL
atctacccctcctccggcaagacctactacgccacctgggtgaagggccg pssgktyyatwvkgrfti
scFv-Fc
gttcaccatctcccgggacaactccaagaacaccctgtacctgcagatga srdnskntlylqmnslra
(H1L1)
actccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactcct edtavyfcardsyaddg
acgccgacgacggcgccctgttcaacatctggggccagggcaccctggtg alfniwgqgtlvtvssgg
accgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtgg ggsggggsggggsgggg
cggctccggtggcggcggatctcagctggtgctgacccagtccccctccgc sqlvltqspsasaslgasv
ctccgcctccctgggcgcctccgtgaagctgacctgcaccctgtcctccgc kltctlssahktdtidwyq
ccacaagaccgacaccatcgactggtaccagcagcagcccgagaaggg qqpekgprylmqvqsd
cccccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggc gsytkrpgvpdrfsgsss
ccggcgtgcccgaccgcttctccggctcctcctccggcgccgagcggtacc gaeryltisslqsedead
tgaccatctcctccctgcagtccgaggacgaggccgactactactgcggc yycgadyiggyvfgggtq
gccgactacatcggcggctacgtgttcggcggcggcacccagctgaccgt Itvlsssepkssdkthtcp
gctgtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccacc pcpapeaagapsvflfp
gtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccc pkpkdtlmisrtpevtcv
aaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcg vvdvshedpevkfnwy
tggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtac vdgvevhnaktkpree
gtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggag qynstyrvvsvltvlhqd
cagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacca wlngkayacavsnkalp
ggactggctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagcc apiektiskakgqprep
ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccc qvytlppsrdeltknqvs
gagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaa Itclvkgfypsdiavewe
gaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgaca sngqpennykttppvld
tcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga sdgsfflyskltvdksrwq
ccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagct qgnvfscsvmhealhn
caccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctcc hytqkslslspgk
gtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccct
gtctccgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR034 gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggct evqlvesggglvqpggsl SEQ ID NO:86 ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:87)
Humanized
gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc wvrqapgkglewiatiy
R12 VH-VL
atctacccctcctccggcaagacctactacgccacctgggtgaagggccg pssgktyyatwvkgrfti
scFv-Fc
gttcaccatctcccgggacaactccaagaacaccctgtacctgcagatga srdnskntlylqmnslra
(H1L2)
actccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactcct edtavyfcardsyaddg
acgccgacgacggcgccctgttcaacatctggggccagggcaccctggtg alfniwgqgtlvtvssgg
accgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtgg ggsggggsggggsgggg
cggctccggtggcggcggatctcagcccgtgctgacccagtcctcctccgc sqpvltqsssasaslgssv
ctccgcctccctgggctcctccgtgaagctgacctgcaccctgtcctccgcc kltctlssahktdtidwyq
cacaagaccgacaccatcgactggtaccagcagcagcccggcaaggccc qqpgkaprylmqvqsd
cccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggccc gsytkrpgvpdrfsgsss
ggcgtgcccgaccgcttctccggctcctcctccggcgccgaccgctacctg gadryltisnlqsedead
accatctccaacctgcagtccgaggacgaggccgactactactgcggcgc yycgadyiggyvfgggtq
cgactacatcggcggctacgtgttcggcggcggcacccagctgaccgtgc Itvlsssepkssdkthtcp
tgtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt pcpapeaagapsvflfp
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pkpkdtlmisrtpevtcv
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg vvdvshedpevkfnwy
gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vdgvevhnaktkpree
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca qynstyrvvsvltvlhqd
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg wlngkayacavsnkalp
actggctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagccctc apiektiskakgqprep
ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag qvytlppsrdeltknqvs
aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa Itclvkgfypsdiavewe
ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg sngqpennykttppvld
ccgtggagtgggagagcaatgggcagccggagaacaactacaagacca sdgsfflyskltvdksrwq
cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac qgnvfscsvmhealhn
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga hytqkslslspgk
tgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc
cgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR035 caggtgcagctggtggagtccggcggcggcctggtgaagcccggcggct qvqlvesggglvkpggsl SEQ ID NO:88 ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:89)
Humanized
gtcctggattcggcaggcccccggcaagggcctggagtggatcgccacca wirqapgkglewiatiyp
R12 VH-VL
tctacccctcctccggcaagacctactacgccacctgggtgaagggccgg ssgktyyatwvkgrftisr
scFv-Fc
ttcaccatctcccgggacaacgccaagaactccctgtacctgcagatgaa dnaknslylqmnslrae
(H2L1)
ctccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactccta dtavyfcardsyaddgal cgccgacgacggcgccctgttcaacatctggggccagggcaccctggtga fniwgqgtlvtvssgggg
ccgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtggc sggggsggggsggggsq
ggctccggtggcggcggatctcagctggtgctgacccagtccccctccgcc Ivltqspsasaslgasvklt
tccgcctccctgggcgcctccgtgaagctgacctgcaccctgtcctccgcc ctlssahktdtidwyqq
cacaagaccgacaccatcgactggtaccagcagcagcccgagaagggc qpekgprylmqvqsdg
ccccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggcc sytkrpgvpdrfsgsssg
cggcgtgcccgaccgcttctccggctcctcctccggcgccgagcggtacct aeryltisslqsedeady
gaccatctcctccctgcagtccgaggacgaggccgactactactgcggcg ycgadyiggyvfgggtql
ccgactacatcggcggctacgtgttcggcggcggcacccagctgaccgtg tvlsssepkssdkthtcp
ctgtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccg pcpapeaagapsvflfp
tgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttcccccca pkpkdtlmisrtpevtcv
aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgt vvdvshedpevkfnwy
ggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtac vdgvevhnaktkpree
gtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggag qynstyrvvsvltvlhqd
cagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacca wlngkayacavsnkalp
ggactggctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagcc apiektiskakgqprep
ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccc qvytlppsrdeltknqvs
gagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaa Itclvkgfypsdiavewe
gaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgaca sngqpennykttppvld
tcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga sdgsfflyskltvdksrwq
ccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagct qgnvfscsvmhealhn
caccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctcc hytqkslslspgk
gtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccct
gtctccgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR036 caggtgcagctggtggagtccggcggcggcctggtgaagcccggcggct qvqlvesggglvkpggsl SEQ ID NO:90 ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID NO:91)
Humanized
gtcctggattcggcaggcccccggcaagggcctggagtggatcgccacca wirqapgkglewiatiyp
R12 VH-VL
tctacccctcctccggcaagacctactacgccacctgggtgaagggccgg ssgktyyatwvkgrftisr
scFv-Fc
ttcaccatctcccgggacaacgccaagaactccctgtacctgcagatgaa dnaknslylqmnslrae
(H2L2)
ctccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactccta dtavyfcardsyaddgal cgccgacgacggcgccctgttcaacatctggggccagggcaccctggtga fniwgqgtlvtvssgggg
ccgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtggc sggggsggggsggggsq
ggctccggtggcggcggatctcagcccgtgctgacccagtcctcctccgcc pvltqsssasaslgssvkl
tccgcctccctgggctcctccgtgaagctgacctgcaccctgtcctccgccc tctlssahktdtidwyqq
acaagaccgacaccatcgactggtaccagcagcagcccggcaaggcccc qpgkaprylmqvqsdg
ccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggcccg sytkrpgvpdrfsgsssg
gcgtgcccgaccgcttctccggctcctcctccggcgccgaccgctacctga adryltisnlqsedeady
ccatctccaacctgcagtccgaggacgaggccgactactactgcggcgcc ycgadyiggyvfgggtql
gactacatcggcggctacgtgttcggcggcggcacccagctgaccgtgct tvlsssepkssdkthtcp
gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt pcpapeaagapsvflfp
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pkpkdtlmisrtpevtcv
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg vvdvshedpevkfnwy
gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vdgvevhnaktkpree
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca qynstyrvvsvltvlhqd
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg wlngkayacavsnkalp
actggctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagccctc apiektiskakgqprep
ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag qvytlppsrdeltknqvs
aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa Itclvkgfypsdiavewe
ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg sngqpennykttppvld
ccgtggagtgggagagcaatgggcagccggagaacaactacaagacca sdgsfflyskltvdksrwq
cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac qgnvfscsvmhealhn
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga hytqkslslspgk
tgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc
cgggtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR064 cagtccgtgaaggagtccgagggcgacctggtgacccccgccggcaacc qsvkesegdlvtpagnlt SEQ ID NO:92 tgaccctgacctgcaccgcctccggctccgacatcaacgactaccccatct Itctasgsdindypiswv (SEQ ID NO:93)
Rabbit
cctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatc rqapgkglewigfinsgg
Rll heavy aactccggcggctccacctggtacgcctcctgggtgaagggccggttcac stwyaswvkgrftisrtst
chain catctcccggacctccaccaccgtggacctgaagatgacctccctgacca tvdlkmtslttddtatyfc
ccgacgacaccgccacctacttctgcgcccggggctactccacctactacg argystyygdfniwgpg
gcgacttcaacatctggggccccggcaccctggtgaccatctcgagtggg tlvtissgqpkapsvfpla
caacctaaggctccatcagtcttcccactggccccctgctgcggggacaca pccgdtpsstvtlgclvkg
cccagctccacggtgaccctgggctgcctggtcaaaggctacctcccgga ylpepvtvtwnsgtltng
gccagtgaccgtgacctggaactcgggcaccctcaccaatggggtacgca vrtfpsvrqssglyslssv
ccttcccgtccgtccggcagtcctcaggcctctactcgctgagcagcgtgg vsvtsssqpvtcnvahp
tgagcgtgacctcaagcagccagcccgtcacctgcaacgtggcccaccca atntkvdktvapstcskp
gccaccaacaccaaagtggacaagaccgttgcgccctcgacatgcagca tcpppellggpsvfifpp
agcccacgtgcccaccccctgaactcctggggggaccgtctgtcttcatct kpkdtlmisrtpevtcvv
tccccccaaaacccaaggacaccctcatgatctcacgcacccccgaggtc vdvsqddpevqftwyi
acatgcgtggtggtggacgtgagccaggatgaccccgaggtgcagttcac nneqvrtarpplreqqf
atggtacataaacaacgagcaggtgcgcaccgcccggccgccgctacgg nstirvvstlpiahqdwlr
gagcagcagttcaacagcacgatccgcgtggtcagcaccctccccatcgc gkefkckvhnkalpapi
gcaccaggactggctgaggggcaaggagttcaagtgcaaagtccacaac ektiskargqplepkvyt
aaggcactcccggcccccatcgagaaaaccatctccaaagccagagggc mgppreelssrsvsltc
agcccctggagccgaaggtctacaccatgggccctccccgggaggagct mingfypsdisvewek
gagcagcaggtcggtcagcctgacctgcatgatcaacggcttctacccttc ngkaednykttpavlds
cgacatctcggtggagtgggagaagaacgggaaggcagaggacaacta dgsyflysklsvptsewq
caagaccacgccggccgtgctggacagcgacggctcctacttcctctaca rgdvftcsvmhealhnh
gcaagctctcagtgcccacgagtgagtggcagcggggcgacgtcttcacc ytqksisrspgk
tgctccgtgatgcacgaggccttgcacaaccactacacgcagaagtccat
ctcccgctctccgggtaaatga
ROR065 gagctggtgatgacccagaccccctcctccacctccggcgccgtgggcgg elvmtqtpsstsgavggt SEQ ID NO:94 caccgtgaccatcaactgccaggcctcccagtccatcgactccaacctgg vtincqasqsidsnlawf (SEQ ID NO:95)
Rabbit
cctggttccagcagaagcccggccagccccccaccctgctgatctaccgg qqkpgqpptlliyrasnl
Rll light gcctccaacctggcctccggcgtgccctcccggttctccggctcccggtcc asgvpsrfsgsrsgteytl
chain ggcaccgagtacaccctgaccatctccggcgtgcagcgggaggacgccg tisgvqredaatyyclgg
ccacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcct vgnvsyrtsfgggtevvv
tcggcggcggcaccgaggtggtggtgaagcgggatccagttgcgccttct krdpvapsvllfppskee
gtcctcctcttcccaccatctaaggaggagctgacaactggaacagccac Ittgtativcvankfypsd
catcgtgtgcgtggcgaataaattctatcccagtgacatcaccgtcacctg itvtwkvdgttqqsgien
gaaggtggatggcaccacccaacagagcggcatcgagaacagtaaaac sktpqspedntyslsstls
accgcagagccccgaagacaatacctacagcctgagcagcactctgtca Itsaqynshsvytcevvq
ctgaccagcgcacagtacaacagccacagcgtgtacacctgcgaggtgg gsaspivqsfnrgdc
tccaaggctcagcctcaccgatcgtccagagcttcaataggggtgactgct
ag Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR067 caggagcagctggtggagtccggcggccggctggtgacccccggcggct qeqlvesggrlvtpggslt SEQ ID NO:96 ccctgaccctgtcctgcaaggcctccggcttcgacttctccgcctactacat Isckasgfdfsayymsw (SEQ ID NO:97)
Rabbit
gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc vrqapgkglewiatiyps
R12 heavy atctacccctcctccggcaagacctactacgccacctgggtgaacggccg sgktyyatwvngrftiss
chain gttcaccatctcctccgacaacgcccagaacaccgtggacctgcagatga dnaqntvdlqmnsltaa
actccctgaccgccgccgaccgggccacctacttctgcgcccgggactcct dratyfcardsyaddgal
acgccgacgacggcgccctgttcaacatctggggccccggcaccctggtg fniwgpgtlvtissgqpk
accatctcgagtgggcaacctaaggctccatcagtcttcccactggccccc apsvfplapccgdtpsst
tgctgcggggacacacccagctccacggtgaccctgggctgcctggtcaa vtlgclvkgylpepvtvt
aggctacctcccggagccagtgaccgtgacctggaactcgggcaccctca wnsgtltngvrtfpsvrq
ccaatggggtacgcaccttcccgtccgtccggcagtcctcaggcctctact ssglyslssvvsvtsssqp
cgctgagcagcgtggtgagcgtgacctcaagcagccagcccgtcacctgc vtcnvahpatntkvdkt
aacgtggcccacccagccaccaacaccaaagtggacaagaccgttgcgc vapstcskptcpppellg
cctcgacatgcagcaagcccacgtgcccaccccctgaactcctgggggga gpsvfifppkpkdtlmis
ccgtctgtcttcatcttccccccaaaacccaaggacaccctcatgatctcac rtpevtcvvvdvsqddp
gcacccccgaggtcacatgcgtggtggtggacgtgagccaggatgaccc evqftwyinneqvrtar
cgaggtgcagttcacatggtacataaacaacgagcaggtgcgcaccgcc pplreqqfnstirvvstlp
cggccgccgctacgggagcagcagttcaacagcacgatccgcgtggtca iahqdwlrgkefkckvh
gcaccctccccatcgcgcaccaggactggctgaggggcaaggagttcaa nkalpapiektiskargq
gtgcaaagtccacaacaaggcactcccggcccccatcgagaaaaccatc plepkvytmgppreels
tccaaagccagagggcagcccctggagccgaaggtctacaccatgggcc srsvsltcmingfypsdis
ctccccgggaggagctgagcagcaggtcggtcagcctgacctgcatgatc vewekngkaednyktt
aacggcttctacccttccgacatctcggtggagtgggagaagaacgggaa pavldsdgsyflysklsvp
ggcagaggacaactacaagaccacgccggccgtgctggacagcgacgg tsewqrgdvftcsvmh
ctcctacttcctctacagcaagctctcagtgcccacgagtgagtggcagcg ealhnhytqksisrspgk
gggcgacgtcttcacctgctccgtgatgcacgaggccttgcacaaccact
acacgcagaagtccatctcccgctctccgggtaaatga
ROR068 gagctggtgctgacccagtccccctccgtgtccgccgccctgggctccccc elvltqspsvsaalgspa SEQ ID NO:98 gccaagatcacctgcaccctgtcctccgcccacaagaccgacaccatcga kitctlssahktdtidwyq (SEQ ID NO:99)
Rabbit
ctggtaccagcagctgcagggcgaggccccccggtacctgatgcaggtgc qlqgeaprylmqvqsd
R12 light agtccgacggctcctacaccaagcggcccggcgtgcccgaccggttctcc gsytkrpgvpdrfsgsss
chain ggctcctcctccggcgccgaccggtacctgatcatcccctccgtgcaggcc gadryliipsvqaddead
gacgacgaggccgactactactgcggcgccgactacatcggcggctacgt yycgadyiggyvfgggtq
gttcggcggcggcacccagctgaccgtgaccggccgggatcccgcggtg Itvtgrdpavtpsvilfpp
accccctcggtcattctgttcccgccctcctctgaggaactcaaggacaac sseelkdnkatlvclindf
aaggctaccctggtgtgtctgatcaatgacttctaccccagaaccgtgaag yprtvkvnwkadgnsvt
gtaaactggaaggcagatggcaactctgtcacccagggcgtggacacca qgvdttqpskqsnnkya
cccagccgtccaaacagagcaacaacaagtacgcggccagcagcttcct assflslsanqwksyqsv
cagcctgtcagccaatcaatggaaatcctaccagagtgtcacctgccagg tcqvtheghtvekslap
tcacgcacgagggccacactgtggagaagagcctggcccctgcagaatg aecs
ctcctag Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR020 cagtccgtgaaggagtccgagggcgacctggtgacccccgccggcaacc qsvkesegdlvtpagnlt SEQ ID N0:100 tgaccctgacctgcaccgcctccggctccgacatcaacgactaccccatct Itctasgsdindypiswv (SEQ ID
Chimeric
cctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatc rqapgkglewigfinsgg N0:101) scFv-Fc- aactccggcggctccacctggtacgcctcctgggtgaagggccggttcac stwyaswvkgrftisrtst scFv
catctcccggacctccaccaccgtggacctgaagatgacctccctgacca tvdlkmtslttddtatyfc
Rll HLx ccgacgacaccgccacctacttctgcgcccggggctactccacctactacg argystyygdfniwgpg
CD3 gcgacttcaacatctggggccccggcaccctggtgaccatctcctccggtg tlvtissggggsggggsgg
gaggcggttcaggcggaggtggatccggcggtggcggctccggtggcgg ggsggggselvmtqtps
cggatctgagctggtgatgacccagaccccctcctccacctccggcgccgt stsgavggtvtincqasq
gggcggcaccgtgaccatcaactgccaggcctcccagtccatcgactcca sidsnlawfqqkpgqpp
acctggcctggttccagcagaagcccggccagccccccaccctgctgatc tlliyrasnlasgvpsrfsg
taccgggcctccaacctggcctccggcgtgccctcccggttctccggctcc srsgteytltisgvqreda
cggtccggcaccgagtacaccctgaccatctccggcgtgcagcgggagg atyyclggvgnvsyrtsfg acgccgccacctactactgcctgggcggcgtgggcaacgtgtcctaccgg ggtevvvksssepkssd
acctccttcggcggcggcaccgaggtggtggtgaagtcctcgagtgagcc kthtcppcpapeaaga
caaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaag psvflfppkpkdtlmisrt ccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccc pevtcvvvdvshedpe
tcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcc vkfnwyvdgvevhnak
acgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggt tkpreeqynstyrvvsvl gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgta tvlhqdwlngkayacav
ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggca snkalpapiektiskakg
aggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccatcgag qprepqvytlppsrdelt aaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtac knqvsltclvkgfypsdia accctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac vewesngqpennyktt
ctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggaga ppvldsdgsfflyskltvd
gcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgga ksrwqqgnvfscsvmh
ctccgacggctccttcttcctctacagcaagctcaccgtggacaagagcag ealhnhytqkslslspgq
gtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgca rhnnsslntgtqmaghs caaccactacacgcagaagagcctctccctgtctccgggtcagaggcaca pnsqvqlvesgggvvqp acaattcttccctgaatacaggaactcagatggcaggtcattctccgaatt grslrlsckasgytftrst
ctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcgg mhwvrqapgqglewi
tcactgaggctgtcctgcaaggcttctggctacacctttactagatctacga gyinpssaytnynqkfk
tgcactgggtaaggcaggcccctggacaaggtctggaatggattggatac drftisadkskstaflqm
attaatcctagcagtgcttatactaattacaatcagaaattcaaggacagg dslrpedtgvyfcarpqv
ttcacaatcagcgcagacaaatccaagagcacagccttcctgcagatgg hydyngfpywgqgtpv
acagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaa tvssggggsggggsggg
gtccactatgattacaacgggtttccttactggggccaagggactcccgtc gsaqdiqmtqspsslsa
actgtctctagcggtggcggagggtctgggggtggcggatccggaggtgg svgd rvtmtcsasssvsy tggctctgcacaagacatccagatgacccagtctccaagcagcctgtctg mnwyqqkpgkapkr
caagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgt wiydssklasgvparfsg
aagttacatgaactggtaccagcagaagccgggcaaggcccccaaaag sgsgtdytltisslqpedf
atggatttatgactcatccaaactggcttctggagtccctgctcgcttcagt atyycqqwsrnpptfgg
ggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcc gtklqitsss
cgaagatttcgccacttattactgccagcagtggagtcgtaacccacccac
gttcggaggggggaccaagctacaaattacatcctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR070 gagctggtgatgacccagaccccctcctccacctccggcgccgtgggcgg elvmtqtpsstsgavggt SEQ ID N0:102 caccgtgaccatcaactgccaggcctcccagtccatcgactccaacctgg vtincqasqsidsnlawf (SEQ ID
Chimeric
cctggttccagcagaagcccggccagccccccaccctgctgatctaccgg qqkpgqpptlliyrasnl N0:103) scFv-Fc- gcctccaacctggcctccggcgtgccctcccggttctccggctcccggtcc asgvpsrfsgsrsgteytl scFv
ggcaccgagtacaccctgaccatctccggcgtgcagcgggaggacgccg tisgvqredaatyyclgg
Rll ccacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcct vgnvsyrtsfgggtevvv
LHxCD3 tcggcggcggcaccgaggtggtggtgaagggtggaggcggttcaggcgg kggggsggggsggggsg
aggtggatccggcggtggcggctccggtggcggcggatctcagtccgtga gggsqsvkesegdlvtp
aggagtccgagggcgacctggtgacccccgccggcaacctgaccctgac agnltltctasgsdindyp ctgcaccgcctccggctccgacatcaacgactaccccatctcctgggtgcg iswvrqapgkglewigfi gcaggcccccggcaagggcctggagtggatcggcttcatcaactccggcg nsggstwyaswvkgrft
gctccacctggtacgcctcctgggtgaagggccggttcaccatctcccgga isrtsttvdlkmtslttddt cctccaccaccgtggacctgaagatgacctccctgaccaccgacgacacc atyfcargystyygdfni
gccacctacttctgcgcccggggctactccacctactacggcgacttcaac wgpgtlvtisssepkssd
atctggggccccggcaccctggtgaccatctcctcgagtgagcccaaatct kthtcppcpapeaaga
tctgacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgg psvflfppkpkdtlmisrt gtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga pevtcvvvdvshedpe
tctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa vkfnwyvdgvevhnak
gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa tkpreeqynstyrvvsvl tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt tvlhqdwlngkayacav
ggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggcgt snkalpapiektiskakg
acgcgtgcgcggtctccaacaaagccctcccagcccccatcgagaaaac qprepqvytlppsrdelt catctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg knqvsltclvkgfypsdia cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct vewesngqpennyktt
ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaat ppvldsdgsfflyskltvd
gggcagccggagaacaactacaagaccacgcctcccgtgctggactccg ksrwqqgnvfscsvmh
acggctccttcttcctctacagcaagctcaccgtggacaagagcaggtgg ealhnhytqkslslspgq
cagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaa rhnnsslntgtqmaghs ccactacacgcagaagagcctctccctgtctccgggtcagaggcacaaca pnsqvqlvesgggvvqp attcttccctgaatacaggaactcagatggcaggtcattctccgaattctc grslrlsckasgytftrst
aggtccagctggtggagtctgggggcggagtggtgcagcctgggcggtc mhwvrqapgqglewi
actgaggctgtcctgcaaggcttctggctacacctttactagatctacgatg gyinpssaytnynqkfk
cactgggtaaggcaggcccctggacaaggtctggaatggattggatacat drftisadkskstaflqm
taatcctagcagtgcttatactaattacaatcagaaattcaaggacaggtt dslrpedtgvyfcarpqv
cacaatcagcgcagacaaatccaagagcacagccttcctgcagatggac hydyngfpywgqgtpv
agcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaagt tvssggggsggggsggg
ccactatgattacaacgggtttccttactggggccaagggactcccgtcac gsaqdiqmtqspsslsa
tgtctctagcggtggcggagggtctgggggtggcggatccggaggtggtg svgdrvtmtcsasssvsy gctctgcacaagacatccagatgacccagtctccaagcagcctgtctgca mnwyqqkpgkapkr
agcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgtaa wiydssklasgvparfsg
gttacatgaactggtaccagcagaagccgggcaaggcccccaaaagatg sgsgtdytltisslqpedf
gatttatgactcatccaaactggcttctggagtccctgctcgcttcagtggc atyycqqwsrnpptfgg
agtgggtctgggaccgactataccctcacaatcagcagcctgcagcccga gtklqitsss
agatttcgccacttattactgccagcagtggagtcgtaacccacccacgtt
cggaggggggaccaagctacaaattacatcctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR084 gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID N0:104 cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID
Humanized
ctggtaccagcagaagcccggcaagccccccaagctgctgatctaccgg yqqkpgkppklliyrasn N0:105) scFv-Fc- gcctccaacctggcctccggcgtgccctcccggttctccggctccggctcc lasgvpsrfsgsgsgtdftl scFv
ggcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggc tisslqpedvatyyclggv
Rll LH cacctactactgcctgggcggcgtgggcaacgtgtcctaccggacctcctt gnvsyrtsfgggtkveik
(L2H9) cggcggcggcaccaaggtggagatcaagggtggaggcggttcaggcgg ggggsggggsggggsgg
xCD3 aggtggatccggcggtggcggctccggtggcggcggatctgaggtgcag ggsevqlvesggglvqp
ctggtggagtccggcggcggcctggtgcagcccggccggtccctgcggct grslrlsctasgsdindypi gtcctgcaccgcctccggctccgacatcaacgactaccccatctcctgggt swvrqapgkglewigfi
gcggcaggcccccggcaagggcctggagtggatcggcttcatcaactccg nsggstwyaswvkgrft
gcggctccacctggtacgcctcctgggtgaagggccggttcaccatctccc isrddsksiaylqmnslkt gggacgactccaagtccatcgcctacctgcagatgaactccctgaagacc edtavyfctrgystyygd
gaggacaccgccgtgtacttctgcacccggggctactccacctactacgg fniwgqgtlvtvsssepk
cgacttcaacatctggggccagggcaccctggtgaccgtgtcctcgagtg ssdkthtcppcpapeaa agcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacct gapsvflfppkpkdtlmi gaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacccaagga srtpevtcvvvdvshed
caccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgt pevkfnwyvdgvevhn
gagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg aktkpreeqynstyrvvs gaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc vltvlhqdwlngkayac
acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaat avsnkalpapiektiska
ggcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagcccccat kgqprepqvytlppsrd
cgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggt eltknqvsltclvkgfyps
gtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcc diavewesngqpenny
tgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgg kttppvldsdgsfflysklt gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtg vdksrwqqgnvfscsv
ctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag mhealhnhytqkslsls
agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggc Pgsggggsggggsgggg
tctgcacaaccactacacgcagaagagcctctccctgtctccgggttctgg spnsqvqlvesgggvvq
tggaggcggttcaggcggaggtggctccggcggtggcggatcgccgaatt pgrslrlsckasgytftrst
ctcaggtccagctggtggagtctgggggcggagtggtgcagcctgggcgg mhwvrqapgqglewi
tcactgaggctgtcctgcaaggcttctggctacacctttactagatctacga gyinpssaytnynqkfk
tgcactgggtaaggcaggcccctggacaaggtctggaatggattggatac drftisadkskstaflqm
attaatcctagcagtgcttatactaattacaatcagaaattcaaggacagg dslrpedtgvyfcarpqv
ttcacaatcagcgcagacaaatccaagagcacagccttcctgcagatgg hydyngfpywgqgtpv
acagcctgaggcccgaggacaccggcgtctatttctgtgcacggccccaa tvssggggsggggsggg
gtccactatgattacaacgggtttccttactggggccaagggactcccgtc gsaqdiqmtqspsslsa
actgtctctagcggtggcggagggtctgggggtggcggatccggaggtgg svgdrvtmtcsasssvsy tggctctgcacaagacatccagatgacccagtctccaagcagcctgtctg mnwyqqkpgkapkr
caagcgtgggggacagggtcaccatgacctgcagtgccagctcaagtgt wiydssklasgvparfsg
aagttacatgaactggtaccagcagaagccgggcaaggcccccaaaag sgsgtdytltisslqpedf
atggatttatgactcatccaaactggcttctggagtccctgctcgcttcagt atyycqqwsrnpptfgg
ggcagtgggtctgggaccgactataccctcacaatcagcagcctgcagcc gtklqitsss
cgaagatttcgccacttattactgccagcagtggagtcgtaacccacccac
gttcggaggggggaccaagctacaaattacatcctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR021 caggagcagctggtggagtccggcggccggctggtgacccccggcggct qeqlvesggrlvtpggslt SEQ ID N0:106 ccctgaccctgtcctgcaaggcctccggcttcgacttctccgcctactacat Isckasgfdfsayymsw (SEQ ID
Chimeric
gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc vrqapgkglewiatiyps N0:107) scFv-Fc- atctacccctcctccggcaagacctactacgccacctgggtgaacggccg sgktyyatwvngrftiss
scFv
gttcaccatctcctccgacaacgcccagaacaccgtggacctgcagatga dnaqntvdlqmnsltaa
R12HLxCD3 actccctgaccgccgccgaccgggccacctacttctgcgcccgggactcct dratyfcardsyaddgal
acgccgacgacggcgccctgttcaacatctggggccccggcaccctggtg fniwgpgtlvtissggggs accatctcctccggtggaggcggttcaggcggaggtggatccggcggtgg ggggsggggsggggsel
cggctccggtggcggcggatctgagctggtgctgacccagtccccctccgt vltqspsvsaalgspakit gtccgccgccctgggctcccccgccaagatcacctgcaccctgtcctccgc ctlssahktdtidwyqql ccacaagaccgacaccatcgactggtaccagcagctgcagggcgaggcc qgeaprylmqvqsdgs
ccccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggcc ytkrpgvpdrfsgsssga
cggcgtgcccgaccggttctccggctcctcctccggcgccgaccggtacct dryliipsvqaddeadyy
gatcatcccctccgtgcaggccgacgacgaggccgactactactgcggcg cgadyiggyvfgggtqlt
ccgactacatcggcggctacgtgttcggcggcggcacccagctgaccgtg vtgsssepkssdkthtcp
accggctcctcgagtgagcccaaatcttctgacaaaactcacacatgccc pcpapeaagapsvflfp
accgtgcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccc pkpkdtlmisrtpevtcv
cccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacat vvdvshedpevkfnwy
gcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactg vdgvevhnaktkpree
gtacgtggacggcgtggaggtgcataatgccaagacaaagccgcggga qynstyrvvsvltvlhqd
ggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgc wlngkayacavsnkalp
accaggactggctgaatggcaaggcgtacgcgtgcgcggtctccaacaa apiektiskakgqprep
agccctcccagcccccatcgagaaaaccatctccaaagccaaagggcag qvytlppsrdeltknqvs
ccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac Itclvkgfypsdiavewe
caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcg sngqpennykttppvld
acatcgccgtggagtgggagagcaatgggcagccggagaacaactaca sdgsfflyskltvdksrwq agaccacgcctcccgtgctggactccgacggctccttcttcctctacagca qgnvfscsvmhealhn
agctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatg hytqkslslspgqrhnns ctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctct slntgtqmaghspnsqv ccctgtctccgggtcagaggcacaacaattcttccctgaatacaggaactc qlvesgggvvqpgrslrls agatggcaggtcattctccgaattctcaggtccagctggtggagtctgggg ckasgytftrstmhwvr
gcggagtggtgcagcctgggcggtcactgaggctgtcctgcaaggcttct qapgqglewigyinpss
ggctacacctttactagatctacgatgcactgggtaaggcaggcccctgg aytnynqkfkdrftisad
acaaggtctggaatggattggatacattaatcctagcagtgcttatactaa kskstaflqmdslrpedt ttacaatcagaaattcaaggacaggttcacaatcagcgcagacaaatcc gvyf ca rpqvhydyngf
aagagcacagccttcctgcagatggacagcctgaggcccgaggacaccg pywgqgtpvtvssggg
gcgtctatttctgtgcacggccccaagtccactatgattacaacgggtttcc gsggggsggggsaqdiq
ttactggggccaagggactcccgtcactgtctctagcggtggcggagggt mtqspsslsasvgdrvt
ctgggggtggcggatccggaggtggtggctctgcacaagacatccagatg mtcsasssvsymnwyq acccagtctccaagcagcctgtctgcaagcgtgggggacagggtcaccat qkpgkapkrwiydsskl gacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcaga asgvparfsgsgsgtdytl agccgggcaaggcccccaaaagatggatttatgactcatccaaactggct tisslqpedfatyycqqw
tctggagtccctgctcgcttcagtggcagtgggtctgggaccgactatacc srnpptfgggtklqitsss
ctcacaatcagcagcctgcagcccgaagatttcgccacttattactgccag
cagtggagtcgtaacccacccacgttcggaggggggaccaagctacaaa
ttacatcctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR050 gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggct evqlvesggglvqpggsl SEQ ID N0:108 ccctgcggctgtcctgcaaggcctccggcttcgacttctccgcctactacat rlsckasgfdfsayyms (SEQ ID
Humanized
gtcctgggtgcggcaggcccccggcaagggcctggagtggatcgccacc wvrqapgkglewiatiy N0:109) scFv-Fc- atctacccctcctccggcaagacctactacgccacctgggtgaagggccg pssgktyyatwvkgrfti scFv
gttcaccatctcccgggacaactccaagaacaccctgtacctgcagatga srdnskntlylqmnslra
R12 HL actccctgcgggccgaggacaccgccgtgtacttctgcgcccgggactcct edtavyfcardsyaddg
(H1L2) acgccgacgacggcgccctgttcaacatctggggccagggcaccctggtg alfniwgqgtlvtvssgg
xCD3 accgtgtcctccggtggaggcggttcaggcggaggtggatccggcggtgg ggsggggsggggsgggg
cggctccggtggcggcggatctcagcccgtgctgacccagtcctcctccgc sqpvltqsssasaslgssv ctccgcctccctgggctcctccgtgaagctgacctgcaccctgtcctccgcc kltctlssahktdtidwyq cacaagaccgacaccatcgactggtaccagcagcagcccggcaaggccc qqpgkaprylmqvqsd
cccggtacctgatgcaggtgcagtccgacggctcctacaccaagcggccc gsytkrpgvpdrfsgsss
ggcgtgcccgaccgcttctccggctcctcctccggcgccgaccgctacctg gadryltisnlqsedead
accatctccaacctgcagtccgaggacgaggccgactactactgcggcgc yycgadyiggyvfgggtq cgactacatcggcggctacgtgttcggcggcggcacccagctgaccgtgc Itvlsssepkssdkthtcp
tgtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt pcpapeaagapsvflfp
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pkpkdtlmisrtpevtcv
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg vvdvshedpevkfnwy
gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vdgvevhnaktkpree
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca qynstyrvvsvltvlhqd
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg wlngkayacavsnkalp
actggctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagccctc apiektiskakgqprep
ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag qvytlppsrdeltknqvs
aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa Itclvkgfypsdiavewe
ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg sngqpennykttppvld
ccgtggagtgggagagcaatgggcagccggagaacaactacaagacca sdgsfflyskltvdksrwq cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac qgnvfscsvmhealhn
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga hytqkslslspgqrhnns tgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc slntgtqmaghspnsqv cgggtcagaggcacaacaattcttccctgaatacaggaactcagatggca qlvesgggvvqpgrslrls ggtcattctccgaattctcaggtccagctggtggagtctgggggcggagtg ckasgytftrstmhwvr
gtgcagcctgggcggtcactgaggctgtcctgcaaggcttctggctacacc qapgqglewigyinpss
tttactagatctacgatgcactgggtaaggcaggcccctggacaaggtct aytnynqkfkdrftisad
ggaatggattggatacattaatcctagcagtgcttatactaattacaatca kskstaflqmdslrpedt gaaattcaaggacaggttcacaatcagcgcagacaaatccaagagcaca gvyf ca rpqvhydyngf
gccttcctgcagatggacagcctgaggcccgaggacaccggcgtctattt pywgqgtpvtvssggg
ctgtgcacggccccaagtccactatgattacaacgggtttccttactgggg gsggggsggggsaqdiq
ccaagggactcccgtcactgtctctagcggtggcggagggtctgggggtg mtqspsslsasvgdrvt
gcggatccggaggtggtggctctgcacaagacatccagatgacccagtct mtcsasssvsymnwyq ccaagcagcctgtctgcaagcgtgggggacagggtcaccatgacctgca qkpgkapkrwiydsskl gtgccagctcaagtgtaagttacatgaactggtaccagcagaagccgggc asgvparfsgsgsgtdytl aaggcccccaaaagatggatttatgactcatccaaactggcttctggagt tisslqpedfatyycqqw
ccctgctcgcttcagtggcagtgggtctgggaccgactataccctcacaat srnpptfgggtklqitsss
cagcagcctgcagcccgaagatttcgccacttattactgccagcagtgga
gtcgtaacccacccacgttcggaggggggaccaagctacaaattacatcc
tccagctaa
Cris7 and RSTMH (SEQ ID DRA222 VH NO:110) CDR1
(Kabat) Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Cris7 and YINPSSAYTNYNQKF (SEQ ID DRA222 VH K NO:lll) CDR2
(Kabat)
Cris7 and QVHYDYNGFPY (SEQ ID DRA222 VH NO:112) CDR3
(Kabat)
Cris7 and SASSSVSYMN (SEQ ID DRA222 VL NO:113) CDR1
(Kabat)
Cris7 and DSSKLAS (SEQ ID DRA222 VL NO:114) CDR2
(Kabat)
Cris7 and QQWSRNPPT (SEQ ID DRA222 VL NO:115) CDR3
(Kabat)
Cris7 and GYTFTRST (SEQ ID DRA222 VH NO:269) CDR1
(IMGT)
Cris7 and INPSSAYT (SEQ ID DRA222 VH NO:270) CDR2
(IMGT)
Cris7 and QQWSRNPPT (SEQ ID DRA222 VH NO:271) CDR3
(IMGT)
Cris7 and ASSSVSY (SEQ ID DRA222 VL NO:272) CDR1
(IMGT)
Cris7 and DSS (SEQ ID DRA222 VL NO:273) CDR2
(IMGT)
Cris7 and QQWSRNPPT (SEQ ID DRA222 VL NO:274) CDR3
(IMGT)
I2C VH KYAMN (SEQ ID
CDR1 NO:275)
(Kabat) Name Nucleotide Sequence Amino Acid SEQID NOs:
Sequence nucleotide
(amino acid)
I2CVH RIRSKYNNYATYYAD (SEQID
CDR2 SVKD NO:276)
(Kabat)
I2CVH HGNFGNSYISYWAY (SEQID
CDR3 NO:277)
(Kabat)
I2CVL GSSTGAVTSGNYPN (SEQID
CDRl NO:278)
(Kabat)
I2CVL GTKFLAP (SEQID
CDR2 NO:279)
(Kabat)
I2CVL VLWYSNRWV (SEQID
CDR3 NO:280)
(Kabat)
I2CVH GFTFNKYA (SEQID
CDRl NO:281)
(IMGT)
I2CVH IRSKYNNYAT (SEQID
CDR2 NO:282)
(IMGT)
I2CVH VRHGNFGNSYISYW (SEQID
CDR3 AY NO:283)
(IMGT)
I2CVL TGAVTSGNY (SEQID
CDRl NO:284)
(IMGT)
I2CVL GTK (SEQID
CDR2 NO:285)
(IMGT)
I2CVL VLWYSNRWV (SEQID
CDR3 NO:286)
(IMGT)
HuM291 SYTMH (SEQID VH CDRl NO:287) (Kabat)
HuM291 YINPRSGYTHYNQKL (SEQID VH CDR2 KD NO:288) (Kabat)
HuM291 SAYYDYDGFAY (SEQID VH CDR3 NO:289) (Kabat)
HuM291 VL SASSSVSYMN (SEQID
CDRl NO:290)
(Kabat) Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
HuM291 VL DTSKLAS (SEQ ID
CDR2 NO:291)
(Kabat)
HuM291 VL QQWSSNPPT (SEQ ID
CDR3 NO:292)
(Kabat)
HuM291 GYTFISYT (SEQ ID VH CDR1 NO:293) (IMGT)
HuM291 INPRSGYT (SEQ ID VH CDR2 NO:294) (IMGT)
HuM291 ARSAYYDYDGFAY (SEQ ID VH CDR3 NO:295) (IMGT)
HuM291 VL ASSSVSY (SEQ ID
CDR1 NO:296)
(IMGT)
HuM291 VL DTS (SEQ ID
CDR2 NO:297)
(IMGT)
HuM291 VL QQWSSNPPT (SEQ ID
CDR3 NO:298)
(IMGT)
gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggct evqlvesggglvqpggsl SEQ ID NO:209 ccctgcggctgtcctgcaccgcctccggctccgacatcaacgactacccca rlsctasgsdindypisw (SEQ ID tctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttc vrqapgkglewigfinsg NO:210) atcaactccggcggctccacctggtacgccgactcggtgaagggccggtt gstwyadsvkgrftisrh
caccatctcccggcactcctccaagaacaccctgtacctgcagatgaactc sskntlylqmnslraedt
cctgcgggccgaggacaccgccgtgtacttctgcgcccggggctactcca avyfcargystyygdfni cctactacggcgacttcaacatctggggccagggcaccctggtgaccgtg wgqgtlvtvss
tcctcg
Humanized gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggt evqlvesggglvqpgrsl SEQ ID NO:211 Rll VH ccctgcggctgtcctgcaccgcctccggctccgacatcaacgactacccca rlsctasgsdindypisw (SEQ ID (H15) tctcctgggtgcggcaggcccccggcaagggcctggagtggatcggcttc vrqapgkglewigfinsg NO:212) atcaactccggcggctccacctggtacgcctcctgggtgaagggccggttc gstwyaswvkgrftisrd
(H9 F224Y
accatctcccgggacgactccaagtccatcgcctacctgcagatgaactc dsksiaylqmnslktedt T226A)
cctgaagaccgaggacaccgccgtgtactattgcgcccggggctactcca a vyyca rgystyygdf n i cctactacggcgacttcaacatctggggccagggcaccctggtgaccgtg wgqgtlvtvss
tcctcg Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR112 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:213 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:214)
Rll VLVH
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc
tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv
(L6H10) cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gnvsyrtsfgggtkveik
cgtggccacctactactgcctgggcggcgtgggcaacgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag ggslrlsctasgsdindyp
gtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccct iswvrqapgkglewigfi gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatctc nsggstwyadsvkgrfti ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca srhsskntlylqmnslra
actccggcggctccacctggtacgccgactcggtgaagggccggttcacc edta vyf ca rgystyygd
atctcccggcactcctccaagaacaccctgtacctgcagatgaactccctg fniwgqgtlvtvsssepk
cgggccgaggacaccgccgtgtacttctgcgcccggggctactccaccta ssdkthtcppcpapeaa
ctacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkayac
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagc eltknqvsltclvkgfyps
ccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgk
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg
gtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR134 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:215 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:216) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H10) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(DRA222)
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag ggslrlsctasgsdindyp
gtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccct iswvrqapgkglewigfi gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatctc nsggstwyadsvkgrfti ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca srhsskntlylqmnslra
actccggcggctccacctggtacgccgactcggtgaagggccggttcacc edta vyf ca rgystyygd
atctcccggcactcctccaagaacaccctgtacctgcagatgaactccctg fniwgqgtlvtvsssepk
cgggccgaggacaccgccgtgtacttctgcgcccggggctactccaccta ssdkthtcppcpapeaa ctacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkeykc
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag eltknqvsltclvkgfyps
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgsggggsggggsgggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca spgsqvqlvesgggvvq
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg pgrslrlsckasgytftrst
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg mhwvrqapgqglewi
ccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcc gyinpssaytnynqkfk
tgggcggtcactgaggctgtcctgcaaggcttctggctacacctttactag drftisadkskstaflqm
atctacgatgcactgggtaaggcaggcccctggacaaggtctggaatgg dslrpedtgvyfcarpqv
attggatacattaatcctagcagtgcttatactaattacaatcagaaattc hydyngfpywgqgtpv
aaggacaggttcacaatcagcgcagacaaatccaagagcacagccttcc tvssggggsggggsggg
tgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgca gsaqdiqmtqspsslsa
cggccccaagtccactatgattacaacgggtttccttactggggccaagg svgd rvtmtcsasssvsy gactcccgtcactgtctctagcggtggcggagggtctgggggtggcggat mnwyqqkpgkapkr
ccggaggtggtggctctgcacaagacatccagatgacccagtctccaagc wiydssklasgvparfsg
agcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccag sgsgtdytltisslqpedf
ctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcc atyycqqwsrnpptfgg
cccaaaagatggatttatgactcatccaaactggcttctggagtccctgct gtklqitsss
cgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcag
cctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaa
cccacccacgttcggaggggggaccaagctacaaattacatcctccagct
aa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR111 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:217 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:218)
Rll VLVH
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc
tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv
(L6H15) cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gnvsyrtsfgggtkveik
cgtggccacctactactgcctgggcggcgtgggcaacgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag grslrlsctasgsdindypi gtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccct swvrqapgkglewigfi
gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatctc nsggstwyaswvkgrft
ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca isrddsksiaylqmnslkt actccggcggctccacctggtacgcctcctgggtgaagggccggttcacc edtavyycargystyyg
atctcccgggacgactccaagtccatcgcctacctgcagatgaactccctg dfniwgqgtlvtvsssep
aagaccgaggacaccgccgtgtactattgcgcccggggctactccaccta kssdkthtcppcpapea
ctacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctc agapsvflfppkpkdtl
gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag misrtpevtcvvvdvsh
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca edpevkfnwyvdgvev
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg hnaktkpreeqynstyr
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vvsvltvlhqdwlngkay gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca acavsnkalpapiektis
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kakgqprepqvytlpps
ctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagc rdeltknqvsltclvkgfy
ccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc psdiavewesngqpen
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag nykttppvldsdgsfflys gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg kltvdksrwqqgnvfscs gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct vmhealhnhytqkslsl
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg spgk
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg
gtaaatga
Humanized gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:219 Rll VL (L7) cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID
ctggttccagcagaagcccggcaagccccccaagctgctgatctaccggg fqqkpgkppklliyrasnl NO:220)
(L6 N94A)
cctccaacctggcctccggcgtgccctcccggttctccggctccggctccg asgvpsrfsgsgsgtdftl gcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggcc tisslqpedvatyyclggv acctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttc gavsyrtsfgggtkveik
ggcggcggcaccaaggtggagatcaag
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR119 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO: 221 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:222) Rll VLVH
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl
scFv-Fc
tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv
(L7H15) cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
(L6H15
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
N94A)
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag grslrlsctasgsdindypi gtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccct swvrqapgkglewigfi
gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatctc nsggstwyaswvkgrft
ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca isrddsksiaylqmnslkt
actccggcggctccacctggtacgcctcctgggtgaagggccggttcacc edtavyycargystyyg
atctcccgggacgactccaagtccatcgcctacctgcagatgaactccctg dfniwgqgtlvtvsssep
aagaccgaggacaccgccgtgtactattgcgcccggggctactccaccta kssdkthtcppcpapea
ctacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctc agapsvflfppkpkdtl
gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag misrtpevtcvvvdvsh
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca edpevkfnwyvdgvev
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg hnaktkpreeqynstyr
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vvsvltvlhqdwlngkay
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca acavsnkalpapiektis
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kakgqprepqvytlpps
ctgaatggcaaggcgtacgcgtgcgcggtctccaacaaagccctcccagc rdeltknqvsltclvkgfy
ccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc psdiavewesngqpen
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag nykttppvldsdgsfflys
gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg kltvdksrwqqgnvfscs
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct vmhealhnhytqkslsl
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg spgk
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg
gtaaatga
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR133 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:223 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:224) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H15) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(DRA222)
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag grslrlsctasgsdindypi gtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccct swvrqapgkglewigfi
gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatctc nsggstwyaswvkgrft
ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca isrddsksiaylqmnslkt actccggcggctccacctggtacgcctcctgggtgaagggccggttcacc edtavyycargystyyg
atctcccgggacgactccaagtccatcgcctacctgcagatgaactccctg dfniwgqgtlvtvsssep
aagaccgaggacaccgccgtgtactattgcgcccggggctactccaccta kssdkthtcppcpapea
ctacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctc agapsvflfppkpkdtl
gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag misrtpevtcvvvdvsh
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca edpevkfnwyvdgvev
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg hnaktkpreeqynstyr gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vvsvltvlhqdwlngkey gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca kcavsnkalpapiektis
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kakgqprepqvytlpps
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag rdeltknqvsltclvkgfy
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc psdiavewesngqpen
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag nykttppvldsdgsfflys gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg kltvdksrwqqgnvfscs gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct vmhealhnhytqkslsl
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg spgsggggsggggsggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca gspgsqvqlvesgggvv
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg qpgrslrlsckasgytftrs gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg tmhwvrqapgqglewi ccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcc gyinpssaytnynqkfk
tgggcggtcactgaggctgtcctgcaaggcttctggctacacctttactag drftisadkskstaflqm
atctacgatgcactgggtaaggcaggcccctggacaaggtctggaatgg dslrpedtgvyfcarpqv
attggatacattaatcctagcagtgcttatactaattacaatcagaaattc hydyngfpywgqgtpv
aaggacaggttcacaatcagcgcagacaaatccaagagcacagccttcc tvssggggsggggsggg
tgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgca gsaqdiqmtqspsslsa
cggccccaagtccactatgattacaacgggtttccttactggggccaagg svgd rvtmtcsasssvsy gactcccgtcactgtctctagcggtggcggagggtctgggggtggcggat mnwyqqkpgkapkr
ccggaggtggtggctctgcacaagacatccagatgacccagtctccaagc wiydssklasgvparfsg
agcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccag sgsgtdytltisslqpedf
ctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcc atyycqqwsrnpptfgg
cccaaaagatggatttatgactcatccaaactggcttctggagtccctgct gtklqitsss
cgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcag
cctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaa
cccacccacgttcggaggggggaccaagctacaaattacatcctccagct
aa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR193 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:225 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:226) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H15) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(TSC394
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag grslrlsctasgsdindypi F87Y)
gtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccct swvrqapgkglewigfi
gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatctc nsggstwyaswvkgrft
ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca isrddsksiaylqmnslkt actccggcggctccacctggtacgcctcctgggtgaagggccggttcacc edtavyycargystyyg
atctcccgggacgactccaagtccatcgcctacctgcagatgaactccctg dfniwgqgtlvtvsssep
aagaccgaggacaccgccgtgtactattgcgcccggggctactccaccta kssdkthtcppcpapea
ctacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctc agapsvflfppkpkdtl
gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag misrtpevtcvvvdvsh
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca edpevkfnwyvdgvev
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg hnaktkpreeqynstyr gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vvsvltvlhqdwlngkey gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca kcavsnkalpapiektis
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kakgqprepqvytlpps
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag rdeltknqvsltclvkgfy
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc psdiavewesngqpen
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag nykttppvldsdgsfflys gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg kltvdksrwqqgnvfscs gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct vmhealhnhytqkslsl
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg spgsggggsggggsggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca gspgsqvqlvqsgpevk
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg kpgssvkvsckasgytfs
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg rstmhwvrqapgqgle
ccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcc wigyinpssaytnynqk
tgggtcctcggtgaaggtctcctgcaaggcttctggatataccttcagcag fkdrvtitadkststaym
atctacgatgcactgggtgcgacaggcccctggacaagggcttgagtgga elsslrsedtavyycarp
taggatacattaatcctagcagtgcttatactaattacaatcagaaattca qvhydyngfpywgqgt aggacagagtcacgattaccgcggacaaatccacgagcacagcctacat Ivtvssggggsggggsgg
ggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcga ggsggggsdiqmtqsps gaccccaagtccactatgattacaacgggtttccttactggggccaagga tlsasvgdrvtmtcsass
accctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatc svsymnwyqqkpgka
cggcggtggcggatcgggtggcggcggatctgacatccagatgacccag pkrwiydssklasgvpsr tctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgca fsgsgsgteytltisslqpd gtgccagctcaagtgtaagttacatgaactggtatcagcagaaaccaggg dfatyycqqwsrnpptf aaagcccctaagagatggatttatgactcatccaaactggcttctggggtc gggtkveikrsss
ccatcaaggttcagcggcagtggatctgggacagagtatactctcaccat
cagcagcctgcagcctgatgattttgcaacttattactgccaacagtggag
tcgtaacccacccactttcggcggagggaccaaggtggagatcaaacgg
tcctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR183 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:227 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:228) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H15) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(TSC394
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag grslrlsctasgsdindypi E86D F87Y)
gtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccct swvrqapgkglewigfi
gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatctc nsggstwyaswvkgrft
ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca isrddsksiaylqmnslkt actccggcggctccacctggtacgcctcctgggtgaagggccggttcacc edtavyycargystyyg
atctcccgggacgactccaagtccatcgcctacctgcagatgaactccctg dfniwgqgtlvtvsssep
aagaccgaggacaccgccgtgtactattgcgcccggggctactccaccta kssdkthtcppcpapea
ctacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctc agapsvflfppkpkdtl
gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag misrtpevtcvvvdvsh
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca edpevkfnwyvdgvev
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg hnaktkpreeqynstyr gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vvsvltvlhqdwlngkey gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca kcavsnkalpapiektis
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kakgqprepqvytlpps
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag rdeltknqvsltclvkgfy
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc psdiavewesngqpen
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag nykttppvldsdgsfflys gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg kltvdksrwqqgnvfscs gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct vmhealhnhytqkslsl
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg spgsggggsggggsggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca gspgsqvqlvqsgpevk
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg kpgssvkvsckasgytfs
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg rstmhwvrqapgqgle
ccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcc wigyinpssaytnynqk
tgggtcctcggtgaaggtctcctgcaaggcttctggatataccttcagcag fkdrvtitadkststaym
atctacgatgcactgggtgcgacaggcccctggacaagggcttgagtgga elsslrsedtavyycarp
taggatacattaatcctagcagtgcttatactaattacaatcagaaattca qvhydyngfpywgqgt aggacagagtcacgattaccgcggacaaatccacgagcacagcctacat Ivtvssggggsggggsgg
ggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcga ggsggggsdiqmtqsps gaccccaagtccactatgattacaacgggtttccttactggggccaagga tlsasvgdrvtmtcsass
accctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatc svsymnwyqqkpgka
cggcggtggcggatcgggtggcggcggatctgacatccagatgacccag pkrwiydssklasgvpsr tctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgca fsgsgsgtdytltisslqp
gtgccagctcaagtgtaagttacatgaactggtatcagcagaaaccaggg ddfatyycqqwsrnppt aaagcccctaagagatggatttatgactcatccaaactggcttctggggtc fgggtkveikrsss
ccatcaaggttcagcggcagtggatctgggacagattatactctcaccatc
agcagcctgcagcctgatgattttgcaacttattactgccaacagtggagt
cgtaacccacccactttcggcggagggaccaaggtggagatcaaacggt
cctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR189 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:229 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:230) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H10) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(TSC394
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag ggslrlsctasgsdindyp
E86D F87Y)
gtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccct iswvrqapgkglewigfi gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatctc nsggstwyadsvkgrfti ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca srhsskntlylqmnslra
actccggcggctccacctggtacgccgactcggtgaagggccggttcacc edta vyf ca rgystyygd
atctcccggcactcctccaagaacaccctgtacctgcagatgaactccctg fniwgqgtlvtvsssepk
cgggccgaggacaccgccgtgtacttctgcgcccggggctactccaccta ssdkthtcppcpapeaa ctacggcgacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkeykc
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag eltknqvsltclvkgfyps
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgsggggsggggsgggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca spgsqvqlvqsgpevkk
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg pgssvkvsckasgytfsr
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg stmhwvrqapgqgle
ccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcc wigyinpssaytnynqk
tgggtcctcggtgaaggtctcctgcaaggcttctggatataccttcagcag fkdrvtitadkststaym
atctacgatgcactgggtgcgacaggcccctggacaagggcttgagtgga elsslrsedtavyycarp
taggatacattaatcctagcagtgcttatactaattacaatcagaaattca qvhydyngfpywgqgt aggacagagtcacgattaccgcggacaaatccacgagcacagcctacat Ivtvssggggsggggsgg
ggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcga ggsggggsdiqmtqsps gaccccaagtccactatgattacaacgggtttccttactggggccaagga tlsasvgdrvtmtcsass
accctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatc svsymnwyqqkpgka
cggcggtggcggatcgggtggcggcggatctgacatccagatgacccag pkrwiydssklasgvpsr tctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgca fsgsgsgtdytltisslqp
gtgccagctcaagtgtaagttacatgaactggtatcagcagaaaccaggg ddfatyycqqwsrnppt aaagcccctaagagatggatttatgactcatccaaactggcttctggggtc fgggtkveikrsss
ccatcaaggttcagcggcagtggatctgggacagattatactctcaccatc
agcagcctgcagcctgatgattttgcaacttattactgccaacagtggagt
cgtaacccacccactttcggcggagggaccaaggtggagatcaaacggt
cctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggt evqlvesggglvqpgrsl SEQ ID NO:231
Rll VH ccctgcggctg tcctgcaccgcctccggctccgacatcaacg actacccca rlsctasgsdindypitw (SEQ ID
(H16) tcacctgggtg cggcaggcccccggcaagggcctggagtgj *atcggcttc vrqapgkglewigfinsg NO:232) atcaactccgf 'cggctccacctggtacgcctcctgggtgaaj >ggccggttc gstwyaswvkgrftisrd
(H15 S40T
accatctcccg ggacgactccaagtccatcgcctacctgcaj *atgaactc dsksiaylqmnslktedt G113R)
cctgaagacc gaggacaccgccgtgtactattgcgcccggg gctactcca a vyyca rgystyy rdf n i cctactaccgg ;gacttcaacatctggggccagggcaccctg gtgaccgtg wgqgtlvtvss
tcctcg
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR154 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:233 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:234) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H16) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(DRA222)
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag grslrlsctasgsdindypi gtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccct twvrqapgkglewigfi
gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatcac nsggstwyaswvkgrft
ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca isrddsksiaylqmnslkt actccggcggctccacctggtacgcctcctgggtgaagggccggttcacc edtavyycargystyyrd atctcccgggacgactccaagtccatcgcctacctgcagatgaactccctg fniwgqgtlvtvsssepk
aagaccgaggacaccgccgtgtactattgcgcccggggctactccaccta ssdkthtcppcpapeaa ctaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkeykc
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag eltknqvsltclvkgfyps
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgsggggsggggsgggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca spgsqvqlvesgggvvq
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg pgrslrlsckasgytftrst
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg mhwvrqapgqglewi
ccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcc gyinpssaytnynqkfk
tgggcggtcactgaggctgtcctgcaaggcttctggctacacctttactag drftisadkskstaflqm
atctacgatgcactgggtaaggcaggcccctggacaaggtctggaatgg dslrpedtgvyfcarpqv
attggatacattaatcctagcagtgcttatactaattacaatcagaaattc hydyngfpywgqgtpv
aaggacaggttcacaatcagcgcagacaaatccaagagcacagccttcc tvssggggsggggsggg
tgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgca gsaqdiqmtqspsslsa
cggccccaagtccactatgattacaacgggtttccttactggggccaagg svgd rvtmtcsasssvsy gactcccgtcactgtctctagcggtggcggagggtctgggggtggcggat mnwyqqkpgkapkr
ccggaggtggtggctctgcacaagacatccagatgacccagtctccaagc wiydssklasgvparfsg
agcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccag sgsgtdytltisslqpedf
ctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcc atyycqqwsrnpptfgg
cccaaaagatggatttatgactcatccaaactggcttctggagtccctgct gtklqitsss
cgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcag
cctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaa
cccacccacgttcggaggggggaccaagctacaaattacatcctccagct
aa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR185 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:235 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:236) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H15) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(TSC394
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag grslrlsctasgsdindypi E86D F87Y)
gtgcagctggtggagtccggcggcggcctggtgcagcccggccggtccct twvrqapgkglewigfi
gcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatcac nsggstwyaswvkgrft
ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca isrddsksiaylqmnslkt actccggcggctccacctggtacgcctcctgggtgaagggccggttcacc edtavyycargystyyrd atctcccgggacgactccaagtccatcgcctacctgcagatgaactccctg fniwgqgtlvtvsssepk
aagaccgaggacaccgccgtgtactattgcgcccggggctactccaccta ssdkthtcppcpapeaa ctaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkeykc
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag eltknqvsltclvkgfyps
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgsggggsggggsgggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca spgsqvqlvqsgpevkk
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg pgssvkvsckasgytfsr
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg stmhwvrqapgqgle
ccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcc wigyinpssaytnynqk
tgggtcctcggtgaaggtctcctgcaaggcttctggatataccttcagcag fkdrvtitadkststaym
atctacgatgcactgggtgcgacaggcccctggacaagggcttgagtgga elsslrsedtavyycarp
taggatacattaatcctagcagtgcttatactaattacaatcagaaattca qvhydyngfpywgqgt aggacagagtcacgattaccgcggacaaatccacgagcacagcctacat Ivtvssggggsggggsgg
ggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcga ggsggggsdiqmtqsps gaccccaagtccactatgattacaacgggtttccttactggggccaagga tlsasvgdrvtmtcsass
accctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatc svsymnwyqqkpgka
cggcggtggcggatcgggtggcggcggatctgacatccagatgacccag pkrwiydssklasgvpsr tctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgca fsgsgsgtdytltisslqp
gtgccagctcaagtgtaagttacatgaactggtatcagcagaaaccaggg ddfatyycqqwsrnppt aaagcccctaagagatggatttatgactcatccaaactggcttctggggtc fgggtkveikrsss
ccatcaaggttcagcggcagtggatctgggacagattatactctcaccatc
agcagcctgcagcctgatgattttgcaacttattactgccaacagtggagt
cgtaacccacccactttcggcggagggaccaaggtggagatcaaacggt
cctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR185 diqmtqspsslsasvgd (SEQ ID
rvtincqasqsidsnlaw NO:299)
(variant)
fqqkpgkppklliyrasnl
asgvpsrfsgsgsgtdftl
tisslqpedvatyyclggv
gavsyrtsfgggtkveik
ggggsggggsggggsgg
ggsevqlvesggglvqp
grslrlsctasgsdindypi
twvrqapgkglewigfi
nsggstwyaswvkgrft
isrddsksiaylqmnslkt
edtavyycargystyyrd
fniwgqgtlvtvsssepk
ssdkthtcppcpapeaa
gapsvflfppkpkdtlmi
srtpevtcvvvdvshed
pevkfnwyvdgvevhn
aktkpreeqynstyrvvs
vltvlhqdwlngkeykc
avsnkalpapiektiska
kgqprepqvytlppsrd
eltknqvsltclvkgfyps
diavewesngqpenny
kttppvldsdgsfflysklt
vdksrwqqgnvfscsv
mhealhnhytqkslsls
Pgsggggsggggsgggg
spgsqvqlvqsgpevkk
pgssvkvsckasgytfsr
stmhwvrqapgqgle
wigyinpssaytnynqk
fkdrvtitadkststaym
elsslrsedtavyycarp
qvhydyngfpywgqgt
Ivtvssggggsggggsgg
ggsggggsdiqmtqsps
tlsasvgdrvtmtcsass
svsymnwyqqkpgka
pkrwiydssklasgvpsr
fsgsgsgtdytltisslqp
ddfatyycqqwsrnppt
fgggtkveikrs
Humanized gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:237 Rll VL cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID
ctggttccagcagaagcccggcaagccccccaagctgctgatctaccggg fqqkpgkppklliyrasnl NO:238)
(L8)
cctccaacctggcctccggcgtgccctcccggttctccggctccggctccg asgvpsrfsgsgsgtdftl
(L7 R98W) gcaccgacttcaccctgaccatctcctccctgcagcccgaggacgtggcc tisslqpedvatyyclggv
acctactactgcctgggcggcgtgggcgccgtgtcctactggacctccttc gavsywtsfgggtkveik ggcggcggcaccaaggtggagatcaag Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR179 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:239 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:240) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsywtsfgggtkveik
(L8H16) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctactggac ggggsggggsggggsgg
ctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttca ggsevqlvesggglvqp
(DRA222)
ggcggaggtggatccggcggtggcggctccggtggcggcggatctgagg grslrlsctasgsdindypi tgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctg twvrqapgkglewigfi
cggctgtcctgcaccgcctccggctccgacatcaacgactaccccatcacc nsggstwyaswvkgrft tgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaa isrddsksiaylqmnslkt ctccggcggctccacctggtacgcctcctgggtgaagggccggttcaccat edtavyycargystyyrd ctcccgggacgactccaagtccatcgcctacctgcagatgaactccctga fniwgqgtlvtvsssepk
agaccgaggacaccgccgtgtactattgcgcccggggctactccacctac ssdkthtcppcpapeaa taccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkeykc
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag eltknqvsltclvkgfyps
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgsggggsggggsgggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca spgsqvqlvesgggvvq
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg pgrslrlsckasgytftrst
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg mhwvrqapgqglewi
ccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcc gyinpssaytnynqkfk
tgggcggtcactgaggctgtcctgcaaggcttctggctacacctttactag drftisadkskstaflqm
atctacgatgcactgggtaaggcaggcccctggacaaggtctggaatgg dslrpedtgvyfcarpqv
attggatacattaatcctagcagtgcttatactaattacaatcagaaattc hydyngfpywgqgtpv
aaggacaggttcacaatcagcgcagacaaatccaagagcacagccttcc tvssggggsggggsggg
tgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgca gsaqdiqmtqspsslsa
cggccccaagtccactatgattacaacgggtttccttactggggccaagg svgd rvtmtcsasssvsy gactcccgtcactgtctctagcggtggcggagggtctgggggtggcggat mnwyqqkpgkapkr
ccggaggtggtggctctgcacaagacatccagatgacccagtctccaagc wiydssklasgvparfsg
agcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccag sgsgtdytltisslqpedf
ctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcc atyycqqwsrnpptfgg
cccaaaagatggatttatgactcatccaaactggcttctggagtccctgct gtklqitsss
cgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcag
cctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaa
cccacccacgttcggaggggggaccaagctacaaattacatcctccagct
aa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR186 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:241 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:242) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsywtsfgggtkveik
(L8H16) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctactggac ggggsggggsggggsgg
ctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttca ggsevqlvesggglvqp
(TSC394
ggcggaggtggatccggcggtggcggctccggtggcggcggatctgagg grslrlsctasgsdindypi E86D F87Y)
tgcagctggtggagtccggcggcggcctggtgcagcccggccggtccctg twvrqapgkglewigfi
cggctgtcctgcaccgcctccggctccgacatcaacgactaccccatcacc nsggstwyaswvkgrft tgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatcaa isrddsksiaylqmnslkt ctccggcggctccacctggtacgcctcctgggtgaagggccggttcaccat edtavyycargystyyrd ctcccgggacgactccaagtccatcgcctacctgcagatgaactccctga fniwgqgtlvtvsssepk
agaccgaggacaccgccgtgtactattgcgcccggggctactccacctac ssdkthtcppcpapeaa taccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkeykc
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag eltknqvsltclvkgfyps
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgsggggsggggsgggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca spgsqvqlvqsgpevkk
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg pgssvkvsckasgytfsr
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg stmhwvrqapgqgle
ccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcc wigyinpssaytnynqk
tgggtcctcggtgaaggtctcctgcaaggcttctggatataccttcagcag fkdrvtitadkststaym
atctacgatgcactgggtgcgacaggcccctggacaagggcttgagtgga elsslrsedtavyycarp
taggatacattaatcctagcagtgcttatactaattacaatcagaaattca qvhydyngfpywgqgt aggacagagtcacgattaccgcggacaaatccacgagcacagcctacat Ivtvssggggsggggsgg
ggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcga ggsggggsdiqmtqsps gaccccaagtccactatgattacaacgggtttccttactggggccaagga tlsasvgdrvtmtcsass
accctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatc svsymnwyqqkpgka
cggcggtggcggatcgggtggcggcggatctgacatccagatgacccag pkrwiydssklasgvpsr tctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgca fsgsgsgtdytltisslqp
gtgccagctcaagtgtaagttacatgaactggtatcagcagaaaccaggg ddfatyycqqwsrnppt aaagcccctaagagatggatttatgactcatccaaactggcttctggggtc fgggtkveikrsss
ccatcaaggttcagcggcagtggatctgggacagattatactctcaccatc
agcagcctgcagcctgatgattttgcaacttattactgccaacagtggagt
cgtaacccacccactttcggcggagggaccaaggtggagatcaaacggt
cctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized gaggtgcagctggtggagtccggcggcggcctggtgcagcccggcggct evqlvesggglvqpggsl SEQ ID NO:243 Rll VH ccctgcggctgtcctgcaccgtgtccggcaccgacatcaacgactacccc rlsctvsgtdindypisw (SEQ ID
atctcctgggtgcggcaggcccccggcaagggcctggagtggatcggctt vrqapgkglewigfinsg NO:244)
(H17)
catcaactccggcggctccacctggtacgccgactcggtgaagggccggt gstwyadsvkgrftisrh
(H10 A25V tcaccatctcccggcactcctccaagaacaccctgtacctgcagatgaact sskntlylqmnslraedt
S28T ccctgcgggccgaggacaccgccgtgtacttctgcgcccggggctactcc avyfcargystyyrdfni
G113R) acctactaccgggacttcaacatctggggccagggcaccctggtgaccgt wgqgtlvtvss
gtcctcg
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR181 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:245 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:246) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H17) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(DRA222)
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag ggslrlsctvsgtdindyp
gtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccct iswvrqapgkglewigfi gcggctgtcctgcaccgtgtccggcaccgacatcaacgactaccccatctc nsggstwyadsvkgrfti ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca srhsskntlylqmnslra
actccggcggctccacctggtacgccgactcggtgaagggccggttcacc edta vyf ca rgystyy rd
atctcccggcactcctccaagaacaccctgtacctgcagatgaactccctg fniwgqgtlvtvsssepk
cgggccgaggacaccgccgtgtacttctgcgcccggggctactccaccta ssdkthtcppcpapeaa ctaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkeykc
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag eltknqvsltclvkgfyps
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgsggggsggggsgggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca spgsqvqlvesgggvvq
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg pgrslrlsckasgytftrst
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg mhwvrqapgqglewi
ccgggctctcaggtccagctggtggagtctgggggcggagtggtgcagcc gyinpssaytnynqkfk
tgggcggtcactgaggctgtcctgcaaggcttctggctacacctttactag drftisadkskstaflqm
atctacgatgcactgggtaaggcaggcccctggacaaggtctggaatgg dslrpedtgvyfcarpqv
attggatacattaatcctagcagtgcttatactaattacaatcagaaattc hydyngfpywgqgtpv
aaggacaggttcacaatcagcgcagacaaatccaagagcacagccttcc tvssggggsggggsggg
tgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtgca gsaqdiqmtqspsslsa
cggccccaagtccactatgattacaacgggtttccttactggggccaagg svgd rvtmtcsasssvsy gactcccgtcactgtctctagcggtggcggagggtctgggggtggcggat mnwyqqkpgkapkr
ccggaggtggtggctctgcacaagacatccagatgacccagtctccaagc wiydssklasgvparfsg
agcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgccag sgsgtdytltisslqpedf
ctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggcc atyycqqwsrnpptfgg
cccaaaagatggatttatgactcatccaaactggcttctggagtccctgct gtklqitsss
cgcttcagtggcagtgggtctgggaccgactataccctcacaatcagcag
cctgcagcccgaagatttcgccacttattactgccagcagtggagtcgtaa
cccacccacgttcggaggggggaccaagctacaaattacatcctccagct
aa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR191 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:247 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgkppklliyrasnl NO:248) Rll x CD3
caacctggcctggttccagcagaagcccggcaagccccccaagctgctga asgvpsrfsgsgsgtdftl scFv-Fc- tctaccgggcctccaacctggcctccggcgtgccctcccggttctccggctc tisslqpedvatyyclggv scFv
cggctccggcaccgacttcaccctgaccatctcctccctgcagcccgagga gavsyrtsfgggtkveik
(L7H17) cgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccgga ggggsggggsggggsgg
cctccttcggcggcggcaccaaggtggagatcaagggtggaggcggttc ggsevqlvesggglvqp
(TSC394
aggcggaggtggatccggcggtggcggctccggtggcggcggatctgag ggslrlsctvsgtdindyp
E86D F87Y)
gtgcagctggtggagtccggcggcggcctggtgcagcccggcggctccct iswvrqapgkglewigfi gcggctgtcctgcaccgtgtccggcaccgacatcaacgactaccccatctc nsggstwyadsvkgrfti ctgggtgcggcaggcccccggcaagggcctggagtggatcggcttcatca srhsskntlylqmnslra
actccggcggctccacctggtacgccgactcggtgaagggccggttcacc edta vyf ca rgystyy rd
atctcccggcactcctccaagaacaccctgtacctgcagatgaactccctg fniwgqgtlvtvsssepk
cgggccgaggacaccgccgtgtacttctgcgcccggggctactccaccta ssdkthtcppcpapeaa ctaccgggacttcaacatctggggccagggcaccctggtgaccgtgtcctc gapsvflfppkpkdtlmi gagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag srtpevtcvvvdvshed
cacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaaccca pevkfnwyvdgvevhn
aggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg aktkpreeqynstyrvvs gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg vltvlhqdwlngkeykc
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca avsnkalpapiektiska
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg kgqprepqvytlppsrd
ctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcccag eltknqvsltclvkgfyps
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc diavewesngqpenny acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag kttppvldsdgsfflysklt gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtg vdksrwqqgnvfscsv
gagtgggagagcaatgggcagccggagaacaactacaagaccacgcct mhealhnhytqkslsls
cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg Pgsggggsggggsgggg
gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgca spgsqvqlvqsgpevkk
tgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg pgssvkvsckasgytfsr
gttctggtggaggcggttcaggcggaggtggctccggcggtggcggatcg stmhwvrqapgqgle
ccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaagcc wigyinpssaytnynqk
tgggtcctcggtgaaggtctcctgcaaggcttctggatataccttcagcag fkdrvtitadkststaym
atctacgatgcactgggtgcgacaggcccctggacaagggcttgagtgga elsslrsedtavyycarp
taggatacattaatcctagcagtgcttatactaattacaatcagaaattca qvhydyngfpywgqgt aggacagagtcacgattaccgcggacaaatccacgagcacagcctacat Ivtvssggggsggggsgg
ggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcga ggsggggsdiqmtqsps gaccccaagtccactatgattacaacgggtttccttactggggccaagga tlsasvgdrvtmtcsass
accctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggatc svsymnwyqqkpgka
cggcggtggcggatcgggtggcggcggatctgacatccagatgacccag pkrwiydssklasgvpsr tctccttccaccctgtctgcatctgtaggagacagagtcaccatgacttgca fsgsgsgtdytltisslqp
gtgccagctcaagtgtaagttacatgaactggtatcagcagaaaccaggg ddfatyycqqwsrnppt aaagcccctaagagatggatttatgactcatccaaactggcttctggggtc fgggtkveikrsss
ccatcaaggttcagcggcagtggatctgggacagattatactctcaccatc
agcagcctgcagcctgatgattttgcaacttattactgccaacagtggagt
cgtaacccacccactttcggcggagggaccaaggtggagatcaaacggt
cctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgac diqmtqspsslsasvgd SEQ ID NO:249 Rll VL, pi cgggtgaccatcaactgccaggcctcccagtccatcgactccaacctggc rvtincqasqsidsnlaw (SEQ ID variant ctggttccagcagaagcccggccagccccccaagctgctgatctaccggg fqqkpgqppklliyrasn NO:250) cctccaacctggcctccggcgtgcccgaccggttctccggctccggctccg lasgvpdrfsgsgsgtdft
(L9)
gcaccgacttcaccctgaccatctcctccctggaggccgaggacgtggcc Itissleaedvatyyclgg
(L7 K42Q acctactactgcctgggcggcgtgggcgccgtgtcctaccggacctccttc vgavsyrtsfgggtkveik
S60D Q79E ggcggcggcaccaaggtggagatcaag
P80A)
Humanized gaggtgcagctggtggagtccggcggcggcctggtgcagcccggccggt evqlvesggglvqpgrsl SEQ ID NO:251 Rll VH, pi ccctgcggctgtcctgcaccgcctccggctccgacatcaacgactacccca rlsctasgsdindypitw (SEQ ID variant tcacctgggtgcggcaggcccccggccagggcctggagtggatcggcttc vrqapgqglewigfinsg NO:252) atcaactccggcggctccacctggtacgcctcctgggtgaagggccggttc gstwyaswvkgrftisrd
(H18)
accatctcccgggacgactccaagtccatcgcctacctgcagatgaactc dsksiaylqmnslktedt
(H17 K43Q) cctgaagaccgaggacaccgccgtgtactattgcgcccggggctactcca a vyyca rgystyy rdf n i
cctactaccgggacttcaacatctggggccagggcaccctggtgaccgtg wgqgtlvtvss
tcctcg
Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR182 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:253 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgqppklliyrasn NO:254) Rll x CD3
caacctggcctggttccagcagaagcccggccagccccccaagctgctga lasgvpdrfsgsgsgtdft scFv-Fc- tctaccgggcctccaacctggcctccggcgtgcccgaccggttctccggct Itissleaedvatyyclgg
scFv
ccggctccggcaccgacttcaccctgaccatctcctccctggaggccgag vgavsyrtsfgggtkveik
(L9H18) gacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccg ggggsggggsggggsgg
gacctccttcggcggcggcaccaaggtggagatcaagggtggaggcggt ggsevqlvesggglvqp
(DRA222)
tcaggcggaggtggatccggcggtggcggctccggtggcggcggatctg grslrlsctasgsdindypi aggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtcc twvrqapgqglewigfi
ctgcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatc nsggstwyaswvkgrft acctgggtgcggcaggcccccggccagggcctggagtggatcggcttcat isrddsksiaylqmnslkt caactccggcggctccacctggtacgcctcctgggtgaagggccggttca edtavyycargystyyrd ccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccc fniwgqgtlvtvsssepk
tgaagaccgaggacaccgccgtgtactattgcgcccggggctactccacc ssdkthtcppcpapeaa
tactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtc gapsvflfppkpkdtlmi ctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcc srtpevtcvvvdvshed
cagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaac pevkfnwyvdgvevhn
ccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtg aktkpreeqynstyrvvs gtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtgg vltvlhqdwlngkeykc
acggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagt avsnkalpapiektiska
acaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggac kgqprepqvytlppsrd
tggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctccc eltknqvsltclvkgfyps
agcccccatcgagaaaaccatctccaaagccaaagggcagccccgaga diavewesngqpenny accacaggtgtacaccctgcccccatcccgggatgagctgaccaagaac kttppvldsdgsfflysklt caggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgc vdksrwqqgnvfscsv
cgtggagtgggagagcaatgggcagccggagaacaactacaagaccac mhealhnhytqkslsls
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcacc Pgsggggsggggsgggg
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat spgsqvqlvesgggvvq
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc pgrslrlsckasgytftrst
cgggttctggtggaggcggttcaggcggaggtggctccggcggtggcgga mhwvrqapgqglewi
tcgccgggctctcaggtccagctggtggagtctgggggcggagtggtgca gyinpssaytnynqkfk
gcctgggcggtcactgaggctgtcctgcaaggcttctggctacacctttact drftisadkskstaflqm
agatctacgatgcactgggtaaggcaggcccctggacaaggtctggaat dslrpedtgvyfcarpqv
ggattggatacattaatcctagcagtgcttatactaattacaatcagaaat hydyngfpywgqgtpv
tcaaggacaggttcacaatcagcgcagacaaatccaagagcacagcctt tvssggggsggggsggg
cctgcagatggacagcctgaggcccgaggacaccggcgtctatttctgtg gsaqdiqmtqspsslsa
cacggccccaagtccactatgattacaacgggtttccttactggggccaag svgd rvtmtcsasssvsy ggactcccgtcactgtctctagcggtggcggagggtctgggggtggcgga mnwyqqkpgkapkr
tccggaggtggtggctctgcacaagacatccagatgacccagtctccaag wiydssklasgvparfsg
cagcctgtctgcaagcgtgggggacagggtcaccatgacctgcagtgcca sgsgtdytltisslqpedf
gctcaagtgtaagttacatgaactggtaccagcagaagccgggcaaggc atyycqqwsrnpptfgg
ccccaaaagatggatttatgactcatccaaactggcttctggagtccctgc gtklqitsss
tcgcttcagtggcagtgggtctgggaccgactataccctcacaatcagca
gcctgcagcccgaagatttcgccacttattactgccagcagtggagtcgta
acccacccacgttcggaggggggaccaagctacaaattacatcctccagc
taa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR192 atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagat diqmtqspsslsasvgd SEQ ID NO:255 accaccggtgacatccagatgacccagtccccctcctccctgtccgcctcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgaccgggtgaccatcaactgccaggcctcccagtccatcgactc fqqkpgqppklliyrasn NO:256) Rll x CD3
caacctggcctggttccagcagaagcccggccagccccccaagctgctga lasgvpdrfsgsgsgtdft scFv-Fc- tctaccgggcctccaacctggcctccggcgtgcccgaccggttctccggct Itissleaedvatyyclgg
scFv
ccggctccggcaccgacttcaccctgaccatctcctccctggaggccgag vgavsyrtsfgggtkveik
(L9H18) gacgtggccacctactactgcctgggcggcgtgggcgccgtgtcctaccg ggggsggggsggggsgg
gacctccttcggcggcggcaccaaggtggagatcaagggtggaggcggt ggsevqlvesggglvqp
(TSC394
tcaggcggaggtggatccggcggtggcggctccggtggcggcggatctg grslrlsctasgsdindypi E86D F87Y)
aggtgcagctggtggagtccggcggcggcctggtgcagcccggccggtcc twvrqapgqglewigfi
ctgcggctgtcctgcaccgcctccggctccgacatcaacgactaccccatc nsggstwyaswvkgrft acctgggtgcggcaggcccccggccagggcctggagtggatcggcttcat isrddsksiaylqmnslkt caactccggcggctccacctggtacgcctcctgggtgaagggccggttca edtavyycargystyyrd ccatctcccgggacgactccaagtccatcgcctacctgcagatgaactccc fniwgqgtlvtvsssepk
tgaagaccgaggacaccgccgtgtactattgcgcccggggctactccacc ssdkthtcppcpapeaa
tactaccgggacttcaacatctggggccagggcaccctggtgaccgtgtc gapsvflfppkpkdtlmi ctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgcc srtpevtcvvvdvshed
cagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaac pevkfnwyvdgvevhn
ccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtg aktkpreeqynstyrvvs gtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtgg vltvlhqdwlngkeykc
acggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagt avsnkalpapiektiska
acaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggac kgqprepqvytlppsrd
tggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctccc eltknqvsltclvkgfyps
agcccccatcgagaaaaccatctccaaagccaaagggcagccccgaga diavewesngqpenny accacaggtgtacaccctgcccccatcccgggatgagctgaccaagaac kttppvldsdgsfflysklt caggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgc vdksrwqqgnvfscsv
cgtggagtgggagagcaatgggcagccggagaacaactacaagaccac mhealhnhytqkslsls
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcacc Pgsggggsggggsgggg
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat spgsqvqlvqsgpevkk
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc pgssvkvsckasgytfsr
cgggttctggtggaggcggttcaggcggaggtggctccggcggtggcgga stmhwvrqapgqgle
tcgccgggctctcaggtccagctggtgcaatctgggcctgaggtgaagaa wigyinpssaytnynqk
gcctgggtcctcggtgaaggtctcctgcaaggcttctggatataccttcag fkdrvtitadkststaym
cagatctacgatgcactgggtgcgacaggcccctggacaagggcttgagt elsslrsedtavyycarp
ggataggatacattaatcctagcagtgcttatactaattacaatcagaaat qvhydyngfpywgqgt tcaaggacagagtcacgattaccgcggacaaatccacgagcacagccta Ivtvssggggsggggsgg
catggagctgagcagcctgagatctgaggacacggccgtgtattactgtg ggsggggsdiqmtqsps cgagaccccaagtccactatgattacaacgggtttccttactggggccaa tlsasvgdrvtmtcsass
ggaaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtgg svsymnwyqqkpgka
atccggcggtggcggatcgggtggcggcggatctgacatccagatgaccc pkrwiydssklasgvpsr agtctccttccaccctgtctgcatctgtaggagacagagtcaccatgactt fsgsgsgtdytltisslqp
gcagtgccagctcaagtgtaagttacatgaactggtatcagcagaaacca ddfatyycqqwsrnppt gggaaagcccctaagagatggatttatgactcatccaaactggcttctgg fgggtkveikrsss
ggtcccatcaaggttcagcggcagtggatctgggacagattatactctcac
catcagcagcctgcagcctgatgattttgcaacttattactgccaacagtg
gagtcgtaacccacccactttcggcggagggaccaaggtggagatcaaa
cggtcctccagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR192 diqmtqspsslsasvgd (SEQ ID
rvtincqasqsidsnlaw NO:300)
(Variant)
fqqkpgqppklliyrasn
lasgvpdrfsgsgsgtdft
Itissleaedvatyyclgg
vgavsyrtsfgggtkveik
ggggsggggsggggsgg
ggsevqlvesggglvqp
grslrlsctasgsdindypi
twvrqapgqglewigfi
nsggstwyaswvkgrft
isrddsksiaylqmnslkt
edtavyycargystyyrd
fniwgqgtlvtvsssepk
ssdkthtcppcpapeaa
gapsvflfppkpkdtlmi
srtpevtcvvvdvshed
pevkfnwyvdgvevhn
aktkpreeqynstyrvvs
vltvlhqdwlngkeykc
avsnkalpapiektiska
kgqprepqvytlppsrd
eltknqvsltclvkgfyps
diavewesngqpenny
kttppvldsdgsfflysklt
vdksrwqqgnvfscsv
mhealhnhytqkslsls
Pgsggggsggggsgggg
spgsqvqlvqsgpevkk
pgssvkvsckasgytfsr
stmhwvrqapgqgle
wigyinpssaytnynqk
fkdrvtitadkststaym
elsslrsedtavyycarp
qvhydyngfpywgqgt
Ivtvssggggsggggsgg
ggsggggsdiqmtqsps
tlsasvgdrvtmtcsass
svsymnwyqqkpgka
pkrwiydssklasgvpsr
fsgsgsgtdytltisslqp
ddfatyycqqwsrnppt
fgggtkveikrs Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
TSC455 QVQLVQSGPEVKKP (SEQ ID
(anti-CD3) GSSVKVSCKASGYTF NO:257)
SRSTMHWVRQAPG
TSC394
QGLEWIGYINPSSAY
F87Y scFv
TNYNQKFKDRVTIT
ADKSTSTAYMELSSL
RSEDTAVYYCARPQ
VHYDYNGFPYWGQ
GTLVTVSSGGGGSG
GGGSGGGGSGGGG
SDIQMTQSPSTLSAS
VGDRVTMTCSASSS
VSYM N WYQQKPG
KAPKRWIYDSSKLAS
GVPSRFSGSGSGTEY
TLTISSLQPDDFATYY
CQQWSRNPPTFGG
GTKVEIKRSSS
TSC456 QVQLVQSGPEVKKP (SEQ ID
(anti-CD3) GSSVKVSCKASGYTF NO:258)
SRSTMHWVRQAPG
TSC394
QGLEWIGYINPSSAY
E86D F87Y
TNYNQKFKDRVTIT
scFv
ADKSTSTAYMELSSL
RSEDTAVYYCARPQ
VHYDYNGFPYWGQ
GTLVTVSSGGGGSG
GGGSGGGGSGGGG
SDIQMTQSPSTLSAS
VGDRVTMTCSASSS
VSYM N WYQQKPG
KAPKRWIYDSSKLAS
GVPSRFSGSGSGTD
YTLTISSLQPDDFATY
YCQQWSRNPPTFG
GGTKVEIKRSSS
TSC455 and QVQLVQSGPEVKKP (SEQ ID
TSC456 GSSVKVSCKASGYTF NO:259) variable SRSTMHWVRQAPG
heavy QGLEWIGYINPSSAY
domain TNYNQKFKDRVTIT
ADKSTSTAYMELSSL
RSEDTAVYYCARPQ
VHYDYNGFPYWGQ
GTLVTVSS Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
TSC455 DIQMTQSPSTLSAS (SEQ ID variable VGDRVTMTCSASSS NO:260) light VSYM N WYQQKPG
domain KAPKRWIYDSSKLAS
GVPSRFSGSGSGTEY
TLTISSLQPDDFATYY
CQQWSRNPPTFGG
GTKVEIKRS
TSC456 DIQMTQSPSTLSAS (SEQ ID variable VGDRVTMTCSASSS NO:261) light VSYM N WYQQKPG
domain KAPKRWIYDSSKLAS
GVPSRFSGSGSGTD YTLTISSLQPDDFATY YCQQWSRNPPTFG G GTKVEIKRS
DRA222 QVQLVESGGGVVQ (SEQ ID
(anti-CD3) PGRSLRLSCKASGYT NO:262)
FTRSTMHWVRQAP
scFv
GQGLEWIGYINPSS
AYTNYNQKFKDRFTI
SADKSKSTAFLQMD
SLRPEDTGVYFCARP
QVHYDYNGFPYWG
QGTPVTVSSGGGGS
GGGGSGGGGSAQD
IQMTQSPSSLSASV
GDRVTMTCSASSSV
SYMN WYQQKPG K
APKRWIYDSSKLAS
GVPARFSGSGSGTD
YTLTISSLQPEDFATY
YCQQWSRNPPTFG
GGTKLQITSSS
DRA222 QVQLVESGGGVVQ (SEQ ID variable PGRSLRLSCKASGYT NO:263) heavy FTRSTMHWVRQAP
domain GQGLEWIGYINPSS
AYTNYNQKFKDRFTI
SADKSKSTAFLQMD
SLRPEDTGVYFCARP
QVHYDYNGFPYWG
QGTPVTVSS
DRA222 DIQMTQSPSSLSAS (SEQ ID variable VGDRVTMTCSASSS NO:264) light VSYM N WYQQKPG
domain KAPKRWIYDSSKLAS
GVPARFSGSGSGTD
YTLTISSLQPEDFATY
YCQQWSRNPPTFG
GGTKLQITS Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR238 atggaggctcccgctcagctgctgttcctcctgctgctctggctgcccgaca diqmtqspsslsasvgd SEQ ID NO:304 ccacaggcgacatccagatgacccagtccccttcctccctgtccgctagcg rvtincqasqsidsnlaw (SEQ ID
Humanized
tgggcgatagggtgaccatcaactgccaggcctcccagtccattgactcc fqqkpgqppklliyrasn NO:305) Rll x CD3
aatctggcctggttccagcagaagcccggacagccccccaagctgctgat lasgvpdrfsgsgsgtdft scFv-Fc- ttacagggcctccaacctggcttccggcgtgcctgacaggttctccggatc Itissleaedvatyyclgg
scFv
cggcagcggcaccgacttcaccctgaccatctcctccctggaggccgagg vgavsyrtsfgggtkveik
Codon atgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgga ggggsggggsggggsgg
optimized cctccttcggcggcggcaccaaggtggagatcaagggcggcggcggcag ggsevqlvesggglvqp
cggcggcggcggcagcggcggcggaggctccggcggcggcggcagcga grslrlsctasgsdindypi ggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtcc twvrqapgqglewigfi
ctgaggctgtcctgcacagccagcggctccgacatcaacgactaccccat nsggstwyaswvkgrft cacctgggtgaggcaggctcctggccagggcctggaatggatcggcttta isrddsksiaylqmnslkt tcaacagcggcggcagcacctggtatgcttcctgggtgaagggccggttc edtavyycargystyyrd
accattagcagggacgactccaagtccattgcctacctgcagatgaactc fniwgqgtlvtvsssepk
cctgaagaccgaggacaccgccgtgtactactgcgccaggggctacagc ssdkthtcppcpapeaa acctattaccgggactttaacatctggggccagggcacactggtcaccgt gapsvflfppkpkdtlmi gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt srtpevtcvvvdvshed
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pevkfnwyvdgvevhn
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg aktkpreeqynstyrvvs gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vltvlhqdwlngkeykc
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca avsnkalpapiektiska
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg kgqprepqvytlppsrd
actggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccct eltknqvsltclvkgfyps
cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga diavewesngqpenny gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga kttppvldsdgsfflysklt accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc vdksrwqqgnvfscsv
gccgtggagtgggagagcaatgggcagccggagaacaactacaagacc mhealhnhytqkslsls
acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctca Pgsggggsggggsgggg
ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg spgsqvqlvqsgpevkk
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc pgssvkvsckasgytfsr
tccgggttccggaggagggggttcaggtgggggaggttctggcggcggg stmhwvrqapgqgle
ggaagccctgggtcacaggtgcaactggtgcagagtggacccgaggtta wigyinpssaytnynqk
aaaaaccagggtcctccgttaaggttagctgcaaagcctctggctacaca fkdrvtitadkststaym
ttttccaggagtacaatgcactgggtgaggcaggctcctggacagggact elsslrsedtavyycarp
cgagtggatcgggtatatcaacccatctagcgcctataccaattacaacc qvhydyngfpywgqgt aaaagtttaaggaccgagttaccattaccgctgacaaatccaccagtaca Ivtvssggggsggggsgg
gcttatatggagctgtcatctcttaggtccgaggacactgctgtttattact ggsggggsdiqmtqsps gcgctcgtcctcaggttcactatgactataatggttttccctactggggtca tlsasvgdrvtmtcsass
gggaaccctggtgactgtctcttctggcggtggaggcagcggtgggggtg svsymnwyqqkpgka
ggtctggaggcggtggcagtggcggcggaggctctgatattcagatgact pkrwiydssklasgvpsr cagtctcctagcactctcagcgccagcgtgggggatcgtgtgacaatgac fsgsgsgtdytltisslqp
ttgctccgctagcagtagtgtgtcttacatgaattggtatcagcagaagcc ddfatyycqqwsrnppt cgggaaagcacctaagcgctggatctatgactcttccaagctggcaagtg fgggtkveikrs
gtgtcccctcacggttctctggctcaggttctggtactgactatactttgact
atctcctccctccagcccgatgatttcgctacctattattgtcagcagtgga
gccgtaacccacccactttcggaggcggtaccaaagtggagatcaagag
gtcatga Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR239 atggaggctcccgctcagctgctgttcctcctgctgctctggctgcccgaca diqmtqspsslsasvgd SEQ ID NO:306 ccacaggcgacatccagatgacccagtccccttcctccctgtccgctagcg rvtincqasqsidsnlaw (SEQ ID
Humanized
tgggcgatagggtgaccatcaactgccaggcctcccagtccattgactcc fqqkpgqppklliyrasn NO:307) Rll x CD3
aatctggcctggttccagcagaagcccggacagccccccaagctgctgat lasgvpdrfsgsgsgtdft scFv-Fc- ttacagggcctccaacctggcttccggcgtgcctgacaggttctccggctc Itissleaedvatyyclgg
scFv
cggcagcggcaccgacttcaccctgaccatctcctccctggaggccgagg vgavsyrtsfgggtkveik
Codon atgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgga ggggsggggsggggsgg
optimized cctccttcggcggcggcaccaaggtggagatcaagggcggcggcggctc ggsevqlvesggglvqp
cggcggcggcggctccggcggcggcggctccggcggcggtggctccgag grslrlsctasgsdindypi gtgcagctggtggaaagcggaggtggcctggtgcagcctggaaggtccct twvrqapgqglewigfi
gaggctgtcctgcacagccagcggctccgacatcaacgactaccccatca nsggstwyaswvkgrft
cctgggtgaggcaggctcctggccagggcctggaatggatcggctttatc isrddsksiaylqmnslkt aacagcggcggcagcacctggtatgcttcctgggtgaagggccggttcac edtavyycargystyyrd cattagcagggacgactccaagtccattgcctacctgcagatgaactccct fniwgqgtlvtvsssepk
gaagaccgaggacaccgccgtgtactactgcgccaggggctacagcacc ssdkthtcppcpapeaa
tattaccgggactttaacatctggggccagggcacactggtcaccgtgtcc gapsvflfppkpkdtlmi tcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgccc srtpevtcvvvdvshed
agcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaacc pevkfnwyvdgvevhn
caaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg aktkpreeqynstyrvvs tggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggac vltvlhqdwlngkeykc
ggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac avsnkalpapiektiska
aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactg kgqprepqvytlppsrd
gctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctccca eltknqvsltclvkgfyps
gcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaa diavewesngqpenny
ccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaacc kttppvldsdgsfflysklt aggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgcc vdksrwqqgnvfscsv
gtggagtgggagagcaatgggcagccggagaacaactacaagaccacg mhealhnhytqkslsls
cctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccg Pgsggggsggggsgggg
tggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatg spgsqvqlvqsgpevkk
catgaggctctgcacaaccactacacgcagaagagcctctccctgtctcc pgssvkvsckasgytfsr
gggttccggaggtggcggttcgggaggtggcgggtcaggaggtgggggc stmhwvrqapgqgle
tctcccggatcccaggtgcagctggtgcagtctggtcctgaggtgaaaaa wigyinpssaytnynqk
gcctggctccagcgtgaaggtgtcctgcaaggccagcggatacaccttta fkdrvtitadkststaym
gccggtccaccatgcattgggtgaggcaggctcctggacagggcctggag elsslrsedtavyycarp
tggatcggctacatcaaccccagcagcgcttataccaactacaatcagaa qvhydyngfpywgqgt gtttaaggaccgggtgaccatcaccgccgataagtccaccagcaccgcct Ivtvssggggsggggsgg
acatggagctgtccagcctgaggagcgaggataccgccgtgtactattgc ggsggggsdiqmtqsps gcccggccccaggtccattacgactacaacggcttcccctattggggcca tlsasvgdrvtmtcsass
gggaaccctggtgaccgtgtccagcggtggcggtggcagcggcggcggc svsymnwyqqkpgka
ggctctggcggaggtggcagcggcggagggggctccgacattcagatga pkrwiydssklasgvpsr cccagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccatga fsgsgsgtdytltisslqp
cctgcagcgccagcagctccgtgtcctacatgaactggtaccagcagaag ddfatyycqqwsrnppt cccggcaaggctcccaagaggtggatttacgactccagcaagctggcctc fgggtkveikrs
tggtgtccccagcaggttctctggtagcggcagcggcacagactacaccc
tgaccatctcctccctgcagcccgacgatttcgccacctactattgccagc
agtggtcccggaatccccctacctttggcggcggcaccaaggtggagatc
aagaggagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR241 atggaggctcccgctcagctcctgttcctgctgctgctgtggctgcctgaca diqmtqspsslsasvgd SEQ ID NO:308 ccaccggcgacatccagatgacccagagcccctccagcctgagcgcttcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtcggagacagggtgaccatcaactgccaggcctcccagagcatcgact fqqkpgkppklliyrasnl NO:309) Rll x CD3
ccaatctggcctggttccagcagaagcctggcaagccccccaaactgctg asgvpsrfsgsgsgtdftl scFv-Fc- atctatagggcctccaacctggcctctggagtgccttccaggtttagcggc tisslqpedvatyyclggv scFv
tccggctccggcacagacttcaccctgaccatctcctccctccaacctgag gavsyrtsfgggtkveik
Codon gacgtcgccacctactattgcctgggtggcgtgggcgctgtgtcctatagg ggggsggggsggggsgg
optimized acctcctttggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaatccggtggtggcctggtgcagcctggaaggtccc twvrqapgkglewigfi
tgaggctgtcctgtaccgccagcggcagcgacatcaacgactaccctatc nsggstwyaswvkgrft
acctgggtgaggcaggctcccggcaaaggcctggagtggatcggctttat isrddsksiaylqmnslkt caactccggcggaagcacctggtacgccagctgggtgaagggccggttc edtavyycargystyyrd accatctcccgggatgactccaagagcatcgcctacctgcagatgaacag fniwgqgtlvtvsssepk
cctgaagacagaggacaccgccgtgtactactgcgccaggggctattcca ssdkthtcppcpapeaa cctactaccgggacttcaacatctggggccagggcaccctggtcaccgtgt gapsvflfppkpkdtlmi cctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgc srtpevtcvvvdvshed
ccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaa pevkfnwyvdgvevhn
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt aktkpreeqynstyrvvs ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg vltvlhqdwlngkeykc
gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag avsnkalpapiektiska
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagga kgqprepqvytlppsrd
ctggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcc eltknqvsltclvkgfyps
cagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag diavewesngqpenny aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa kttppvldsdgsfflysklt ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg vdksrwqqgnvfscsv
ccgtggagtgggagagcaatgggcagccggagaacaactacaagacca mhealhnhytqkslsls
cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac Pgsggggsggggsgggg
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga spgsqvqlvqsgpevkk
tgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc pgssvkvsckasgytfsr
cgggttccggaggtggcggttcgggaggtggcgggtcaggaggtggggg stmhwvrqapgqgle
ctctcccggatcccaggtgcagctggtgcagtctggtcctgaggtgaaaa wigyinpssaytnynqk
agcctggctccagcgtgaaggtgtcctgcaaggccagcggatacaccttt fkdrvtitadkststaym
agccggtccaccatgcattgggtgaggcaggctcctggacagggcctgga elsslrsedtavyycarp
gtggatcggctacatcaaccccagcagcgcttataccaactacaatcaga qvhydyngfpywgqgt agtttaaggaccgggtgaccatcaccgccgataagtccaccagcaccgcc Ivtvssggggsggggsgg
tacatggagctgtccagcctgaggagcgaggataccgccgtgtactattg ggsggggsdiqmtqsps cgcccggccccaggtccattacgactacaacggcttcccctattggggcca tlsasvgdrvtmtcsass
gggaaccctggtgaccgtgtccagcggtggcggtggcagcggcggcggc svsymnwyqqkpgka
ggctctggcggaggtggcagcggcggagggggctccgacattcagatga pkrwiydssklasgvpsr cccagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccatga fsgsgsgtdytltisslqp
cctgcagcgccagcagctccgtgtcctacatgaactggtaccagcagaag ddfatyycqqwsrnppt cccggcaaggctcccaagaggtggatttacgactccagcaagctggcctc fgggtkveikrs
tggtgtccccagcaggttctctggtagcggcagcggcacagactacaccc
tgaccatctcctccctgcagcccgacgatttcgccacctactattgccagc
agtggtcccggaatccccctacctttggcggcggcaccaaggtggagatc
aagaggagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR242 atggaggctcccgctcagctgctgttcctcctgctgctctggctgcccgaca diqmtqspsslsasvgd SEQ ID NO:310 ccacaggcgacatccagatgacccagtccccttcctccctgtccgctagcg rvtincqasqsidsnlaw (SEQ ID
Humanized
tgggcgatagggtgaccatcaactgccaggcctcccagtccattgactcc fqqkpgqppklliyrasn NO:311) Rll x CD3
aatctggcctggttccagcagaagcccggacagccccccaagctgctgat lasgvpdrfsgsgsgtdft scFv-Fc- ttacagggcctccaacctggcttccggcgtgcctgacaggttctccggatc Itissleaedvatyyclgg
scFv
cggcagcggcaccgacttcaccctgaccatctcctccctggaggccgagg vgavsyrtsfgggtkveik
Codon atgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgga ggggsggggsggggsgg
optimized cctccttcggcggcggcaccaaggtggagatcaagggcggcggcggcag ggsevqlvesggglvqp
cggcggcggcggcagcggcggcggaggctccggcggcggcggcagcga grslrlsctasgsdindypi ggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtcc twvrqapgqglewigfi
ctgaggctgtcctgcacagccagcggctccgacatcaacgactaccccat nsggstwyaswvkgrft cacctgggtgaggcaggctcctggccagggcctggaatggatcggcttta isrddsksiaylqmnslkt tcaacagcggcggcagcacctggtatgcttcctgggtgaagggccggttc edtavyycargystyyrd
accattagcagggacgactccaagtccattgcctacctgcagatgaactc fniwgqgtlvtvsssepk
cctgaagaccgaggacaccgccgtgtactactgcgccaggggctacagc ssdkthtcppcpapeaa acctattaccgggactttaacatctggggccagggcacactggtcaccgt gapsvflfppkpkdtlmi gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt srtpevtcvvvdvshed
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pevkfnwyvdgvevhn
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg aktkpreeqynstyrvvs gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vltvlhqdwlngkeykc
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca avsnkalpapiektiska
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg kgqprepqvytlppsrd
actggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccct eltknqvsltclvkgfyps
cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga diavewesngqpenny gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga kttppvldsdgsfflysklt accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc vdksrwqqgnvfscsv
gccgtggagtgggagagcaatgggcagccggagaacaactacaagacc mhealhnhytqkslsls
acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctca Pgsggggsggggsgggg
ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg spasqvqlvqsgpevkk
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc pgssvkvsckasgytfsr
tccgggttccggaggagggggttcaggtgggggaggttctggcggcggg stmhwvrqapgqgle
ggaagccctgcttcacaggtgcaactggtgcagagtggacccgaggttaa wigyinpssaytnynqk
aaaaccagggtcctccgttaaggttagctgcaaagcctctggctacacatt fkdrvtitadkststaym
ttccaggagtacaatgcactgggtgaggcaggctcctggacagggactcg elsslrsedtavyycarp
agtggatcgggtatatcaacccatctagcgcctataccaattacaaccaa qvhydyngfpywgqgt aagtttaaggaccgagttaccattaccgctgacaaatccaccagtacagc Ivtvssggggsggggsgg
ttatatggagctgtcatctcttaggtccgaggacactgctgtttattactgc ggsggggsdiqmtqsps gctcgtcctcaggttcactatgactataatggttttccctactggggtcagg tlsasvgdrvtmtcsass
gaaccctggtgactgtctcttctggcggtggaggcagcggtgggggtggg svsymnwyqqkpgka
tctggaggcggtggcagtggcggcggaggctctgatattcagatgactca pkrwiydssklasgvpsr gtctcctagcactctcagcgccagcgtgggggatcgtgtgacaatgacttg fsgsgsgtdytltisslqp
ctccgctagcagtagtgtgtcttacatgaattggtatcagcagaagcccgg ddfatyycqqwsrnppt gaaagcacctaagcgctggatctatgactcttccaagctggcaagtggtg fgggtkveikrs
tcccctcacggttctctggctcaggttctggtactgactatactttgactatc
tcctccctccagcccgatgatttcgctacctattattgtcagcagtggagcc
gtaacccacccactttcggaggcggtaccaaagtggagatcaagaggtc
atga Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR243 atggaggctcccgctcagctgctgttcctcctgctgctctggctgcccgaca diqmtqspsslsasvgd SEQ ID NO:312 ccacaggcgacatccagatgacccagtccccttcctccctgtccgctagcg rvtincqasqsidsnlaw (SEQ ID NO:313
Humanized
tgggcgatagggtgaccatcaactgccaggcctcccagtccattgactcc fqqkpgqppklliyrasn
Rll x CD3
aatctggcctggttccagcagaagcccggacagccccccaagctgctgat lasgvpdrfsgsgsgtdft
scFv-Fc- ttacagggcctccaacctggcttccggcgtgcctgacaggttctccggatc Itissleaedvatyyclgg
scFv
cggcagcggcaccgacttcaccctgaccatctcctccctggaggccgagg vgavsyrtsfgggtkveik
Codon atgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgga ggggsggggsggggsgg
optimized cctccttcggcggcggcaccaaggtggagatcaagggcggcggcggcag ggsevqlvesggglvqp
cggcggcggcggcagcggcggcggaggctccggcggcggcggcagcga grslrlsctasgsdindypi ggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtcc twvrqapgqglewigfi
ctgaggctgtcctgcacagccagcggctccgacatcaacgactaccccat nsggstwyaswvkgrft
cacctgggtgaggcaggctcctggccagggcctggaatggatcggcttta isrddsksiaylqmnslkt
tcaacagcggcggcagcacctggtatgcttcctgggtgaagggccggttc edtavyycargystyyrd
accattagcagggacgactccaagtccattgcctacctgcagatgaactc fniwgqgtlvtvsssepk
cctgaagaccgaggacaccgccgtgtactactgcgccaggggctacagc ssdkthtcppcpapeaa
acctattaccgggactttaacatctggggccagggcacactggtcaccgt gapsvflfppkpkdtlmi
gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt srtpevtcvvvdvshed
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pevkfnwyvdgvevhn
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg aktkpreeqynstyrvvs
gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vltvlhqdwlngkeykc
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca avsnkalpapiektiska
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg kgqprepqvytlppsrd
actggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccct eltknqvsltclvkgfyps
cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga diavewesngqpenny
gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga kttppvldsdgsfflysklt
accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc vdksrwqqgnvfscsv
gccgtggagtgggagagcaatgggcagccggagaacaactacaagacc mhealhnhytqkslsls
acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctca Pgsggggsggggsgggg
ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg spsqvqlvqsgpevkkp
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc gssvkvsckasgytfsrst
tccgggttccggaggagggggttcaggtgggggaggttctggcggcggg mhwvrqapgqglewi
ggaagcccttcacaggtgcaactggtgcagagtggacccgaggttaaaa gyinpssaytnynqkfk
aaccagggtcctccgttaaggttagctgcaaagcctctggctacacatttt drvtitadkststaymel
ccaggagtacaatgcactgggtgaggcaggctcctggacagggactcga sslrsedtavyycarpqv
gtggatcgggtatatcaacccatctagcgcctataccaattacaaccaaa hydyngfpywgqgtlvt
agtttaaggaccgagttaccattaccgctgacaaatccaccagtacagctt vssggggsggggsgggg
atatggagctgtcatctcttaggtccgaggacactgctgtttattactgcgc sggggsdiqmtqspstls
tcgtcctcaggttcactatgactataatggttttccctactggggtcaggga asvgdrvtmtcsasssvs
accctggtgactgtctcttctggcggtggaggcagcggtgggggtgggtct ymnwyqqkpgkapkr
ggaggcggtggcagtggcggcggaggctctgatattcagatgactcagtc wiydssklasgvpsrfsg
tcctagcactctcagcgccagcgtgggggatcgtgtgacaatgacttgctc sgsgtdytltisslqpddf
cgctagcagtagtgtgtcttacatgaattggtatcagcagaagcccggga atyycqqwsrnpptfgg
aagcacctaagcgctggatctatgactcttccaagctggcaagtggtgtcc gtkveikrs
cctcacggttctctggctcaggttctggtactgactatactttgactatctcc
tccctccagcccgatgatttcgctacctattattgtcagcagtggagccgta
acccacccactttcggaggcggtaccaaagtggagatcaagaggtcatg
a Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR244 atggaggctcccgctcagctgctgttcctcctgctgctctggctgcccgaca diqmtqspsslsasvgd SEQ ID NO: 314 ccacaggcgacatccagatgacccagtccccttcctccctgtccgctagcg rvtincqasqsidsnlaw (SEQ ID
Humanized
tgggcgatagggtgaccatcaactgccaggcctcccagtccattgactcc fqqkpgqppklliyrasn NO:315) Rll x CD3
aatctggcctggttccagcagaagcccggacagccccccaagctgctgat lasgvpdrfsgsgsgtdft
scFv-Fc- ttacagggcctccaacctggcttccggcgtgcctgacaggttctccggatc Itissleaedvatyyclgg
scFv
cggcagcggcaccgacttcaccctgaccatctcctccctggaggccgagg vgavsyrtsfgggtkveik
Codon atgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgga ggggsggggsggggsgg
optimized cctccttcggcggcggcaccaaggtggagatcaagggcggcggcggcag ggsevqlvesggglvqp
cggcggcggcggcagcggcggcggaggctccggcggcggcggcagcga grslrlsctasgsdindypi ggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtcc twvrqapgqglewigfi
ctgaggctgtcctgcacagccagcggctccgacatcaacgactaccccat nsggstwyaswvkgrft
cacctgggtgaggcaggctcctggccagggcctggaatggatcggcttta isrddsksiaylqmnslkt tcaacagcggcggcagcacctggtatgcttcctgggtgaagggccggttc edtavyycargystyyrd
accattagcagggacgactccaagtccattgcctacctgcagatgaactc fniwgqgtlvtvsssepk
cctgaagaccgaggacaccgccgtgtactactgcgccaggggctacagc ssdkthtcppcpapeaa
acctattaccgggactttaacatctggggccagggcacactggtcaccgt gapsvflfppkpkdtlmi gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt srtpevtcvvvdvshed
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pevkfnwyvdgvevhn
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg aktkpreeqynstyrvvs gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vltvlhqdwlngkeykc
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca avsnkalpapiektiska
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg kgqprepqvytlppsrd
actggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccct eltknqvsltclvkgfyps
cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga diavewesngqpenny
gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga kttppvldsdgsfflysklt
accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc vdksrwqqgnvfscsv
gccgtggagtgggagagcaatgggcagccggagaacaactacaagacc mhealhnhytqkslsls
acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctca Pgsggggsggggsgggg
ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg spssqvqlvqsgpevkk
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc pgssvkvsckasgytfsr
tccgggttccggaggagggggttcaggtgggggaggttctggcggcggg stmhwvrqapgqgle
ggaagccctagttcacaggtgcaactggtgcagagtggacccgaggtta wigyinpssaytnynqk
aaaaaccagggtcctccgttaaggttagctgcaaagcctctggctacaca fkdrvtitadkststaym
ttttccaggagtacaatgcactgggtgaggcaggctcctggacagggact elsslrsedtavyycarp
cgagtggatcgggtatatcaacccatctagcgcctataccaattacaacc qvhydyngfpywgqgt
aaaagtttaaggaccgagttaccattaccgctgacaaatccaccagtaca Ivtvssggggsggggsgg
gcttatatggagctgtcatctcttaggtccgaggacactgctgtttattact ggsggggsdiqmtqsps
gcgctcgtcctcaggttcactatgactataatggttttccctactggggtca tlsasvgdrvtmtcsass
gggaaccctggtgactgtctcttctggcggtggaggcagcggtgggggtg svsymnwyqqkpgka
ggtctggaggcggtggcagtggcggcggaggctctgatattcagatgact pkrwiydssklasgvpsr
cagtctcctagcactctcagcgccagcgtgggggatcgtgtgacaatgac fsgsgsgtdytltisslqp
ttgctccgctagcagtagtgtgtcttacatgaattggtatcagcagaagcc ddfatyycqqwsrnppt cgggaaagcacctaagcgctggatctatgactcttccaagctggcaagtg fgggtkveikrs
gtgtcccctcacggttctctggctcaggttctggtactgactatactttgact
atctcctccctccagcccgatgatttcgctacctattattgtcagcagtgga
gccgtaacccacccactttcggaggcggtaccaaagtggagatcaagag
gtcatga Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR246 atggaagcaccagcgcagctgctgttcctcctgctgctctggctgcccgac diqmtqspsslsasvgd SEQ ID NO:316 accacaggcgacatccagatgacccagtccccttcctccctgtccgctagc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgatagggtgaccatcaactgccaggcctcccagtccattgactc fqqkpgqppklliyrasn NO:317) Rll x CD3
caatctggcctggttccagcagaagcccggacagccccccaagctgctga lasgvpdrfsgsgsgtdft scFv-Fc- tttacagggcctccaacctggcttccggcgtgcctgacaggttctccggat Itissleaedvatyyclgg
scFv
ccggcagcggcaccgacttcaccctgaccatctcctccctggaggccgag vgavsyrtsfgggtkveik
Codon gatgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgg ggggsggggsggggsgg
optimized acctccttcggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtcc twvrqapgqglewigfi
ctgaggctgtcctgcacagccagcggctccgacatcaacgactaccccat nsggstwyaswvkgrft cacctgggtgaggcaggctcctggccagggcctggaatggatcggcttta isrddsksiaylqmnslkt tcaacagcggcggcagcacctggtatgcttcctgggtgaagggccggttc edtavyycargystyyrd
accattagcagggacgactccaagtccattgcctacctgcagatgaactc fniwgqgtlvtvsssepk
cctgaagaccgaggacaccgccgtgtactactgcgccaggggctacagc ssdkthtcppcpapeaa acctattaccgggactttaacatctggggccagggcacactggtcaccgt gapsvflfppkpkdtlmi gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt srtpevtcvvvdvshed
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pevkfnwyvdgvevhn
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg aktkpreeqynstyrvvs gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vltvlhqdwlngkeykc
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca avsnkalpapiektiska
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg kgqprepqvytlppsrd
actggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccct eltknqvsltclvkgfyps
cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga diavewesngqpenny gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga kttppvldsdgsfflysklt accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc vdksrwqqgnvfscsv
gccgtggagtgggagagcaatgggcagccggagaacaactacaagacc mhealhnhytqkslsls
acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctca Pgsggggsggggsgggg
ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg sqvqlvqsgpevkkpgs atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc svkvsckasgytfsrstm
tccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtggg hwvrqapgqglewigyi ggctctcaggtgcagctggtgcagtctggtcctgaggtgaaaaagcctgg npssaytnynqkfkdrv
ctccagcgtgaaggtgtcctgcaaggccagcggatacacctttagccggt titadkststaymelsslr
ccaccatgcattgggtgaggcaggctcctggacagggcctggagtggatc sedtavyycarpqvhyd
ggctacatcaaccccagcagcgcttataccaactacaatcagaagtttaa yngfpywgqgtlvtvss
ggaccgggtgaccatcaccgccgataagtccaccagcaccgcctacatg ggggsggggsggggsgg
gagctgtccagcctgaggagcgaggataccgccgtgtactattgcgcccg ggsdiqmtqspstlsasv gccccaggtccattacgactacaacggcttcccctattggggccagggaa gdrvtmtcsasssvsym
ccctggtgaccgtgtccagcggtggcggtggcagcggcggcggcggctct nwyqqkpgkapkrwiy ggcggaggtggcagcggcggagggggctccgacattcagatgacccagt dssklasgvpsrfsgsgs
ccccctccaccctgtccgctagcgtgggcgatcgggtgaccatgacctgca gtdytltisslqpddfaty
gcgccagcagctccgtgtcctacatgaactggtaccagcagaagcccggc ycqqwsrnpptfgggtk aaggctcccaagaggtggatttacgactccagcaagctggcctctggtgt veikrs
ccccagcaggttctctggtagcggcagcggcacagactacaccctgacca
tctcctccctgcagcccgacgatttcgccacctactattgccagcagtggtc
ccggaatccccctacctttggcggcggcaccaaggtggagatcaagagg
agctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR247 atggaagcaccagcgcagctgctgttcctcctgctgctctggctgcccgac diqmtqspsslsasvgd SEQ ID NO:318 accacaggcgacatccagatgacccagtccccttcctccctgtccgctagc rvtincqasqsidsnlaw (SEQ ID NO:
Humanized
gtgggcgatagggtgaccatcaactgccaggcctcccagtccattgactc fqqkpgqppklliyrasn 319) Rll x CD3
caatctggcctggttccagcagaagcccggacagccccccaagctgctga lasgvpdrfsgsgsgtdft scFv-Fc- tttacagggcctccaacctggcttccggcgtgcctgacaggttctccggat Itissleaedvatyyclgg
scFv
ccggcagcggcaccgacttcaccctgaccatctcctccctggaggccgag vgavsyrtsfgggtkveik
Codon gatgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgg ggggsggggsggggsgg
optimized acctccttcggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtcc twvrqapgqglewigfi
ctgaggctgtcctgcacagccagcggctccgacatcaacgactaccccat nsggstwyaswvkgrft cacctgggtgaggcaggctcctggccagggcctggaatggatcggcttta isrddsksiaylqmnslkt tcaacagcggcggcagcacctggtatgcttcctgggtgaagggccggttc edtavyycargystyyrd
accattagcagggacgactccaagtccattgcctacctgcagatgaactc fniwgqgtlvtvsssepk
cctgaagaccgaggacaccgccgtgtactactgcgccaggggctacagc ssdkthtcppcpapeaa acctattaccgggactttaacatctggggccagggcacactggtcaccgt gapsvflfppkpkdtlmi gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt srtpevtcvvvdvshed
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pevkfnwyvdgvevhn
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg aktkpreeqynstyrvvs gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vltvlhqdwlngkeykc
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca avsnkalpapiektiska
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg kgqprepqvytlppsrd
actggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccct eltknqvsltclvkgfyps
cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga diavewesngqpenny gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga kttppvldsdgsfflysklt accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc vdksrwqqgnvfscsv
gccgtggagtgggagagcaatgggcagccggagaacaactacaagacc mhealhnhytqkslsls
acgccccccgtgctggactccgacggctccttcttcctctacagcaagctc Pgsggggsggggsgggg
accgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgt spasqvqlvqsgpevkk
gatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgt pgssvkvsckasgytfsr
ctccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtgg stmhwvrqapgqgle
gggctctcctgcttcacaggtgcagctggtgcagtctggtcctgaggtgaa wigyinpssaytnynqk
aaagcctggctccagcgtgaaggtgtcctgcaaggccagcggatacacct fkdrvtitadkststaym
ttagccggtccaccatgcattgggtgaggcaggctcctggacagggcctg elsslrsedtavyycarp
gagtggatcggctacatcaaccccagcagcgcttataccaactacaatca qvhydyngfpywgqgt gaagtttaaggaccgggtgaccatcaccgccgataagtccaccagcacc Ivtvssggggsggggsgg
gcctacatggagctgtccagcctgaggagcgaggataccgccgtgtacta ggsggggsdiqmtqsps ttgcgcccggccccaggtccattacgactacaacggcttcccctattgggg tlsasvgdrvtmtcsass
ccagggaaccctggtgaccgtgtccagcggtggcggtggcagcggcggc svsymnwyqqkpgka
ggcggctctggcggaggtggcagcggcggagggggctccgacattcaga pkrwiydssklasgvpsr tgacccagtccccctccaccctgtccgctagcgtgggcgatcgggtgacca fsgsgsgtdytltisslqp
tgacctgcagcgccagcagctccgtgtcctacatgaactggtaccagcag ddfatyycqqwsrnppt aagcccggcaaggctcccaagaggtggatttacgactccagcaagctgg fgggtkveikrs
cctctggtgtccccagcaggttctctggtagcggcagcggcacagactac
accctgaccatctcctccctgcagcccgacgatttcgccacctactattgcc
agcagtggtcccggaatccccctacctttggcggcggcaccaaggtggag
atcaaaaggagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR248 atggaagcaccagcgcagctgctgttcctcctgctgctctggctgcccgac diqmtqspsslsasvgd SEQ ID NO:320 accacaggcgacatccagatgacccagtccccttcctccctgtccgctagc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgatagggtgaccatcaactgccaggcctcccagtccattgactc fqqkpgqppklliyrasn NO:321) Rll x CD3
caatctggcctggttccagcagaagcccggacagccccccaagctgctga lasgvpdrfsgsgsgtdft scFv-Fc- tttacagggcctccaacctggcttccggcgtgcctgacaggttctccggat Itissleaedvatyyclgg
scFv
ccggcagcggcaccgacttcaccctgaccatctcctccctggaggccgag vgavsyrtsfgggtkveik
Codon gatgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgg ggggsggggsggggsgg
optimized acctccttcggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtcc twvrqapgqglewigfi
ctgaggctgtcctgcacagccagcggctccgacatcaacgactaccccat nsggstwyaswvkgrft cacctgggtgaggcaggctcctggccagggcctggaatggatcggcttta isrddsksiaylqmnslkt tcaacagcggcggcagcacctggtatgcttcctgggtgaagggccggttc edtavyycargystyyrd
accattagcagggacgactccaagtccattgcctacctgcagatgaactc fniwgqgtlvtvsssepk
cctgaagaccgaggacaccgccgtgtactactgcgccaggggctacagc ssdkthtcppcpapeaa acctattaccgggactttaacatctggggccagggcacactggtcaccgt gapsvflfppkpkdtlmi gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt srtpevtcvvvdvshed
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pevkfnwyvdgvevhn
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg aktkpreeqynstyrvvs gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vltvlhqdwlngkeykc
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca avsnkalpapiektiska
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg kgqprepqvytlppsrd
actggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccct eltknqvsltclvkgfyps
cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga diavewesngqpenny gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga kttppvldsdgsfflysklt accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc vdksrwqqgnvfscsv
gccgtggagtgggagagcaatgggcagccggagaacaactacaagacc mhealhnhytqkslsls
acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctca Pgsggggsggggsgggg
ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg spsqvqlvqsgpevkkp
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc gssvkvsckasgytfsrst tccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtggg mhwvrqapgqglewi
ggctctccttcacaggtgcagctggtgcagtctggtcctgaggtgaaaaa gyinpssaytnynqkfk
gcctggctccagcgtgaaggtgtcctgcaaggccagcggatacaccttta drvtitadkststaymel gccggtccaccatgcattgggtgaggcaggctcctggacagggcctggag sslrsedtavyycarpqv
tggatcggctacatcaaccccagcagcgcttataccaactacaatcagaa hydyngfpywgqgtlvt gtttaaggaccgggtgaccatcaccgccgataagtccaccagcaccgcct vssggggsggggsgggg
acatggagctgtccagcctgaggagcgaggataccgccgtgtactattgc sggggsdiqmtqspstls gcccggccccaggtccattacgactacaacggcttcccctattggggcca asvgdrvtmtcsasssvs gggaaccctggtgaccgtgtccagcggtggcggtggcagcggcggcggc ymnwyqqkpgkapkr ggctctggcggaggtggcagcggcggagggggctccgacattcagatga wiydssklasgvpsrfsg
cccagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccatga sgsgtdytltisslqpddf
cctgcagcgccagcagctccgtgtcctacatgaactggtaccagcagaag atyycqqwsrnpptfgg
cccggcaaggctcccaagaggtggatttacgactccagcaagctggcctc gtkveikrs
tggtgtccccagcaggttctctggtagcggcagcggcacagactacaccc
tgaccatctcctccctgcagcccgacgatttcgccacctactattgccagc
agtggtcccggaatccccctacctttggcggcggcaccaaggtggagatc
aagaggagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR249 atggaagcaccagcgcagctgctgttcctcctgctgctctggctgcccgac diqmtqspsslsasvgd SEQ ID NO:322 accacaggcgacatccagatgacccagtccccttcctccctgtccgctagc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtgggcgatagggtgaccatcaactgccaggcctcccagtccattgactc fqqkpgqppklliyrasn NO:323) Rll x CD3
caatctggcctggttccagcagaagcccggacagccccccaagctgctga lasgvpdrfsgsgsgtdft scFv-Fc- tttacagggcctccaacctggcttccggcgtgcctgacaggttctccggat Itissleaedvatyyclgg
scFv
ccggcagcggcaccgacttcaccctgaccatctcctccctggaggccgag vgavsyrtsfgggtkveik
Codon gatgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgg ggggsggggsggggsgg
optimized acctccttcggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaaagcggaggaggcctggtgcagcctggaaggtcc twvrqapgqglewigfi
ctgaggctgtcctgcacagccagcggctccgacatcaacgactaccccat nsggstwyaswvkgrft cacctgggtgaggcaggctcctggccagggcctggaatggatcggcttta isrddsksiaylqmnslkt tcaacagcggcggcagcacctggtatgcttcctgggtgaagggccggttc edtavyycargystyyrd
accattagcagggacgactccaagtccattgcctacctgcagatgaactc fniwgqgtlvtvsssepk
cctgaagaccgaggacaccgccgtgtactactgcgccaggggctacagc ssdkthtcppcpapeaa acctattaccgggactttaacatctggggccagggcacactggtcaccgt gapsvflfppkpkdtlmi gtcctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgt srtpevtcvvvdvshed
gcccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaa pevkfnwyvdgvevhn
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg aktkpreeqynstyrvvs gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt vltvlhqdwlngkeykc
ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca avsnkalpapiektiska
gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg kgqprepqvytlppsrd
actggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccct eltknqvsltclvkgfyps
cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga diavewesngqpenny gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga kttppvldsdgsfflysklt accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc vdksrwqqgnvfscsv
gccgtggagtgggagagcaatgggcagccggagaacaactacaagacc mhealhnhytqkslsls
acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctca Pgsggggsggggsgggg
ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg spssqvqlvqsgpevkk
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc pgssvkvsckasgytfsr
tccgggttccggaggtggcggttcgggaggtggcgggtcaggaggtggg stmhwvrqapgqgle
ggctctcctagttcacaggtgcagctggtgcagtctggtcctgaggtgaaa wigyinpssaytnynqk
aagcctggctccagcgtgaaggtgtcctgcaaggccagcggatacacctt fkdrvtitadkststaym
tagccggtccaccatgcattgggtgaggcaggctcctggacagggcctgg elsslrsedtavyycarp
agtggatcggctacatcaaccccagcagcgcttataccaactacaatcag qvhydyngfpywgqgt aagtttaaggaccgggtgaccatcaccgccgataagtccaccagcaccg Ivtvssggggsggggsgg
cctacatggagctgtccagcctgaggagcgaggataccgccgtgtactat ggsggggsdiqmtqsps tgcgcccggccccaggtccattacgactacaacggcttcccctattggggc tlsasvgdrvtmtcsass
cagggaaccctggtgaccgtgtccagcggtggcggtggcagcggcggcg svsymnwyqqkpgka
gcggctctggcggaggtggcagcggcggagggggctccgacattcagat pkrwiydssklasgvpsr gacccagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccat fsgsgsgtdytltisslqp
gacctgcagcgccagcagctccgtgtcctacatgaactggtaccagcaga ddfatyycqqwsrnppt agcccggcaaggctcccaagaggtggatttacgactccagcaagctggc fgggtkveikrs
ctctggtgtccccagcaggttctctggtagcggcagcggcacagactaca
ccctgaccatctcctccctgcagcccgacgatttcgccacctactattgcca
gcagtggtcccggaatccccctacctttggcggcggcaccaaggtggaga
tcaagaggagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR250 atggaggctcccgctcagctcctgttcctgctgctgctgtggctgcctgaca diqmtqspsslsasvgd SEQ ID NO:324 ccaccggcgacatccagatgacccagagcccctccagcctgagcgcttcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtcggagacagggtgaccatcaactgccaggcctcccagagcatcgact fqqkpgkppklliyrasnl NO:325) Rll x CD3
ccaatctggcctggttccagcagaagcctggcaagccccccaaactgctg asgvpsrfsgsgsgtdftl scFv-Fc- atctatagggcctccaacctggcctctggagtgccttccaggtttagcggc tisslqpedvatyyclggv scFv
tccggctccggcacagacttcaccctgaccatctcctccctccaacctgag gavsyrtsfgggtkveik
Codon gacgtcgccacctactattgcctgggtggcgtgggcgctgtgtcctatagg ggggsggggsggggsgg
optimized acctcctttggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaatccggtggtggcctggtgcagcctggaaggtccc twvrqapgkglewigfi
tgaggctgtcctgtaccgccagcggcagcgacatcaacgactaccctatc nsggstwyaswvkgrft
acctgggtgaggcaggctcccggcaaaggcctggagtggatcggctttat isrddsksiaylqmnslkt caactccggcggaagcacctggtacgccagctgggtgaagggccggttc edtavyycargystyyrd accatctcccgggatgactccaagagcatcgcctacctgcagatgaacag fniwgqgtlvtvsssepk
cctgaagacagaggacaccgccgtgtactactgcgccaggggctattcca ssdkthtcppcpapeaa cctactaccgggacttcaacatctggggccagggcaccctggtcaccgtgt gapsvflfppkpkdtlmi cctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgc srtpevtcvvvdvshed
ccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaa pevkfnwyvdgvevhn
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt aktkpreeqynstyrvvs ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg vltvlhqdwlngkeykc
gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag avsnkalpapiektiska
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagga kgqprepqvytlppsrd
ctggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcc eltknqvsltclvkgfyps
cagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag diavewesngqpenny aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa kttppvldsdgsfflysklt ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg vdksrwqqgnvfscsv
ccgtggagtgggagagcaatgggcagccggagaacaactacaagacca mhealhnhytqkslsls
cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac Pgsggggsggggsgggg
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga sqvqlvqsgpevkkpgs tgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc svkvsckasgytfsrstm
cgggttccggaggtggcggttcgggaggtggcgggtcaggaggtggggg hwvrqapgqglewigyi ctctcaggtgcagctggtgcagtctggtcctgaggtgaaaaagcctggctc npssaytnynqkfkdrv
cagcgtgaaggtgtcctgcaaggccagcggatacacctttagccggtcca titadkststaymelsslr
ccatgcattgggtgaggcaggctcctggacagggcctggagtggatcggc sedtavyycarpqvhyd
tacatcaaccccagcagcgcttataccaactacaatcagaagtttaagga yngfpywgqgtlvtvss
ccgggtgaccatcaccgccgataagtccaccagcaccgcctacatggagc ggggsggggsggggsgg
tgtccagcctgaggagcgaggataccgccgtgtactattgcgcccggccc ggsdiqmtqspstlsasv caggtccattacgactacaacggcttcccctattggggccagggaaccct gdrvtmtcsasssvsym
ggtgaccgtgtccagcggtggcggtggcagcggcggcggcggctctggc nwyqqkpgkapkrwiy ggaggtggcagcggcggagggggctccgacattcagatgacccagtccc dssklasgvpsrfsgsgs
cctccaccctgtccgctagcgtgggcgatcgggtgaccatgacctgcagc gtdytltisslqpddfaty
gccagcagctccgtgtcctacatgaactggtaccagcagaagcccggca ycqqwsrnpptfgggtk aggctcccaagaggtggatttacgactccagcaagctggcctctggtgtc veikrs
cccagcaggttctctggtagcggcagcggcacagactacaccctgaccat
ctcctccctgcagcccgacgatttcgccacctactattgccagcagtggtcc
cggaatccccctacctttggcggcggcaccaaggtggagatcaagagga
gctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR251 atggaggctcccgctcagctcctgttcctgctgctgctgtggctgcctgaca diqmtqspsslsasvgd SEQ ID NO:326 ccaccggcgacatccagatgacccagagcccctccagcctgagcgcttcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtcggagacagggtgaccatcaactgccaggcctcccagagcatcgact fqqkpgkppklliyrasnl NO:327) Rll x CD3
ccaatctggcctggttccagcagaagcctggcaagccccccaaactgctg asgvpsrfsgsgsgtdftl scFv-Fc- atctatagggcctccaacctggcctctggagtgccttccaggtttagcggc tisslqpedvatyyclggv scFv
tccggctccggcacagacttcaccctgaccatctcctccctccaacctgag gavsyrtsfgggtkveik
Codon gacgtcgccacctactattgcctgggtggcgtgggcgctgtgtcctatagg ggggsggggsggggsgg
optimized acctcctttggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaatccggtggtggcctggtgcagcctggaaggtccc twvrqapgkglewigfi
tgaggctgtcctgtaccgccagcggcagcgacatcaacgactaccctatc nsggstwyaswvkgrft
acctgggtgaggcaggctcccggcaaaggcctggagtggatcggctttat isrddsksiaylqmnslkt caactccggcggaagcacctggtacgccagctgggtgaagggccggttc edtavyycargystyyrd accatctcccgggatgactccaagagcatcgcctacctgcagatgaacag fniwgqgtlvtvsssepk
cctgaagacagaggacaccgccgtgtactactgcgccaggggctattcca ssdkthtcppcpapeaa cctactaccgggacttcaacatctggggccagggcaccctggtcaccgtgt gapsvflfppkpkdtlmi cctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgc srtpevtcvvvdvshed
ccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaa pevkfnwyvdgvevhn
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt aktkpreeqynstyrvvs ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg vltvlhqdwlngkeykc
gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag avsnkalpapiektiska
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagga kgqprepqvytlppsrd
ctggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcc eltknqvsltclvkgfyps
cagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag diavewesngqpenny aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa kttppvldsdgsfflysklt ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg vdksrwqqgnvfscsv
ccgtggagtgggagagcaatgggcagccggagaacaactacaagacca mhealhnhytqkslsls
cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac Pgsggggsggggsgggg
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga spasqvqlvqsgpevkk
tgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc pgssvkvsckasgytfsr
cgggttccggaggtggcggttcgggaggtggcgggtcaggaggtggggg stmhwvrqapgqgle
ctctcctgcttcacaggtgcagctggtgcagtctggtcctgaggtgaaaaa wigyinpssaytnynqk
gcctggctccagcgtgaaggtgtcctgcaaggccagcggatacaccttta fkdrvtitadkststaym
gccggtccaccatgcattgggtgaggcaggctcctggacagggcctggag elsslrsedtavyycarp
tggatcggctacatcaaccccagcagcgcttataccaactacaatcagaa qvhydyngfpywgqgt gtttaaggaccgggtgaccatcaccgccgataagtccaccagcaccgcct Ivtvssggggsggggsgg
acatggagctgtccagcctgaggagcgaggataccgccgtgtactattgc ggsggggsdiqmtqsps gcccggccccaggtccattacgactacaacggcttcccctattggggcca tlsasvgdrvtmtcsass
gggaaccctggtgaccgtgtccagcggtggcggtggcagcggcggcggc svsymnwyqqkpgka
ggctctggcggaggtggcagcggcggagggggctccgacattcagatga pkrwiydssklasgvpsr cccagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccatga fsgsgsgtdytltisslqp
cctgcagcgccagcagctccgtgtcctacatgaactggtaccagcagaag ddfatyycqqwsrnppt cccggcaaggctcccaagaggtggatttacgactccagcaagctggcctc fgggtkveikrs
tggtgtccccagcaggttctctggtagcggcagcggcacagactacaccc
tgaccatctcctccctgcagcccgacgatttcgccacctactattgccagc
agtggtcccggaatccccctacctttggcggcggcaccaaggtggagatc
aagaggagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR252 atggaggctcccgctcagctcctgttcctgctgctgctgtggctgcctgaca diqmtqspsslsasvgd SEQ ID NO:328 ccaccggcgacatccagatgacccagagcccctccagcctgagcgcttcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtcggagacagggtgaccatcaactgccaggcctcccagagcatcgact fqqkpgkppklliyrasnl NO:329) Rll x CD3
ccaatctggcctggttccagcagaagcctggcaagccccccaaactgctg asgvpsrfsgsgsgtdftl scFv-Fc- atctatagggcctccaacctggcctctggagtgccttccaggtttagcggc tisslqpedvatyyclggv scFv
tccggctccggcacagacttcaccctgaccatctcctccctccaacctgag gavsyrtsfgggtkveik
Codon gacgtcgccacctactattgcctgggtggcgtgggcgctgtgtcctatagg ggggsggggsggggsgg
optimized acctcctttggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaatccggtggtggcctggtgcagcctggaaggtccc twvrqapgkglewigfi
tgaggctgtcctgtaccgccagcggcagcgacatcaacgactaccctatc nsggstwyaswvkgrft
acctgggtgaggcaggctcccggcaaaggcctggagtggatcggctttat isrddsksiaylqmnslkt caactccggcggaagcacctggtacgccagctgggtgaagggccggttc edtavyycargystyyrd accatctcccgggatgactccaagagcatcgcctacctgcagatgaacag fniwgqgtlvtvsssepk
cctgaagacagaggacaccgccgtgtactactgcgccaggggctattcca ssdkthtcppcpapeaa cctactaccgggacttcaacatctggggccagggcaccctggtcaccgtgt gapsvflfppkpkdtlmi cctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgc srtpevtcvvvdvshed
ccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaa pevkfnwyvdgvevhn
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt aktkpreeqynstyrvvs ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg vltvlhqdwlngkeykc
gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag avsnkalpapiektiska
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagga kgqprepqvytlppsrd
ctggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcc eltknqvsltclvkgfyps
cagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag diavewesngqpenny aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa kttppvldsdgsfflysklt ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg vdksrwqqgnvfscsv
ccgtggagtgggagagcaatgggcagccggagaacaactacaagacca mhealhnhytqkslsls
cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac Pgsggggsggggsgggg
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga spsqvqlvqsgpevkkp
tgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc gssvkvsckasgytfsrst cgggttccggaggtggcggttcgggaggtggcgggtcaggaggtggggg mhwvrqapgqglewi
ctctccttcacaggtgcagctggtgcagtctggtcctgaggtgaaaaagcc gyinpssaytnynqkfk
tggctccagcgtgaaggtgtcctgcaaggccagcggatacacctttagcc drvtitadkststaymel ggtccaccatgcattgggtgaggcaggctcctggacagggcctggagtgg sslrsedtavyycarpqv
atcggctacatcaaccccagcagcgcttataccaactacaatcagaagtt hydyngfpywgqgtlvt taaggaccgggtgaccatcaccgccgataagtccaccagcaccgcctac vssggggsggggsgggg
atggagctgtccagcctgaggagcgaggataccgccgtgtactattgcgc sggggsdiqmtqspstls ccggccccaggtccattacgactacaacggcttcccctattggggccagg asvgdrvtmtcsasssvs gaaccctggtgaccgtgtccagcggtggcggtggcagcggcggcggcgg ymnwyqqkpgkapkr ctctggcggaggtggcagcggcggagggggctccgacattcagatgacc wiydssklasgvpsrfsg
cagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccatgacc sgsgtdytltisslqpddf
tgcagcgccagcagctccgtgtcctacatgaactggtaccagcagaagcc atyycqqwsrnpptfgg
cggcaaggctcccaagaggtggatttacgactccagcaagctggcctctg gtkveikrs
gtgtccccagcaggttctctggtagcggcagcggcacagactacaccctg
accatctcctccctgcagcccgacgatttcgccacctactattgccagcag
tggtcccggaatccccctacctttggcggcggcaccaaggtggagatcaa
gaggagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
ROR253 atggaggctcccgctcagctcctgttcctgctgctgctgtggctgcctgaca diqmtqspsslsasvgd SEQ ID NO:330 ccaccggcgacatccagatgacccagagcccctccagcctgagcgcttcc rvtincqasqsidsnlaw (SEQ ID
Humanized
gtcggagacagggtgaccatcaactgccaggcctcccagagcatcgact fqqkpgkppklliyrasnl NO:331) Rll x CD3
ccaatctggcctggttccagcagaagcctggcaagccccccaaactgctg asgvpsrfsgsgsgtdftl scFv-Fc- atctatagggcctccaacctggcctctggagtgccttccaggtttagcggc tisslqpedvatyyclggv scFv
tccggctccggcacagacttcaccctgaccatctcctccctccaacctgag gavsyrtsfgggtkveik
Codon gacgtcgccacctactattgcctgggtggcgtgggcgctgtgtcctatagg ggggsggggsggggsgg
optimized acctcctttggcggcggcaccaaggtggagatcaagggcggtggcggat ggsevqlvesggglvqp
cgggtggcggtggaagcggtggaggtggctccggcggtggtggatcaga grslrlsctasgsdindypi ggtgcagctggtggaatccggtggtggcctggtgcagcctggaaggtccc twvrqapgkglewigfi
tgaggctgtcctgtaccgccagcggcagcgacatcaacgactaccctatc nsggstwyaswvkgrft
acctgggtgaggcaggctcccggcaaaggcctggagtggatcggctttat isrddsksiaylqmnslkt caactccggcggaagcacctggtacgccagctgggtgaagggccggttc edtavyycargystyyrd accatctcccgggatgactccaagagcatcgcctacctgcagatgaacag fniwgqgtlvtvsssepk
cctgaagacagaggacaccgccgtgtactactgcgccaggggctattcca ssdkthtcppcpapeaa cctactaccgggacttcaacatctggggccagggcaccctggtcaccgtgt gapsvflfppkpkdtlmi cctcgagtgagcccaaatcttctgacaaaactcacacatgcccaccgtgc srtpevtcvvvdvshed
ccagcacctgaagccgcgggtgcaccgtcagtcttcctcttccccccaaaa pevkfnwyvdgvevhn
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt aktkpreeqynstyrvvs ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg vltvlhqdwlngkeykc
gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag avsnkalpapiektiska
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagga kgqprepqvytlppsrd
ctggctgaatggcaaggaatacaagtgcgcggtctccaacaaagccctcc eltknqvsltclvkgfyps
cagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag diavewesngqpenny aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa kttppvldsdgsfflysklt ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcg vdksrwqqgnvfscsv
ccgtggagtgggagagcaatgggcagccggagaacaactacaagacca mhealhnhytqkslsls
cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcac Pgsggggsggggsgggg
cgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga spssqvqlvqsgpevkk
tgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc pgssvkvsckasgytfsr
cgggttccggaggtggcggttcgggaggtggcgggtcaggaggtggggg stmhwvrqapgqgle
ctctcctagttcacaggtgcagctggtgcagtctggtcctgaggtgaaaaa wigyinpssaytnynqk
gcctggctccagcgtgaaggtgtcctgcaaggccagcggatacaccttta fkdrvtitadkststaym
gccggtccaccatgcattgggtgaggcaggctcctggacagggcctggag elsslrsedtavyycarp
tggatcggctacatcaaccccagcagcgcttataccaactacaatcagaa qvhydyngfpywgqgt gtttaaggaccgggtgaccatcaccgccgataagtccaccagcaccgcct Ivtvssggggsggggsgg
acatggagctgtccagcctgaggagcgaggataccgccgtgtactattgc ggsggggsdiqmtqsps gcccggccccaggtccattacgactacaacggcttcccctattggggcca tlsasvgdrvtmtcsass
gggaaccctggtgaccgtgtccagcggtggcggtggcagcggcggcggc svsymnwyqqkpgka
ggctctggcggaggtggcagcggcggagggggctccgacattcagatga pkrwiydssklasgvpsr cccagtccccctccaccctgtccgctagcgtgggcgatcgggtgaccatga fsgsgsgtdytltisslqp
cctgcagcgccagcagctccgtgtcctacatgaactggtaccagcagaag ddfatyycqqwsrnppt cccggcaaggctcccaagaggtggatttacgactccagcaagctggcctc fgggtkveikrs
tggtgtccccagcaggttctctggtagcggcagcggcacagactacaccc
tgaccatctcctccctgcagcccgacgatttcgccacctactattgccagc
agtggtcccggaatccccctacctttggcggcggcaccaaggtggagatc
aagaggagctaa Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtca diqmtqspsslsasvgd SEQ ID NO:332 light chain cgaattcagacatccagatgacccagagcccctccagcctgagcgcttcc rvtincqasqsidsnlaw (SEQ ID
gtcggagacagggtgaccatcaactgccaggcctcccagagcatcgact fqqkpgkppklliyrasnl NO:333)
HuRll mAb
ccaatctggcctggttccagcagaagcctggcaagccccccaaactgctg asgvpsrfsgsgsgtdftl atctatagggcctccaacctggcctctggagtgccttccaggtttagcggc tisslqpedvatyyclggv tccggctccggcacagacttcaccctgaccatctcctccctccaacctgag gavsyrtsfgggtkveikr gacgtcgccacctactattgcctgggtggcgtgggcgctgtgtcctatagg tvaapsvfifppsdeqlk
acctcctttggcggcggcaccaaggtggagatcaagcgtacggtggctgc sgtasvvcllnnfyprea
accatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaact kvqwkvdnalqsgnsq
gcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagta esvteqdskdstyslsstl cagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgt tlskadyekhkvyacevt cacagagcaggacagcaaggacagcacctacagcctcagcagcaccct hqglsspvtksfnrgec
gacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcga
agtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg
ggagagtgttag
Humanized atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtca evqlvesggglvqpgrsl SEQ ID NO:334 heavy chain cgaattcggaggtgcagctggtggaatccggtggtggcctggtgcagcct rlsctasgsdindypitw (SEQ ID
ggaaggtccctgaggctgtcctgtaccgccagcggcagcgacatcaacg vrqapgkglewigfinsg NO:335)
HuRll mAb
actaccctatcacctgggtgaggcaggctcccggcaaaggcctggagtgg gstwyaswvkgrftisrd
atcggctttatcaactccggcggaagcacctggtacgccagctgggtgaa dsksiaylqmnslktedt gggccggttcaccatctcccgggatgactccaagagcatcgcctacctgc a vyyca rgystyy rdf n i
agatgaacagcctgaagacagaggacaccgccgtgtactactgcgccag wgqgtlvtvssastkgps gggctattccacctactaccgggacttcaacatctggggccagggcaccc vfplapsskstsggtaalg
tggtcaccgtgtcctcggctagcaccaagggcccatcggtcttccccctgg clvkdyfpepvtvswns
caccctcctccaagagcacctctgggggcacagcggccctgggctgcctg galtsgvhtfpavlqssgl gtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgc yslssvvtvpssslgtqtyi cctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggact cnvnhkpsntkvdkkv
ctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcaccca epkscdkthtcppcpap gacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggac ellggpsvflfppkpkdtl aagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtg misrtpevtcvvvdvsh
cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaa edpevkfnwyvdgvev
acccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtgg hnaktkpreeqynstyr
tggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg vvsvltvlhqdwlngkey gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag kckvsnkalpapiektis
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagga kakgqprepqvytlpps
ctggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc reemtknqvsltclvkgf ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag ypsdiavewesngqpe
aaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaa nnykttppvldsdgsffl
ccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc yskltvdksrwqqgnvf
cgtggagtgggagagcaatgggcagccggagaacaactacaagaccac scsvmhealhnhytqk
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcacc slslspgk
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc
cgggtaaatga Name Nucleotide Sequence Amino Acid SEQ ID NOs:
Sequence nucleotide
(amino acid)
Humanized atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtca diqmtqspsslsasvgd SEQ ID NO:336 light chain, cgaattcagacatccagatgacccagtccccttcctccctgtccgctagcg rvtincqasqsidsnlaw (SEQ ID pi variant tgggcgatagggtgaccatcaactgccaggcctcccagtccattgactcc fqqkpgqppklliyrasn NO:337)
aatctggcctggttccagcagaagcccggacagccccccaagctgctgat lasgvpdrfsgsgsgtdft
HuRll mAb
ttacagggcctccaacctggcttccggcgtgcctgacaggttctccggatc Itissleaedvatyyclgg
cggcagcggcaccgacttcaccctgaccatctcctccctggaggccgagg vgavsyrtsfgggtkveik
atgtcgccacctactactgtctgggcggcgtgggcgctgtgagctatcgga rtvaapsvfifppsdeql
cctccttcggcggcggcaccaaggtggagatcaagcgtacggtggctgca ksgtasvvcllnnfypre
ccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactg akvqwkvdnalqsgns
cctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtac qesvteqdskdstyslss
agtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtc tltlskadyekhkvyace
acagagcaggacagcaaggacagcacctacagcctcagcagcaccctg vthqglsspvtksfnrge
acgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa c
gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggg
gagagtgttga
Humanized atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtca evqlvesggglvqpgrsl SEQ ID NO:338 heavy cgaattcggaggtgcagctggtggaaagcggaggaggcctggtgcagcc rlsctasgsdindypitw (SEQ ID chain, pi tggaaggtccctgaggctgtcctgcacagccagcggctccgacatcaacg vrqapgqglewigfinsg NO:339) variant actaccccatcacctgggtgaggcaggctcctggccagggcctggaatgg gstwyaswvkgrftisrd
atcggctttatcaacagcggcggcagcacctggtatgcttcctgggtgaag dsksiaylqmnslktedt
HuRll mAb
ggccggttcaccattagcagggacgactccaagtccattgcctacctgca a vyyca rgystyy rdf n i
gatgaactccctgaagaccgaggacaccgccgtgtactactgcgccagg wgqgtlvtvssastkgps
ggctacagcacctattaccgggactttaacatctggggccagggcacact vfplapsskstsggtaalg
ggtcaccgtgtcctcggctagcaccaagggcccatcggtcttccccctggc clvkdyfpepvtvswns
accctcctccaagagcacctctgggggcacagcggccctgggctgcctgg galtsgvhtfpavlqssgl
tcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgcc yslssvvtvpssslgtqtyi
ctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactc cnvnhkpsntkvdkkv
tactccctcagcagcgtggtgaccgtgccctccagcagcttgggcaccca epkscdkthtcppcpap
gacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggac ellggpsvflfppkpkdtl
aagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtg misrtpevtcvvvdvsh
cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaa edpevkfnwyvdgvev
acccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtgg hnaktkpreeqynstyr
tggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg vvsvltvlhqdwlngkey
gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag kckvsnkalpapiektis
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagga kakgqprepqvytlpps
ctggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc reemtknqvsltclvkgf
ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag ypsdiavewesngqpe
aaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaa nnykttppvldsdgsffl
ccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc yskltvdksrwqqgnvf
cgtggagtgggagagcaatgggcagccggagaacaactacaagaccac scsvmhealhnhytqk
gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcacc slslspgk
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat
gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctc
cgggtaaatga
[00212] ROR1-binding proteins (e.g., an anti-ROR1 x anti-CD3 molecule) may comprise any of the ROR1-binding domains described above. In some aspects, ROR1-binding proteins (e.g., an anti-ROR1 x anti-CD3 molecule) comprise humanized VH or VL amino acid sequences, or both. [00213] ROR1 -binding polypeptides may comprise an amino acid sequence that is at least about 95%, at least about 97% identical, at least about 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:71 , SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91 , SEQ ID NO:214, SEQ ID NO:218, or SEQ ID NO:222. In some embodiments, a ROR1 -binding polypeptide comprises an amino acid sequence that is that is at least about 95%, at least about 97% identical, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321 , SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, or SEQ ID NO:331 . In certain embodiments, a ROR1-binding polypeptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321 , SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, or SEQ ID NO:331 . The disclosure encompasses ROR1 -binding polypeptides (e.g. , an anti-ROR1 x anti-CD3 molecule) that are variants of the disclosed sequences. For example, variants lacking the two carboxyl-terminal serines are included in the disclosure, such as SEQ ID NO:299, which is a variant of SEQ ID NO:236, and SEQ ID NO:300, which is a variant of SEQ ID NO:256. In some embodiments, the ROR-1 binding poritens are codon-optimized bispecific anti-ROR1 proteins. For example, ROR238, ROR239, ROR241 , ROR242, ROR243, ROR244, ROR246, ROR247, ROR248, ROR249, ROR250, ROR251 , ROR252, ROR253 are codon-optimized versions of parental bispecific anti-ROR1 molecules ROR185 or ROR192.
[00214] ROR1 -binding molecules (e.g., an anti-ROR1 x anti-CD3 molecule) may be made using scaffolding as generally disclosed in WO 201 1/090762 (US 2013/0129723) and WO 201 1/090754 (US 2013/0095097), which are each incorporated herein by reference in their entirety. ROR1 -binding proteins (e.g., an anti-ROR1 x anti-CD3 molecule) may comprise two non-identical polypeptide chains, each polypeptide chain comprising an immunoglobulin heterodimerization domain. The interfacing immunoglobulin heterodimerization domains are different. In one embodiment, the immunoglobulin heterodimerization domain comprises a CH1 domain or a derivative thereof. In another embodiment, the immunoglobulin heterodimerization domain comprises a CL domain or a derivative thereof. In one embodiment, the CL domain is a CK or CA isotype or a derivative thereof.
[00215] The ROR1 -binding domains and polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule) disclosed herein may have improved characteristics compared to other ROR1 - binding domains or polypeptides. For example, a ROR1 -binding domain or polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) may exhibit a reduced isoelectric point compared to the isoelectric point of a different ROR1 -binding doman or polypeptide. "Isoelectric point" or "pi" is the pH at which net charge is zero. In one embodiment, a ROR1 -binding domain may have a reduced isoelectric point compared to the isoelectric point of a ROR1 -binding domain comprising an immunoglobulin light chain variable region of SEQ ID NO:220 and an immunoglobulin heavy chain variable region of SEQ ID NO:232. The isoelectric point of a protein may be measured by any suitable method, e.g., analytical capillary isoelectric focusing chromatography.
[00216] A ROR1 -binding domain or protein (e.g., an anti-ROR1 x anti-CD3 molecule) disclosed herein may bind to ROR1 (e.g., human ROR1) with a higher affinity than the parent rabbit R1 1 or R12 antibody or scFv construct. In some embodiments, the dissociation constant of a ROR1 -binding domain or polypeptide (e.g., an anti-ROR1 x anti- CD3 molecule) may be about 2-5 nM. In certain embodiments, the off rate of a ROR1 - binding domain or polypeptide may be 4- to 10-fold reduced compared to the off rate of the parent rabbit R1 1 or R12 antibody or scFv construct. In one example, a ROR1 -binding domain binds to human ROR1 with a dissociation constant that is lower than the dissociation constant of a ROR1 -binding domain comprising SEQ ID NO:73.
[00217] The disclosure also includes nucleic acids (e.g. , DNA or RNA) encoding ROR1 - binding domains, proteins and polypeptides (e.g. , an anti-ROR1 x anti-CD3 molecule) described herein, or one or more polypeptide chains of a dimeric or heterodimeric ROR1 - binding protein as described herein. Nucleic acids of the disclosure include nucleic acids having a region that is substantially identical to a polynucleotide as listed in Table 3, infra. In certain embodiments, a nucleic acid in accordance with the present disclosure has at least 80%, typically at least about 90%, and more typically at least about 95% or at least about 98% identity to a polypeptide-encoding polynucleotide as listed in Table 3. Nucleic acids of the disclosure also include complementary nucleic acids. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there can be up to about a 20% mismatch in the sequences. In some embodiments of the disclosure are provided nucleic acids encoding both first and second polypeptide chains of a heterodimeric ROR1 -binding protein of the disclosure. The nucleic acid sequences provided herein can be exploited using codon optimization, degenerate sequence, silent mutations, and other DNA techniques to optimize expression in a particular host, and the present disclosure encompasses such sequence modifications. For example, nucleotide sequences set forth in SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, and SEQ ID NO:330 contain codon-optimized sequences.
[00218] The disclosure relates to an isolated nucleic acid molecule encoding ROR1 -binding domains, proteins and polypeptides (or portions thereof) described herein (e.g., an anti- ROR1 x anti-CD3 molecule), wherein said nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:209, SEQ ID NO:21 1 , SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221 , SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231 , SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241 , SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251 , SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, or SEQ ID NO:338.
[00219] Polynucleotide molecules comprising a desired polynucleotide sequence are propagated by placing the molecule in a vector. Viral and non-viral vectors are used, including plasmids. The choice of plasmid will depend on the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially. The partial or full- length polynucleotide is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination in vivo. Typically this is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence. Regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example.
[00220] For expression, an expression cassette or system may be employed. To express a nucleic acid encoding a polypeptide disclosed herein, a nucleic acid molecule encoding the polypeptide, operably linked to regulatory sequences that control transcriptional expression in an expression vector, is introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector. The gene product encoded by a polynucleotide of the disclosure is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. In the expression vector, the polypeptide- encoding polynucleotide is linked to a regulatory sequence as appropriate to obtain the desired expression properties. These can include promoters, enhancers, terminators, operators, repressors, and inducers. The promoters can be regulated (e.g. , the promoter from the steroid inducible pIND vector (Invitrogen)) or constitutive (e.g. , promoters from CMV, SV40, Elongation Factor, or LTR sequences). These are linked to the desired nucleotide sequence using the techniques described above for linkage to vectors. Any techniques known in the art can be used. Accordingly, the expression vector will generally provide a transcriptional and translational initiation region, which can be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region.
[00221] An expression cassette ("expression unit") can be introduced into a variety of vectors, e.g. , plasmid, BAC, YAC, bacteriophage such as lambda, P1 , M 13, etc. , plant or animal viral vectors (e.g. , retroviral-based vectors, adenovirus vectors), and the like, where the vectors are normally characterized by the ability to provide selection of cells comprising the expression vectors. The vectors can provide for extrachromosomal maintenance, particularly as plasmids or viruses, or for integration into the host chromosome. Where extrachromosomal maintenance is desired, an origin sequence is provided for the replication of the plasmid, which can be low- or high copy-number. A wide variety of markers are available for selection, particularly those which protect against toxins, more particularly against antibiotics. The particular marker that is chosen is selected in accordance with the nature of the host, where, in some cases, complementation can be employed with auxotrophic hosts. Introduction of the DNA construct can use any convenient method, including, e.g. , conjugation, bacterial transformation, calcium-precipitated DNA,
electroporation, fusion, transfection, infection with viral vectors, biolistics, and the like. The disclosure relates to an expression vector comprising a nucleic acid segment, wherein said nucleic acid segment may comprise a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90,
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:209, SEQ ID NO:21 1 , SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221 , SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231 , SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241 , SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251 , SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, or SEQ ID NO:338.
[00222] Accordingly, proteins for use within the present disclosure can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells (including cultured cells of multicellular organisms), particularly cultured mammalian cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001), and Ausubel et al. , Short Protocols in Molecular Biology (4th ed., John Wiley & Sons, 1999).
[00223] For example, for recombinant expression of a homodimeric ROR1 -binding protein comprising two identical ROR1 -binding polypeptides as described herein (e.g., an anti- ROR1 x anti-CD3 molecule), an expression vector will generally include a nucleic acid segment encoding the ROR1 -binding polypeptide, operably linked to a promoter. For recombinant expression of a heterodimeric ROR1 -binding protein (e.g., an anti-ROR1 x anti- CD3 molecule), comprising different first and second polypeptide chains, the first and second polypeptide chains can be co-expressed from separate vectors in the host cell for expression of the entire heterodimeric protein. Alternatively, for the expression of heterodimeric ROR1 -binding proteins (e.g., an anti-ROR1 x anti-CD3 molecule), the first and second polypeptide chains are co-expressed from separate expression units in the same vector in the host cell for expression of the entire heterodimeric protein. The expression vector(s) are transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the encoded polypeptide(s) to produce the corresponding ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule).
[00224] To direct a recombinant protein into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence) is provided in the expression vector. The secretory signal sequence can be that of the native form of the recombinant protein, or can be derived from another secreted protein or synthesized de novo. The secretory signal sequence is operably linked to the polypeptide-encoding DNA sequence, i.e. , the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences can be positioned elsewhere in the DNA sequence of interest (see, e.g. , Welch et al. , U.S. Patent No. 5,037,743; Holland et al. , U.S. Patent No. 5, 143,830). In certain variations, a secretory signal sequence for use in accordance with the present disclosure has the amino acid sequence
MEAPAQLLFLLLLWLPDTTG (SEQ ID NO:208).
[00225] Cultured mammalian cells are suitable hosts for production of recombinant proteins (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) for use within the present disclosure. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al. , Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981 : Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al. , EMBO J. 1 :841 -845, 1982), DEAE-dextran mediated transfection (Ausubel et al. , supra), and liposome-mediated transfection (Hawley-Nelson et al. , Focus 15:73, 1993; Ciccarone et al. , Focus 15:80, 1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed by, for example, Levinson et al. , U.S. Patent No. 4,713,339; Hagen et al. , U.S. Patent No. 4,784,950; Palmiter et al. , U.S. Patent No. 4,579,821 ; and Ringold, U.S. Patent No. 4,656, 134. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21 , BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1 ; ATCC CCL61 ; CHO DG44; CHO DXB1 1 (Hyclone, Logan, UT); see also, e.g. , Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1 ; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1 ; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658). Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, Virginia. Strong transcription promoters can be used, such as promoters from SV-40 or cytomegalovirus. See, e.g. , U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patents Nos. 4,579,821 and 4,601 ,978) and the adenovirus major late promoter.
[00226] Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants." Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." Exemplary selectable markers include a gene encoding resistance to the antibiotic neomycin, which allows selection to be carried out in the presence of a neomycin-type drug, such as G-418 or the like; the gpt gene for xanthine-guanine phosphoribosyl transferase, which permits host cell growth in the presence of mycophenolic acid/xanthine; and markers that provide resistance to zeocin, bleomycin, blastocidin, and hygromycin (see, e.g. , Gatignol et al., Mol. Gen. Genet. 207:342, 1987; Drocourt et al., Nucl. Acids Res. 18:4009, 1990). Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. , hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used.
[00227] Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al. , J. Biosci. (Bangalore) 1 1 :47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al. , US 5, 162,222 and WO 94/06463.
[00228] Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See King and Possee, The Baculovirus Expression System: A Laboratory Guide (Chapman & Hall, London); O'Reilly et a/. , Baculovirus Expression Vectors: A Laboratory Manual (Oxford University Press., New York 1994); and Baculovirus Expression Protocols. Methods in Molecular Biology (Richardson ed., Humana Press, Totowa, NJ, 1995). Recombinant baculovirus can also be produced through the use of a transposon-based system described by Luckow et al. (J. Virol. 67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, MD). The transfer vector (e.g. , PFASTBAC1 ; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." See Hill-Perkins and Possee, J. Gen. Virol. 71 :971 -976, 1990; Bonning et al. , J. Gen. Virol. 75: 1551 -1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270: 1543-1549, 1995. In addition, transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above. Using techniques known in the art, a transfer vector containing a protein-encoding DNA sequence is transformed into E. coli host cells, and the cells are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells. Recombinant virus that expresses the protein or interest is subsequently produced. Recombinant viral stocks are made by methods commonly used in the art.
[00229] For protein production, the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g. , Sf9 or Sf21 cells) or Trichoplusia ni (e.g. , HIGH FIVE cells; Invitrogen, Carlsbad, CA). See generally Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA (ASM Press, Washington, D.C., 1994). See also U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers. The cells are grown up from an inoculation density of approximately 2-5 x 10s cells to a density of 1 -2 x 106 cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally described in available laboratory manuals (see, e.g. , King and Possee, supra; O'Reilly et al. , supra; Richardson, supra).
[00230] Fungal cells, including yeast cells, can also be used within the present disclosure to produce an anti-ROR1 x anti-CD3 molecule as described herein. Yeast species of in this regard include, e.g., Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,31 1 ; Kawasaki et al. , U.S. Patent No. 4,931 ,373; Brake, U.S. Patent No.
4,870,008; Welch et al. , U.S. Patent No. 5,037,743; and Murray et al. , U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g. , leucine). An exemplary vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931 ,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g. , Kawasaki, U.S. Patent No. 4,599,31 1 ; Kingsman et al. , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S.
Patents Nos. 4,990,446; 5,063, 154; 5, 139,936; and 4,661 ,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,
Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii, and Candida maltosa are known in the art. See, e.g. , Gleeson et al. , J. Gen. Microbiol. 132:3459-3465, 1986; Cregg, U.S. Patent No. 4,882,279; and Raymond et al. , Yeast 14: 1 1 -23, 1998. Aspergillus cells can be utilized according to the methods of McKnight et al. , U.S. Patent No. 4,935,349. Methods for transforming
Acremonium chrysogenum are disclosed by Sumino et al. , U.S. Patent No. 5, 162,228.
Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No.
4,486,533. Production of recombinant proteins in Pichia methanolica is disclosed in U.S. Patents Nos. 5,716,808; 5,736,383; 5,854,039; and 5,888,768.
[00231] Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus, and other genera are also useful host cells within the present disclosure to produce, for example, an anti-ROR1 x anti-CD3 molecule. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well-known in the art (see, e.g. , Sambrook and Russell, supra). When expressing a recombinant protein in bacteria such as E. coli, the protein can be retained in the cytoplasm, typically as insoluble granules, or can be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured protein can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the alternative, the protein can be recovered from the cytoplasm in soluble form and isolated without the use of denaturants. The protein is recovered from the cell as an aqueous extract in, for example, phosphate buffered saline. To capture the protein of interest, the extract is applied directly to a chromatographic medium, such as an immobilized antibody or heparin-Sepharose column. Secreted proteins can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
Antibodies, including single-chain antibodies, can be produced in bacterial host cells according to known methods. See, e.g. , Bird et al. , Science 242:423-426, 1988; Huston et al. , Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; and Pantoliano et al. , Biochem.
30: 101 17-10125, 1991 .
[00232] Transformed or transfected host cells to produce ROR1 -binding proteins (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media can also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
[00233] ROR1 -binding proteins (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) may be purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See generally Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification: Principles and Practice (Springer- Verlag, New York 1994). Proteins comprising an immunoglobulin Fc region can be purified by affinity chromatography on immobilized protein A or protein G. Additional purification steps, such as gel filtration, can be used to obtain the desired level of purity or to provide for desalting, buffer exchange, and the like.
[00234] The present disclosure provides methods for treating a subject with a disorder characterized by expression of ROR1 . Generally, such methods include administering to a subject in need of such treatment a ROR1 -binding protein as described herein (e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or
ROR243). In some embodiments, a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) does not induce or induces minimal antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity. In other embodiments, where the ROR1 - binding protein comprises a second binding domain that specifically binds a T-cell (e.g. , to a TCR complex or component thereof, such as CD3£), the ROR1 -binding protein induces redirected T-cell cytotoxicity (RTCC) against ROR1 -expressing cells in the subject. [00235] In certain variations of the method of treating a subject with a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243), the disorder is a cancer. Exemplary cancers amenable to treatment in accordance with a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) of the present disclosure include, for example, breast cancer (e.g., triple negative breast cancer (TNBC)), pancreatic cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), acute lymphoblastic leukemia (ALL), melanoma, adrenal cancer, bladder cancer or prostate cancer. TNBC is defined as breast cancer with the absence of staining for estrogen receptor, progesterone receptor, and HER2/neu.
[00236] In a further embodiment, the disclosure encompasses a method for inducing redirected T-cell cytotoxicity (RTCC) against a cell expressing ROR1 , the method comprising contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243), wherein said contacting is under conditions whereby RTCC against the ROR1 -expressing cell is induced. In some embodiments, the disclosure relates to a method for inducing redirected T-cell cytotoxicity (RTCC) against a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and a second binding domain that specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; wherein said contacting is under conditions whereby RTCC against the ROR1 -expressing cell is induced. The disclosure encompasses a method for inducing T-cell dependent lysis of a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said
ROR1 -expressing cell with a ROR1 -binding polypeptide or ROR1 -binding protein described herein (for example, ROR248, ROR246, ROR252, ROR250, or ROR243), wherein the second binding domain specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; and wherein said contacting is under conditions whereby T-cell dependent lysis of the ROR1 -expressing cell is induced. In some embodiments, the disclosure relates to a method for inducing T-cell dependent lysis of a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and a second binding domain that specifically binds a T-cell, CD3, CD3£ or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; wherein said contacting is under conditions whereby T-cell dependent lysis of the ROR1 - expressing cell is induced.
[00237] The disclosure also encompasses a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) for the manufacture of a medicament for treatment of a disorder (e.g., cancer) characterized by expression of ROR1 . In one embodiment, the ROR1 -binding polypeptide has RTCC activity, e.g. , it comprises an anti-ROR1 and anti-CD3 binding domain. In one embodiment, the disclosure includes a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) for use in treating a disorder (e.g., cancer) characterized by expression of ROR1 . In certain embodiments, the disclosure relates to a method for treating a disorder in a subject, wherein said disorder is characterized by expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising administering to the subject a therapeutically effective amount of a ROR1 -binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR.
[00238] In some embodiments, the disclosure provides a method of treating a patient with a cancer, comprising administering to the patient a ROR1 -binding polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321 , SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, or SEQ ID NO:331 .
[00239] In some embodiments, for treatment methods and uses described herein, a ROR1 - binding protein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) is delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, a therapeutically effective amount of the ROR1 -binding protein is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.
[00240] Subjects for administration of ROR1 -binding proteins as described herein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) include patients at high risk for developing a particular disorder characterized by ROR1 expression as well as patients presenting with an existing such disorder. Typically, the subject has been diagnosed as having the disorder for which treatment is sought.
Further, subjects can be monitored during the course of treatment for any change in the disorder (e.g. , for an increase or decrease in clinical symptoms of the disorder). Also, in some variations, the subject does not suffer from another disorder requiring treatment that involves targeting ROR1 -expressing cells.
[00241] In prophylactic applications, pharmaceutical compositions or medicants (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) are administered to a patient susceptible to, or otherwise at risk of, a particular disorder in an amount sufficient to eliminate or reduce the risk or delay the onset of the disorder. In therapeutic applications, compositions or medicants (e.g., an anti-ROR1 x anti- CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) are administered to a patient suspected of, or already suffering from such a disorder in an amount sufficient to cure, or at least partially arrest, the symptoms of the disorder and its complications. An amount adequate to accomplish this is referred to as a therapeutically effective dose or amount. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response (e.g. , inhibition of inappropriate angiogenesis activity) has been achieved. Typically, the response is monitored and repeated dosages are given if the desired response starts to fade.
[00242] To identify subject patients for treatment according to the methods of the disclosure (e.g., treated with an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243), accepted screening methods can be employed to determine risk factors associated with specific disorders or to determine the status of an existing disorder identified in a subject. Such methods can include, for example, determining whether an individual has relatives who have been diagnosed with a particular disorder. Screening methods can also include, for example, conventional work-ups to determine familial status for a particular disorder known to have a heritable component. For example, various cancers are also known to have certain inheritable components. Inheritable components of cancers include, for example, mutations in multiple genes that are transforming (e.g. , Ras, Raf, EGFR, cMet, and others), the presence or absence of certain HLA and killer inhibitory receptor (KIR) molecules, or mechanisms by which cancer cells are able to modulate immune suppression of cells like NK cells and T-cells, either directly or indirectly (see, e.g. , Ljunggren and Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann, Clin. Exp. Immunol. 149: 1 -8, 2007). Toward this end, nucleotide probes can be routinely employed to identify individuals carrying genetic markers associated with a particular disorder of interest. In addition, a wide variety of immunological methods are known in the art that are useful to identify markers for specific disorder. For example, various ELISA immunoassay methods are available and well-known in the art that employ monoclonal antibody probes to detect antigens associated with specific tumors. Screening can be implemented as indicated by known patient symptomology, age factors, related risk factors, etc. These methods allow the clinician to routinely select patients in need of the methods described herein for treatment. In accordance with these methods, targeting pathological, ROR1 -expressing cells can be implemented as an independent treatment program or as a follow-up, adjunct, or coordinate treatment regimen to other treatments.
[00243] For administration, a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) may be formulated as a pharmaceutical composition. A pharmaceutical composition may comprise: (i) a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243); and (ii) a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition comprising a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier, diluent or excipient. A carrier is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers, diluents or excipients are well-known to those in the art. (See, e.g. , Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing
Company, 19th ed. 1995).) Formulations can further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. In certain embodiments, a pharmaceutical composition comprises a ROR1 -binding polypeptide (e.g., an anti-ROR1 x anti-CD3 molecule) that is a homodimer or a heterodimer. A "homodimer" may be a dimer formed from two identical polypeptides (e.g., an anti-ROR1 x anti-CD3 molecule as described herein; for example ROR248, ROR246, ROR252, ROR250, or ROR243).
[00244] A pharmaceutical composition comprising a ROR1 -binding protein (e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or
ROR243) may be formulated in a dosage form selected from the group consisting of: an oral unit dosage form, an intravenous unit dosage form, an intranasal unit dosage form, a suppository unit dosage form, an intradermal unit dosage form, an intramuscular unit dosage form, an intraperitoneal unit dosage form, a subcutaneous unit dosage form, an epidural unit dosage form, a sublingual unit dosage form, and an intracerebral unit dosage form. The oral unit dosage form may be selected from the group consisting of: tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained-release formulations, aerosols, and sprays.
[00245] A pharmaceutical composition comprising a ROR1 -binding protein therapeutic (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) may be administered to a subject in a therapeutically effective amount. According to the methods of the present disclosure, a ROR1 -binding protein (e.g., an anti-ROR1 x anti- CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) can be administered to subjects by a variety of administration modes, including, for example, by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral routes of administration. For prevention and treatment purposes, an antagonist (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243)can be administered to a subject in a single bolus delivery, via continuous delivery (e.g. , continuous transdermal delivery) over an extended time period, or in a repeated administration protocol (e.g. , on an hourly, daily, weekly, or monthly basis).
[00246] Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of the subject disorder in model subjects. Effective doses of the compositions of the present disclosure vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, whether treatment is prophylactic or therapeutic, as well as the specific activity of the composition itself and its ability to elicit the desired response in the individual. Usually, the patient is a human, but in some diseases, the patient can be a nonhuman mammal. Typically, dosage regimens are adjusted to provide an optimum therapeutic response, i.e. , to optimize safety and efficacy. Accordingly, a therapeutically effective amount is also one in which any undesired collateral effects are outweighed by the beneficial effects of administering a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) as described herein. For administration of a ROR1 -binding protein, a dosage may range from about 0.1 μg to 100 mg/kg or 1 μg/kg to about 50 mg/kg, and more usually 10 μg to 5 mg/kg of the subject's body weight. In more specific embodiments, an effective amount of the agent is between about 1 μg/kg and about 20 mg/kg, between about 10 μg/kg and about 10 mg/kg, or between about 0.1 mg/kg and about 5 mg/kg. Dosages within this range can be achieved by single or multiple administrations, including, e.g. , multiple administrations per day or daily, weekly, bi-weekly, or monthly administrations. For example, in certain variations, a regimen consists of an initial administration followed by multiple, subsequent administrations at weekly or bi-weekly intervals. Another regimen consists of an initial administration followed by multiple, subsequent administrations at monthly or bi-monthly intervals. Alternatively, administrations can be on an irregular basis as indicated by monitoring clinical symptoms of the disorder.
[00247] Dosage of the pharmaceutical composition comprising a ROR1 -binding protein (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) can be varied by the attending clinician to maintain a desired concentration at a target site. For example, if an intravenous mode of delivery is selected, local concentration of the agent in the bloodstream at the target tissue can be between about 0.01 -50 nanomoles of the composition per liter, sometimes between about 1 .0 nanomole per liter and 10, 15, or 25 nanomoles per liter depending on the subject's status and projected measured response. Higher or lower concentrations can be selected based on the mode of delivery, e.g. , trans-epidermal delivery versus delivery to a mucosal surface. Dosage should also be adjusted based on the release rate of the administered formulation, e.g. , nasal spray versus powder, sustained release oral or injected particles, transdermal formulations, etc. To achieve the same serum concentration level, for example, slow-release particles with a release rate of 5 nanomolar (under standard conditions) would be administered at about twice the dosage of particles with a release rate of 10 nanomolar.
[00248] The anti-ROR1 therapeutic (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) may also be administered at a daily dosage of from about 0.001 to about 10 milligrams (mg) per kilogram (mpk) of body weight, preferably given as a single daily dose or in divided doses about two to six times a day. For administration to a human adult patient, the therapeutically effective amount may be administered in doses in the range of 0.2 mg to 800 mg per dose, including but not limited to 0.2 mg per dose, 0.5 mg per dose, 1 mg per dose, 5 mg per dose, 10 mg per dose, 25 mg per dose, 100 mg per dose, 200 mg per dose, and 400 mg per dose, and multiple, usually consecutive daily doses may be administered in a course of treatment. The anti-ROR1 therapeutic (e.g., an anti-ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or ROR243) can be administered at different times of the day. In one embodiment the optimal therapeutic dose can be administered in the evening. In another embodiment the optimal therapeutic dose can be administered in the morning. The total daily dosage of the anti-ROR1 therapeutic thus can in one embodiment range from about 1 mg to about 2 g, and often ranges from about 100 mg to about 1 .5 g, and most often ranges from about 200 mg to about 1200 mg. In the case of a typical 70 kg adult human, the total daily dose of the anti-ROR1 therapeutic can range from about 2 mg to about 1200 mg and will often range, as noted above, from about 0.2 mg to about 800 mg.
[00249] With particular regard to treatment of solid tumors, protocols for assessing endpoints and anti-tumor activity are well-known in the art. While each protocol may define tumor response assessments differently, the RECIST (Response evaluation Criteria in solid tumors) criteria is currently considered to be the recommended guidelines for assessment of tumor response by the National Cancer Institute (see Therasse et a/., J. Natl. Cancer Inst. 92:205-216, 2000). According to the RECIST criteria tumor response means a reduction or elimination of all measurable lesions or metastases. Disease is generally considered measurable if it comprises lesions that can be accurately measured in at least one dimension as > 20mm with conventional techniques or > 10mm with spiral CT scan with clearly defined margins by medical photograph or X-ray, computerized axial tomography (CT), magnetic resonance imaging (MRI), or clinical examination (if lesions are superficial). Non-measurable disease means the disease comprises of lesions < 20mm with conventional techniques or < 10mm with spiral CT scan, and truly non-measurable lesions (too small to accurately measure). Non-measureable disease includes pleural effusions, ascites, and disease documented by indirect evidence.
[00250] The criteria for objective status are required for protocols to assess solid tumor response. Representative criteria include the following: (1) Complete Response (CR), defined as complete disappearance of all measurable disease; no new lesions; no disease related symptoms; no evidence of non-measurable disease; (2) Partial Response (PR) defined as 30% decrease in the sum of the longest diameter of target lesions (3) Progressive Disease (PD), defined as 20% increase in the sum of the longest diameter of target lesions or appearance of any new lesion; (4) Stable or No Response, defined as not qualifying for CR, PR, or Progressive Disease. (See Therasse et a/., supra.)
[00251] Additional endpoints that are accepted within the oncology art include overall survival (OS), disease-free survival (DFS), objective response rate (ORR), time to progression (TTP), and progression-free survival (PFS) (see Guidance for Industry: Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologies, April 2005, Center for Drug Evaluation and Research, FDA, Rockville, MD.)
[00252] Pharmaceutical compositions comprising an anti-ROR1 therapeutic (e.g., an anti- ROR1 x anti-CD3 molecule; for example ROR248, ROR246, ROR252, ROR250, or
ROR243) can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry- powder disperser, liquid aerosol generator, or nebulizer for administration of a
pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.
[00253] The disclosure will be further clarified by the following examples, which are intended to be purely exemplary of the disclosure and in no way limiting.
EXAMPLES
EXAMPLE 1 : Generation of humanized variants of anti-ROR1 antibodies and construction of monospecific and bispecific ROR1 -binding molecules
Preparation of monospecific ROR1-bindinq molecules
[00254] R1 1 and R12 are two previously described rabbit antibodies that bind to the receptor tyrosine kinase ROR1 (see, e.g., U.S. Patent Application Publication No. 2013/0251642; Yang et al., PLoS ONE 6(6): e21018 (201 1)) (sequences provided in Table 3). Variable domain sequences for the R1 1 and R12 antibodies were identified, and synthetic DNA sequences coding for these variable domain sequences were designed as scFv sequences and cassetted for insertion into molecular scaffolds containing human immunoglobulin constant domains (e.g. Fc regions). The variable domain constructs were then synthesized by Blue Heron (Bothell, WA) and standard, restriction-digest-based cloning techniques were used to produce the chimeric gene sequences corresponding to ROR015 (SEQ ID NO:70; amino acid sequence SEQ ID NO:71), and ROR016 (SEQ ID NO:74; amino acid sequence SEQ ID NO:75). Nucleotide and amino acid sequences referred to in these Examples can be found in Table 3.
Preparation of humanized monospecific ROR1 -binding molecules
[00255] Humanized sequences designed through CDR grafting to human frameworks were similarly synthesized by Blue Heron in the VH-VL scFv orientation and cloned into similar vectors using restriction digests to produce the following gene sequences using two piece ligation utilizing a Hindlll/Xhol fragment and a destination vector cut with Hindlll/Xhol to produce the gene sequences corresponding to ROR033 (SEQ ID NO:84; amino acid sequence SEQ ID NO:85), ROR034 (SEQ ID NO:86; amino acid sequence SEQ ID NO:87), ROR035 (SEQ ID NO:88; amino acid sequence SEQ ID NO:89), and ROR036 (SEQ ID NO:90; amino acid sequence SEQ ID NO:91). For select numbers of constructs, VL-VH scFv molecules were made by PCR amplification of VH and VL fragments after which three piece ligation using a Hind 11 I/Bam HI fragment, a BamHI/Xhol fragment, and a destination vector cut with Hindlll/Xhol was performed to produce the gene sequences corresponding to the chimeric ROR063 (SEQ ID NO:72; amino acid sequence SEQ ID NO:73), and the hemi- humanized or humanized ROR073 (SEQ ID NO:76; amino acid sequence SEQ ID NO:77), ROR091 (SEQ ID NO:78; amino acid sequence SEQ ID NO:79), ROR081 (SEQ ID NO:80; amino acid sequence SEQ ID NO:81), and ROR101 (SEQ ID NO:82; amino acid sequence SEQ ID NO:83). The humanized R1 1 VL region polynucleotide sequences are given in SEQ ID NO:22 and SEQ ID NO:24, and the amino acid sequences are given in SEQ ID NO:23 and SEQ ID NO:25. Humanized R1 1 VH region polynucleotide sequences are given in SEQ ID NO:26, and the amino acid sequences are given in SEQ ID NO:27. The humanized R12 VL region polynucleotide sequences are given in SEQ ID NO:54 and SEQ ID NO:56, and the amino acid sequences are given in SEQ ID NO:55 and SEQ ID NO:57. Humanized R12 VH region polynucleotide sequences are given in SEQ ID NO:58 and SEQ ID NO:60, and the amino acid sequences are given in SEQ ID NO:59 and SEQ ID NO:61 .
Preparation of bispecific ROR1 -binding molecules
[00256] Bispecific ROR1 -binding molecules targeting ROR1 and CD3 epsilon [ROR070 (SEQ ID NO: 102 (nucleic acid), SEQ ID NO: 103 (amino acid); ROR084 (SEQ ID NO: 104 (nucleic acid), SEQ ID NO: 105 (amino acid); ROR021 (SEQ ID NO: 106 (nucleic acid), SEQ ID NO: 107 (amino acid); ROR050 (SEQ ID NO: 108 (nucleic acid), SEQ ID NO: 109 (amino acid)] were made using standard molecular biology techniques, starting with existing bispecific binding molecules as templates and using the methods generally disclosed in, e.g. , PCT Application Publication No. WO 2007/146968, U.S. Patent Application Publication No. 2006/0051844, PCT Application Publication No. WO 2010/040105, PCT Application Publication No. WO 2010/003108, and U.S. Patent No. 7, 166,707 (see also Table 3).
Insertion of the N-terminal anti-ROR1 scFv binding domain was accomplished through digestion of the parental template and scFv insert with either the restriction enzymes Hindlll and Xhol or Agel and Xhol, desired fragments were identified and isolated by agarose gel purification, and ligated. Insertion of the C-terminal anti-CD3 epsilon scFv binding domain was accomplished through digestion of the parental template and scFv insert with the restriction enzymes EcoRI and Notl; desired fragments were identified and isolated by agarose gel purification, and ligated.
Preparation of recombinant rabbit antibodies as controls
[00257] Recombinant R1 1 and R12 antibodies were also prepared as controls. To generate full recombinant rabbit antibodies, pFUSE-CLIg and pFUSE-CHIg vectors from Invivogen were used. These vectors contain the constant regions of the rabbit IgG heavy chain (allele 3/2) and kappa light chain. The VL and VH regions of each scFv were PCR amplified with primers encoding restriction sites allowing for the directional cloning upstream of the constant regions in the appropriate pFUSE vectors. For R12, a constant region
corresponding to a lambda light chain (UNIPROT#P01847) was synthesized by Blue Heron (Bothell, WA) and cloned into the pFUSE-CLIg vector, replacing the kappa 2 constant region.
5'R1 1VH EcoRI gtcacgaattcgcagtccgtgaaggagtccgag (SEQ ID NO: 1 19)
3'R1 1VH Xhol tgcccactcgagatggtcaccagggtgccggggccccagatgt (SEQ ID NO: 120)
5'R1 1VL EcoRI tcacgaattcggagctggtgatgacccagacc (SEQ ID NO: 121)
3'R1 1VL BamHI aactggatcccgcttcaccaccacctcggtgcc (SEQ ID NO: 122)
5'R12VH EcoRI tcacgaattcgcaggagcagctggtggagtcc (SEQ ID NO: 123)
3'R12VH Xhol gcccactcgagatggtcaccagggtgccggggccccagatgtt (SEQ ID NO: 124)
5'R12VL EcoRI tcacgaattcggagctggtgctgacccagtcc (SEQ ID NO: 125)
3'R12VL BamHI aactggatcccggccggtcacggtcagctgggt (SEQ ID NO: 126)
[00258] The polynucleotide sequence for the heavy chain of the R1 1 mAb (ROR064) is given in SEQ ID NO:92, and the amino acid sequence is given in SEQ ID NO:93. The polynucleotide sequence for the light chain of the R1 1 mAb (ROR065) is given in SEQ ID NO:94, and the amino acid sequence is given in SEQ ID NO:95. The polynucleotide sequence for the heavy chain of the R12 mAb (ROR067) is given in SEQ ID NO:96, and the amino acid sequence is given in SEQ ID NO:97. The polynucleotide sequence for the light chain of the R1 1 mAb (ROR068) is given in SEQ ID NO:98, and the amino acid sequence is given in SEQ ID NO:99.
Preparation of recombinant human antibodies
[00259] To generate full length recombinant humanized antibodies, pFUSEss-CLIg-ck and pFUSEss-CHIg-hG 1 vectors from Invivogen were used. These vectors contain the constant regions of the human lgG 1 heavy chain and kappa light chain. The first humanized antibody was created using the variable domain from the humanized R1 1 variable domains L7 (SEQ ID NO: 219) and H15 (SEQ ID NO:21 1). The second humanized antibody was created using the corresponding pi variants of the same humanized R1 1 variable domains, L9 (SEQ ID NO:249) and H18 (SEQ ID NO:250). The VL and VH regions were PCR amplified from scFv sequences with primers encoding restriction sites allowing for the directional cloning upstream of the constant regions in the appropriate pFUSE vectors:
5'huVH EcoRI gcgatcgaattcggaggtgcagctggtggaatccgg (SEQ ID NO:340)
3'huVH Nhel gcgatcgctagccgaggacacggtgaccagggtgcc (SEQ ID NO:341)
5'huVL EcoRI gcgatcgaattcagacatccagatgacccagagcccc (SEQ ID NO:342)
3'huVL BsiWI gcgatccgtacgcttgatctccaccttggtgccgcc (SEQ ID NO:343) 5'plVH EcoRI gcgatcgaattcggaggtgcagctggtggaaagcgg (SEQ ID NO:344)
3'plVH Nhel gcgatcgctagccgaggacacggtgaccagtgtgcc (SEQ ID NO:345)
5'plVL EcoRI gcgatcgaattcagacatccagatgacccagtcccct (SEQ ID NO:346)
3'plVL BsiWI gcgatccgtacgcttgatctccaccttggtgccgcc (SEQ ID NO:347)
[00260] The polynucleotide sequence for the heavy chain of the humanized R1 1 mAb is given in SEQ ID NO:334, and the amino acid sequence is given in SEQ ID NO:335. The polynucleotide sequence for the light chain of the humanized R1 1 mAb is given in SEQ ID NO:332, and the amino acid sequence is given in SEQ ID NO:333. The polynucleotide sequence for the heavy chain of the pi variant humanized mAb is given in SEQ ID NO:338, and the amino acid sequence is given in SEQ ID NO:339. The polynucleotide sequence for the light chain of the pi variant humanized R1 1 mAb is given in SEQ ID NO:336, and the amino acid sequence is given in SEQ ID NO:337.
Expression and purification of ROR1 -binding molecules and antibodies
[00261] Monospecific and bispecific ROR1 -binding molecules disclosed herein were produced by transient transfection of human HEK293 cells, and purified from cell culture supernatants by Protein A affinity chromatography. If aggregates were detected after affinity chromatography, secondary size exclusion chromatography was also performed to ensure homogeneity of the protein.
[00262] Antibodies were also produced by transient transfection of HEK293 cells with plasmid pairs coding for the heavy and light chains of each antibody; the parental R1 1 mAb (ROR066) was produced by co-transfecting ROR064 and ROR065, and the parental R12 mAb (ROR069) was produced by co-transfecting ROR067 and ROR068. Secreted IgG was purified from the supernatant using Protein A affinity chromatography. If aggregates were detected after affinity chromatography, secondary size exclusion chromatography was also performed to ensure homogeneity of the protein.
EXAMPLE 2: Binding of chimeric and humanized molecules to recombinant ROR1 ectodomains by surface plasmon resonance
[00263] To confirm that binding to ROR1 was not lost upon scFv conversion or humanization of the variable domains, surface plasmon resonance (SPR) was used to measure binding kinetics of the constructed ROR1 -binding molecules and parental antibodies to recombinant ROR1 ectodomain (Table 5, SEQ ID NO: 129 (nucleotide sequence), SEQ ID NO: 130 (amino acid sequence)). The secretion/leader sequence was intact in the recombinant ectodomain, and a C-terminal set of affinity tags was present, containing a BirA biotinylation site, a 3xFLAG tag and a 10xHis tag. [00264] SPR binding studies of chimeric & humanized molecules to recombinant monomeric ROR1 ectodomain (ECD) were conducted at 25°C in HBS-EP+ buffer on a BIACORE™ T200 system. AffiniPure F(ab')2 fragment Goat anti-human IgG Fey fragment-specific (Jackson Immuno Research), at 20 μg/ml in 10 mM sodium acetate (pH 4.5), was immobilized at a density of 4,000 response units (RU) on two flow cells of a CM5 research- grade sensor chip (GE) by standard amine coupling chemistry. The chimeric & humanized molecules at 5 μg/ml were captured on one flow cell of immobilized anti-Fc F(ab')2 fragment at a flow rate of 10 μΙ/min for 15-20 sec to reach 200-400 RU response, leaving the other immobilized anti-Fc F(ab')2 surface as the reference. Different concentrations of ROR1 ECD (1 .25-400 nM by 2- or 3- fold dilutions, including buffer as blank) were injected in randomized duplicate runs at 50 μΙ/min for 120-360 sec followed by a 120-360 sec dissociation period. Optimal regeneration was achieved by two injections of 10 mM glycine (pH 1 .5) at a flow rate of 50 μΙ/min for 10 sec followed by HBS-EP+ buffer stabilization for 2 min.
[00265] For binding analyses of rabbit R1 1 (or R12) mAb to monomeric ROR1 ECD, 4 μg/ml rabbit R1 1 (or R12) mAb in 10 mM sodium acetate (pH 4.5) was immobilized on a CM5 chip by direct amine coupling chemistry. A reference flow cell was left blank. Immobilization of a 300-RU rabbit R1 1 (or R12) mAb resulted in optimal responses for subsequent kinetic analyses. Different concentrations of ROR1 ECD sample injections (25-400 nM for R1 1 mAb and 1 .2-100 nM for R12 mAb) were carried out in randomized duplicate runs, at 50 μΙ/min for 120-180 sec followed by a 90-400 sec dissociation period. Regeneration was performed as described above.
[00266] Sensorgrams obtained from kinetic SPR measurements were analyzed by the double subtraction method. The signal from the reference flow cell was subtracted from the analyte binding response obtained from flow cells with immobilized or captured ligands. Buffer reference responses were then averaged from multiple injections. The averaged buffer reference responses were then subtracted from analyte binding responses, and the final double-referenced data were analyzed with BIACORE™ T200 Evaluation software (2.0, GE), globally fitting data to derive kinetic parameters (Table 4). All sensorgrams were fitted using a simple one-to-one binding model.
Table 4. Binding results to monomeric human ROR1 ECD by SPR.
Figure imgf000163_0001
R11 VHVL scFv-Fc ROR015 scFv-Fc 7 3.5E+5 2.6E-3
R11 VLVH scFv-Fc ROR063 scFv-Fc 8 4.4E+5 3.3E-3
R11 L2H0 scFv-Fc ROR073 scFv-Fc 6 4.1 E+5 2.4E-3
R11 L2H9 scFv-Fc ROR081 scFv-Fc 146 2.7E+5 4.0E-2
R11 L6H0 scFv-Fc ROR091 scFv-Fc 2 6.0E+5 1.3E-3
R11 L6H9 scFv-Fc ROR101 scFv-Fc 48 4.3E+5 2.1 E-2
R11 HL.XCD3 scFv-Fc-
ROR020 scFv-Fc-scFv 5 2.7E+5 1.3E-3 scFv
R11 LH X CD3 scFv-Fc-
ROR070 scFv-Fc-scFv 8 4.1 E+5 3.2E-3 scFv
R12 Rabbit mAb ROR069 mAb 2 2.5E+5 5.5E-4
R12 VHVL scFv-Fc ROR016 scFv-Fc 1 3.1 E+5 3.7E-4
R12 HLxCD3 scFv-Fc ROR021 scFv-Fc-scFv 2 2.2E+5 3.8E-4
R12-H1 L2 X CD3 scFv-
ROR050 scFv-Fc-scFv 13 1.5E+5 1.8E-3 Fc-scFv
[00267] The R1 1 mAb (ROR066) bound to the ROR1 ECD with a KD of 1 17 nM.
Surprisingly, both chimeric monospecific molecules (ROR015, ROR063) bound to the ROR1 ECD with a 14-fold lower affinity, with KD values of 7 and 8 nM, respectively. The net decrease in KD appeared to be due to both an increase in on-rate (Ka) by 2.5 to 3 fold over the parental antibody and a decreased off-rate (Kd) by 4.5 to 7 fold over the parental antibody. This dramatic improvement in binding affinity from conversion of an antibody to the scFv-Fc format was unexpected. Binding of chimeric bispecific molecules containing the rabbit R1 1 scFv (ROR020, ROR070) was comparable to the parent chimeric monospecific molecules (ROR015, ROR063). Monospecific, hemi-humanized R1 1 constructs containing a humanized VL domain and a rabbit VH domain (ROR073, ROR091) showed comparable binding to the parental chimeric constructs as well. Fully humanized R1 1 constructs
(ROR081 , ROR101) showed lower affinities to binding ROR1 compared to the chimeric or hemi-humanized constructs.
[00268] In contrast, the R12 mAb (ROR069) bound the ROR1 ECD with a lower KD of 2 nM, and chimeric R12 molecules (ROR016, ROR021) had comparable binding affinities, suggesting no change in binding affinity upon scFv conversion. A bispecific molecule containing a humanized R12 scFv (ROR050) had a higher KD of 13 nM, which primarily appeared to be due to a roughly 5-fold increase in the off-rate (Kd). EXAMPLE 3: Binding of chimeric and humanized molecules to ROR1 (+) cell lines
[00269] To confirm that binding to ROR1 on the surface of cancer cells was not lost upon scFv conversion or humanization of the variable domains, flow cytometry was used to quantitate binding of constructed ROR1 -binding molecules and parental antibodies to cancer cell lines expressing ROR1 .
Binding of chimeric and humanized monospecific proteins to RORK+) cell lines
[00270] Binding studies on ROR1 (+) (MDA-MB-231 (Zhang et al., 2012, PLoS ONE 3: e31 127); KASUMI-2 (Dave et al., 2012, PLoS ONE 12: e52655)) and ROR1 (-) (Ramos, Baskar et al., 2013, mAbs 4:3, 349-361) cancer cell lines were performed by standard flow cytometry- based staining procedures. The MDA-MB-231 cell line was obtained from ATCC (Manassas, VA) and the Kasumi-2 cell line was obtained from DSMZ (Braunschweig, Germany). Both cell lines were cultured according to the provided protocols. A typical experiment would label 200,000 cells per well, in 96- well plates, with a range of 500 nM to 0.1 nM binding molecule in 100 μΙ of saline buffer with 2%BSA and 2mM EDTA, for 30 min on ice, followed by washes and incubation with PE-labeled secondary antibody, goat anti- human IgG Fey (Jackson Laboratory), and 7-Aminoactinomycin D (7-AAD) staining solution (SIGMA™) for 20 minutes on ice. Signal from bound molecules was detected using a LSR- II™ flow cytometer (BD Biosciences) and analyzed by Flow Jo flow cytometry analysis software. Mean fluorescence intensity (MFI) of bound molecules on live cells was determined after exclusion of doublets and 7-AAD+ cells. Nonlinear regression analysis to determine EC50 values was performed in GraphPad Prism 6® graphing and statistics software.
[00271] Figure 1 shows the binding of four humanized R12 molecules (ROR033 to
ROR036) and the chimeric parental R12 molecule (ROR016) to the ROR1 (+) cell line, Kasumi-2. All the humanized molecules had comparable cell binding to ROR016. Binding of the chimeric R1 1 molecule (ROR015) required higher concentrations of ROR1 -binding molecule and saturated at a lower level of fluorescence. Figure 2 shows the binding of two chimeric R1 1 molecules (ROR015, ROR063) to another ROR1 (+) cell line, MDA-MB-231 . Surprisingly, ROR063 with its scFv in the VL-VH orientation showed a five-fold lower EC50 value and a rough doubling of overall binding at saturation when compared to ROR015 that had its scFv in the VH-VL orientation. This was highly unexpected, as both constructs show similar affinity to the recombinant ROR1 ectodomain by surface plasmon resonance (Example 2). Figure 3 shows that the hemi-humanized R1 1 molecule (ROR091) had similar binding to MDA-MB-231 cell line as the chimeric molecule (ROR063). However, the fully humanized molecule (ROR101) had a lower level of binding and saturation, analogous to the lower affinity seen for the molecule in the previous SPR study (Example 2).
Binding of monoclonal antibodies to RORK+) cell line
[00272] Binding studies comparing the rabbit monoclonal to the monospecific ROR1 -binding proteins were performed on the ROR1 (+) cell line, MDA-MB-231 , by standard flow cytometry- based staining procedures. A typical experiment would label 10,000 cells per well, in 96-well plates, with a range of 100 nM to 0.1 nM binding molecule in 100 ul of saline buffer with 1 % BSA and 2mM EDTA, on ice, followed by washes and incubation with one of two fluorescently-labeled secondary antibodies, either goat anti-human IgG (Invitrogen) or goat anti-rabbit IgG (Jackson ImmunoResearch). After washing secondary antibody off cells, signal from bound molecules was detected using a GUAVA flow cytometer (EMD Millipore, Billerica, MA) and analyzed by CytoSoft flow cytometry analysis software to obtain the MFI.
[00273] Figure 4A shows the binding of the parental R1 1 antibody (ROR066) to MDA-MB- 231 cells contrasted with the binding of the chimeric R1 1 molecule with the scFv in the VL- VH orientation (ROR063). Surprisingly, a dramatically higher overall level of binding was observed from the chimeric R1 1 molecule compared to the parental antibody. Figure 4B shows the binding of the parental R12 antibody (ROR069) and the chimeric R12 molecule (ROR016), displaying similar binding patterns.
Binding of bispecific ROR1 -bindinq molecules to RORK+) cell line
[00274] Binding characteristics of the bispecific humanized molecules (ROR050, ROR084) were compared with those of the parental bispecific chimeric molecules (ROR021 and ROR070). Binding studies were performed by standard flow cytometry-based staining procedures used on ROR1 (+) Kasumi-2 B-cell precursor leukemia cell line. All labeling and washes were performed in U-bottom 96-well plates in saline buffer with 0.1 % BSA and 2mM EDTA. Kasumi-2 cells were plated at 200,000 cells per well and incubated with a range of 0.1 nM to 200 nM concentrations of test molecules in 50 μΙ volume/well, for 30 minutes on ice. Cells were washed three times then incubated for another 30 min on ice with fluorescently-labeled secondary polyclonal antibody, F(ab')2 goat anti-human IgG (Jackson ImmunoResearch Laboratories) and 7-AAD. The cells were then washed twice and the samples acquired in a BD LSRII flow cytometer. The sample files were analyzed using FlowJo software; the mean fluorescence intensity (MFI) of the live population of Kasumi-2 cells in each well was calculated after gating on live cells (forward vs side scatter, 7-AAD" cells). [00275] In this assay, the bispecific chimeric R12 molecule ROR021 showed a higher affinity to Kasumi-2 cells compared to the bispecific humanized R12 molecule (ROR050) (Figure 5). This difference was not previously observed with the monospecific chimeric and humanized R12 molecules (Figure 1). Also, ROR070, another chimeric bispecific R1 1 molecule showed a higher affinity to ROR1 (+) cells compared to the humanized R1 1 molecule containing the same binding domain (ROR084) (Figure 5).
EXAMPLE 4: Redirected T-cell cytotoxicity against ROR1 (+) cell lines
Redirected T-cell cytotoxicity assessed by chromium-51 release
[00276] To compare the effectiveness of different bispecific ROR1 -binding molecules at inducing target-dependent T-cell cytotoxicity in the short term (4 hours), four different bispecific anti-ROR1 x anti-CD3 molecules were compared in a chromium-51 release assay.
[00277] MDA-MB-231 and Kasumi-2 cell lines were both cultured according to the provided protocols. Peripheral blood mononuclear cells (PBMC) were isolated from human blood using standard ficoll gradients. The isolated cells were washed in saline buffer. T-cells were additionally isolated using a Pan T-cell Isolation Kit (catalogued 30-096-535, Miltenyi Biotec, Bergisch Gladbach, Germany) using the manufacturer's protocol. Concentrations of bispecific molecules with final concentration ranging from 200 pM to 0.01 pM were added to isolated T-cells (approximately 100,000). A total lysis control was generated by including 0.04% NP-40 as the treatment.
[00278] Approximately 2.5x106 target Kasumi-2 or MDA-MB-231 cells were treated with
0.125 mCi of 51Cr and incubated for 90 minutes at 37°C. After incubation, cells were washed 4 times with the corresponding target cell culture media (RPMI with 10% FBS for Kasumi-2 cell line or DMEM with 10% FBS for MDA-MB-231 cell line) and re-suspended in 12.5 mL of media. From this suspension, 50 μΙ_ was dispensed per well into 96 well U-bottom plates (approximately 10,000 cells/well) to bring the T-cell to target cell ratio to 10:1 .
[00279] Plates were incubated for 4 hours at 37°C, 5% C02 in a humidified incubator, after which they were centrifuged at 178 g for 3 minutes, and 25 μΙ_ of supernatant was transferred from each well to the corresponding well of a 96-well Luma sample plate.
Sample plates were allowed to air dry in a chemical safety hood for 18 hours, and then radioactivity was read on a Topcount scintillation counter using a standard protocol.
[00280] In Figure 6, the humanized bispecific R12 molecule (ROR050) exhibited potent RTCC activity against MDA-MB-231 cells with an EC50 value of 2.79 pM, comparable to the EC50 value of 1.36 pM for the parental chimeric bispecific R12 molecule (ROR021). Figure 7 shows that the bispecific chimeric R1 1 molecule, ROR070, with the R1 1 scFv in the VL-VH orientation displayed about five-fold higher potency in RTCC activity over ROR020 with the R1 1 scFv in the VH-VL orientation, consistent with the cell binding data. This demonstrated that the unusual and unexpected improvement in on-cell binding from the VL-VH orientation of the R1 1 scFv also translated into improved in vitro potency.
Redirected T-cell cytotoxicity assessed by flow cytometry
[00281] To compare the effectiveness of different bispecific ROR1 -binding molecules at inducing target-dependent T-cell cytotoxicity over a longer period (18 hours), four bispecific anti-ROR1 x anti-CD3 molecules were compared. Two different chimeric bispecific molecules (ROR021 and ROR070) were tested along with two different humanized molecules (ROR050 and ROR084).
[00282] Cytotoxicity was assessed using a flow cytometric assay using a ROR1 (+) cell line, Kasumi-2. Kasumi-2 cells (ROR1 (+)) were cultured according to the provided protocol. Peripheral blood mononuclear cells (PBMC) were isolated from human blood using standard density-gradient separation methods. The isolated cells were washed in saline buffer. T- cells were additionally isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE-labeled T-cells were plated in U-bottom 96-well plates at 100,000 cells/well, with 30,000 unlabeled Kasumi-2 target cells/well, to achieve approximate T-cell to tumor cell ratios of 3: 1 . Concentrations of test molecules ranging from 1 nM to 0.1 pM were added to the cell mixtures to a final volume of 200 μΙ/well, in RPMI 1640 media supplemented with 10% human AB serum, sodium pyruvate, antibiotics and non-essential amino acids. Plates were incubated for 18 hours at 37°C, 5% C02 in a humidified incubator. Plates were subsequently centrifuged briefly to tighten cell pellets and remove supernatant; each well was then resuspended in 180 μΙ of saline buffer with 0.1 % bovine serum albumin and 2mM EDTA, containing 7AAD for 30 min on ice. At the end of 30 minutes, 20 μΙ of Counting Beads (CountBright, Invitrogen, Cat #C36950, Lot 987252) were added per well, and 100 μΙ volumes/well were immediately collected in a BD LSRII flow cytometer. The sample files were analyzed using FlowJo software. Percentages of live Kasumi-2 cells were determined as the fraction of 7AAD" events with the CFSE" population. Microsoft Excel was used to calculate the percentages and numbers of live target (Kasumi-2) cells/well and graphs were plotted using GraphPad Prism.
[00283] Results showed a strong T-cell directed cytotoxicity against Kasumi-2 cells in the presence of T-cells and the bispecific R1 1 molecules (ROR070, ROR084), showing only less than 10% live target cells remaining after 18 hours at a concentration between 50 and 1000 pM (Figure 8). The bispecific R12 molecules (ROR021 , ROR050) showed a comparatively lower level of Kasumi-2 cell lysis (ROR1 +) at this timepoint, reaching maximum activity between 50 and 1000 pM (Figure 8). The differences in relative activity between the R1 1 molecules and R12 molecules at 18 hours were more pronounced than those seen at shorter timepoints (Figures 6 and 7).
EXAMPLE 5: Target-dependent T-cell proliferation induced against ROR1 (+) cell line by anti-ROR1 bispecific molecules
[00284] To compare the effectiveness of different bispecific ROR1 -binding molecules at inducing target-dependent T-cell proliferation, four different anti-ROR1 x anti-CD3 bispecific molecules including ROR021 and ROR070 (chimeric molecules), ROR054 and ROR084 (humanized molecules) were tested in parallel.
[00285] T-cell proliferation was assessed by flow cytometry using a ROR1 (+) cell line, Kasumi-2. Peripheral blood mononuclear cells (PBMC) were isolated from human blood using standard density-gradient separation methods. The isolated cells were washed in saline buffer. T-cells were further isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol.
[00286] Proliferation was assessed by labeling isolated T-cell populations with CFSE.
CFSE-labeled T-cells were plated in U-bottom 96-well plates at 100,000 cells/well, respectively, with 30,000 Kasumi-2 tumor cells/well, to achieve approximate T-cell to tumor cell ratios of 3: 1 . Concentrations of test molecules ranging from 10 nM to 0.1 pM were added to the cell mixtures to a final volume of 200 μΙ/well in RPMI 1640 media supplemented with 10% human AB serum, sodium pyruvate, antibiotics and non-essential amino acids. Plates were incubated at 37°C, 5% C02 in humidified incubators. After 4 days, cells were labeled with antibodies for flow cytometric analysis in original plates to minimize cell losses, using saline buffer with 0.1 % bovine serum albumin and 2 mM EDTA. After centrifugation and removal of supernatant, the cell pellets were resuspended with a mixture of the following dye-labeled antibodies in 50 μΙ volumes: CD5-APC, CD8-Pacific Blue, CD25-PE-Cy7, and 7AAD, and incubated for 30 min on ice. Cells were washed twice and resuspended in 100 μΙ volumes immediately prior to acquisition of 50% of each well in a BD LSRII flow cytometer. The sample files were analyzed using FlowJo software to calculate the percentages of CD4+ (CD8")or CD8+ T-cells that had undergone at least one cell division, according to their CFSE profile, by gating sequentially on forward vs side scatter, 7AAD", CD5+, CD4+ or CD8+ T-cells (7AAD", CD5+ CD8" or 7AAD" CD5+ CD8+, respectively). Graphs were plotted using
GraphPad Prism. [00287] Analysis of dividing T-cell populations (Figures 9A and 9B) revealed a significant increase in the percent of proliferating cells in the presence of Kasumi-2 cells. All molecules showed robust induction of T-cell proliferation at low concentrations (10 pM), and proliferation was slightly higher in frequency in the CD8+ (Figure 9B) than the CD4+ (CD8") (Figure 9A) T-cell population.
EXAMPLE 6: Testing inhibition of tumor growth in a mouse xenograft model
[00288] To compare the effectiveness of different bispecific molecules directed against ROR1 at inhibiting tumor growth in a mouse xenograft model, ROR1 -directed molecules are tested in the following experiments.
[00289] Prophylactic treatment, or prevention of tumor engraftment of subcutaneous tumors: Cultured tumor cell lines (e.g., Kasumi-2, MDA-MB-231 , MDA-MB-157, HCC1 187, HCC70, MiaPaCa-2, KP-4, AsPC-1 , PANC1 , MDA-PCa-2b, and/or A375) are mixed with human lymphocytes (e.g., human peripheral blood mononuclear cells or purified T-cells) and injected subcutaneously into immunodeficient mice (such as SCID, NOD/SCID, etc.).
Bispecific molecules are injected intravenously on the day of injection and, optionally, on several subsequent days. Bispecific molecules are evaluated for a dose-dependent inhibition of tumor outgrowth, as assessed by tumor volume, metastasis and/or an increase in median survival when compared to control.
[00290] Therapeutic treatment, or regression of previously established subcutaneous tumors: Cultured tumor cell lines (e.g., Kasumi-2, MDA-MB-231 , MDA-MB-157, HCC1 187, HCC70, MiaPaCa-2, KP-4, AsPC-1 , PANC1 , MDA-PCa-2b, A375) are injected
subcutaneously into immunodeficient mice (such as SCID, NOD/SCID, etc.). Tumor growth is monitored, and the study is initiated when tumors show signs of established growth (typically a volume of ~200 mm3). Human lymphocytes (e.g., human peripheral blood mononuclear cells or purified T-cells) are injected intravenously or intraperitoneally along with, but not mixed with, bispecific molecules on the day of injection and/or may be injected over several subsequent days. Molecules are evaluated for a dose-dependent inhibition of tumor growth, as assessed by tumor volume, and/or an increase in median survival when compared to control.
[00291] Prophylactic treatment, or prevention of tumor engraftment of orthotopic tumors: Cultured tumor cell lines are mixed with human lymphocytes (e.g., human peripheral blood mononuclear cells or purified T-cells) and injected into orthotopic sites in immunodeficient mice (such as SCID, NOD/SCID, etc.). For example, this could include the mammary fat pad of female mice (e.g., MDA-MB-231 , MDA-MB-157, HCC1 187, HCC70 cell lines), the prostate of male mice (e.g., MDA-PCa-2b cell lines), the pancreas (e.g., MiaPaCa- 2, KP-4, AsPC-1 , PANC1) or the tibia (e.g., MDA-MB-231 , M DA- MB- 157, HCC1 187, HCC70, MDA-PCa-2b cell lines). Bispecific molecules are injected intravenously on the day of injection and/or on several subsequent days. Bispecific molecules are evaluated for a dose-dependent inhibition of tumor growth, as assessed by serum biomarkers, radiography, fluorescent imaging, luminescence, weight loss, and other proxy measurements of tumor volume or disease progression, and/or an increase in median survival when compared to control.
[00292] Therapeutic treatment, or regression of previously established orthotopic tumors: Cultured tumor cell lines are injected into orthotopic sites in immunodeficient mice (such as SCID, NOD/SCID, etc.). For example, this could include the mammary fat pad of female mice (e.g., MDA-MB-231 , MDA-MB-157, HCC1 187, HCC70 cell lines), the prostate of male mice (e.g., MDA-PCa-2b cell lines), the pancreas (e.g., MiaPaCa-2, KP-4, AsPC-1 , PANC1) or the tibia (e.g., MDA-MB-231 , MDA-MB-157, HCC1 187, HCC70, MDA-PCa-2b cell lines). Tumor growth is monitored, and the study is initiated when tumors show signs of established growth (typically a volume of -200 mm3). Human lymphocytes (e.g., human peripheral blood mononuclear cells or purified T-cells) are injected intravenously and/or intraperitoneally along with bispecific molecules on the day of injection and/or bispecific molecules are injected several subsequent days. Bispecific molecules are evaluated for a dose-dependent inhibition of tumor growth, as assessed by serum biomarkers, radiography, fluorescent imaging, luminescence, weight loss, and other proxy measurements of tumor volume or disease progression and/or an increase in median survival when compared to control.
EXAMPLE 7: Determination of pharmacokinetics in a representative non-clinical species
[00293] To determine the pharmacokinetics in a relevant non-clinical species, a bispecific anti-ROR1 x anti-CD3 molecule is tested in a Non-Human Primate (NHP) species (e.g., cynomolgus monkeys) for pharmacokinetic (PK) and toxicology assessment of the anti- ROR1 x anti-CD3 molecule. Cross-reactivity of the anti-ROR1 binding domain to NHP ROR1 is tested by ELISA or surface plasmon resonance assays (similar to Example 2). Since there is 100% sequence identity between NHP and human ROR1 , equivalent binding is expected. PK and immunogenicity assays can be developed and used to detect and quantify the anti-ROR1 x anti-CD3 molecule in the presence of cyno serum, as well as to evaluate the immunogenicity response in NHP. Upon successful completion of these tasks to justify cynomolgus monkeys as a relevant tox species for ROR1 -targeting molecules, a single dose PK/tolerability experiment is performed. The study can be performed at a qualified clinical research organization (CRO). The study results are examined for PK parameters and any adverse events including unexplainable adverse events that cannot be attributed to the mechanism of action of the drug or to anti-drug antibodies (ADA). A toxicologist verifies and interprets the study results and supports continued development of the lead. PK parameters derived from this study are used to design future toxicology studies in non-human primates and/or clinical studies in human patients.
EXAMPLE 8: Testing of safety and tolerability upon human clinical administration
[00294] To determine the safety and tolerability in humans of a lead bispecific molecule targeting ROR1 and CD3, the following phase 1 clinical trial could be conducted. The first-inhuman study of an anti-ROR1 x anti-CD3 bispecific molecule can be a dose-escalation study to identify the maximum tolerated dose (MTD) in human administration.
[00295] As bispecific anti-ROR1 x anti-CD3 molecules are agonistic, the starting dose is set at a dose producing the Minimum Anticipated Biological Effect Level (MABEL) based on in vitro activity of anti-ROR1 x anti-CD3. Human pharmacokinetics (PK) are estimated using PK values determined in non-clinical models (mice or non-human primates) and allometric scaling to predict a dose yielding the MABEL. Bispecific molecule is dosed using either intravenous infusion or subcutaneous injection. Dose escalation follows a standard 3+3 design, with an anticipated 12 dose cohorts (N=24-72 patients). Dose frequency is dependent on observed PK in non-clinical studies, but could be weekly (QW), every other week (Q2W), every third week (Q3W), or monthly (Q4W). Dosing continues until disease progression (as defined by either the immune-related response criteria (irRC) or the response criteria in solid tumors (RECIST)).
[00296] Primary endpoint of the study is safety, defining an MTD and any dose limiting toxicities. Secondary endpoints of the study is PK, immunogenicity, and objective responses in tumor volume assessed either by irRC or RECIST criteria. For patients with hematologic malignancies, additional criteria may be assessed, such as the presence of minimal residual disease (MRD status). As appropriate or feasible, biomarker samples are taken from whole blood as well as from solid tumor biopsies to monitor the effects on the immune system as well as effects on the tumor. Where applicable, other indication-specific biomarkers are measured, including circulating tumor cells (CTC), prostate serum antigen (PSA), serum TAG 72, etc.
[00297] Inclusion criteria for a phase 1 study may be broad and allow for inclusion of patients with refractory or relapsed disease from multiple indications where ROR1 expression has been previously shown to be high, such as chronic lymphocytic leukemia, mantle cell lymphoma, triple negative breast cancer, pancreatic cancer, non-small cell lung cancer, bladder cancer, melanoma, adrenal cancer, and/or castration-resistant prostate cancer. Patients entering the study require evidence of ROR1 expression from
immunohistochemical (IHC) analysis from either archival biopsy samples of the primary tumor or from a pre-treatment biopsy of a recurrent tumor from a metastatic site.
[00298] A bispecific anti-ROR1 x anti-CD3 molecule is determined to be sufficiently safe if patients dosed at the MTD or dose levels below the MTD show evidence of clinical benefit, either from objective responses from the irRC or RECIST criteria or changes in potentially prognostic serum biomarkers, such as CTC, PSA, or TAG72.
Table 5. Human ROR1 sequences
Figure imgf000173_0001
gctcgcaatattttaatcggagagcaacttcatgtaaagatttcagactt ggggctttccagagaaatttactccgctgattactacagggtccagagta agtccttgctgcccattcgctggatgccccctgaagccatcatgtatggc aaattctcttctgattcagatatctggtcctttggggttgtcttgtggga gattttcagttttggactccagccatattatggattcagtaaccaggaag tgattgagatggtgagaaaacggcagctcttaccatgctctgaagactgc ccacccagaatgtacagcctcatgacagagtgctggaatgagattccttc taggagaccaagatttaaagatattcacgtccggcttcggtcctgggagg gactctcaagtcacacaagctctactactccttcagggggaaatgccacc acacagacaacctccctcagtgccagcccagtgagtaatctcagtaaccc cagatatcctaattacatgttcccgagccagggtattacaccacagggcc agattgctggtttcattggcccgccaatacctcagaaccagcgattcatt cccatcaatggatacccaatacctcctggatatgcagcgtttccagctgc ccactaccagccaacaggtcctcccagagtgattcagcactgcccacctc ccaagagtcggtccccaagcagtgccagtgggtcgactagcactggccat gtgactagcttgccctcatcaggatccaatcaggaagcaaatattccttt actaccacacatgtcaattccaaatcatcctggtggaatgggtatcaccg tttttggcaacaaatctcaaaaaccctacaaaattgactcaaagcaagca tctttactaggagacgccaatattcatggacacaccgaatctatgatttc tgcagaactgtaaaatgcacaacttttgtaaatgtggtatacaggacaaa ctagacggccgtagaaaagatttatattcaaatgtttttattaaagtaag gttctcatttagcagacatcgcaacaagtaccttctgtgaagtttcactg tgtcttaccaagcaggacagacactcggccagaaaaaaaaaaaaaaaaaa aaaacaagcaaacaaaaacattgtgggatgtgcactccattggagtgcat gacatggcattgggattggaacatgtggtttcgagcactgaaagctgcaa accagtgaagaggaaaagaaccttgtgattaaatataaaaccaaaagtca aatggtgctttgtgttttagccttcagtcaccatgactggtctctccccc agatgtatatataccatagcatttgtctacctgctgtcttttcttcagga cagatgttcaggaattatattgattgaatttagactctgtgcatgttctt atggaaatgatgttcagaatccatgaagaaacttcaggccaaatttgaaa ccctggagggaaatgagccataagggaagtataacaagccctgaagcctt ttatgtcgttgtgcttctttgggaaggtgtagagtgtgcctttttgtgaa tcctcctctgatcatgagggtctttcccacagtttctcacagtgtgttta cactgcccttggaataacacagcgcatcagaccataagaaggctagatgt ggatgctagaattgattgttggttgatagttctctttgctggattaggaa tgaggegccaaaggaagcacagccaggaaaatggccccacagcctagate agcatctgtgggaaaggaaaaagggttcccatggggacagcccccatagg agttttctggaaccagtaacactgaaaaataagtgtgtggctacagatga gcacgcccaccccttgcaactccctgtttacaagttgtccgaggcattgg agtgcttatggtcaatgggctctagggaagtaggaaactccatcatgata caaatgtctagtaattttaaagtttctttccctttttttctgtgctggaa atgttcacagatttgattcccgccccaagaattacaacaatgtattcata cagtcagttggaatccaaaggcaattaattctattttgcaaaatatgatg gtcttcctaaaaaacaagtactgagttctcatttcaaaagttaccaagaa ctgaattctttaaactagcaaaccgaagtaacatgaccatttttgcctta gtgggaggtttcaaatgtgcagctcatggcatttacctgccgaccatctt ttgccaagtttagaattcttatgcgtttcaagttctatatagaaagtaat tttacttttgatttttctcttgttaaaaaaaaactcctttattctaagaa caatgtctcaaagtctcatttttactttaaaggtataagagacttctaaa gagacttacgggatataaaagtaattcctggaaatgatatttgatgagga gaatgtaagagaatgaaaaacacattgatgttttcatttttaaaaaatac aactagcatgagaaatcaagaaaatatgtttaccaaaatgcattgcaatt ttcccaaacctgagtcttcaaataacaaacatgaacttataggtactgtg aactagaagaattggttatatccagatttctgggagataaattaaaacat atttttgtggcataaataagctgattcagaagttacatttcttaacttta ggtagaggagaatatttgtatccttttgttgctatgcaactgtttaatga tgaagcttcaaaccacaattttgtatatcatatgacaatcaattgttttc caagaatatttattattttaagtacacaatttcagtgaatcttgagtttt tctaggagtcccttaaagggaaattagcattccatgagccagtaaaatac ctactctggtaaaacattattatggttaaaaagtaaattcaagttagttt tttataaagaactataacatttattttaaacattttataataactgaaaa cattaaagtgagcaaatgaaatttcaaaaccacttgtaataatgtatttt ataatcgcactgtgatactatataacacagtctcttttgtattaaaatag tatttttttcacaagctgaaaaatatcttttcctttgttgacaatgtttt gtaagatgactttattttcagatctttttctccttttttgcacaaaacta tgcttatggtttgtgtcacagaagtgaaaatatatctttgcatttttata tctggtctgtttttcttgtttcctttgtttttaacttgatatagattttc ttcaaatatataatggcaattttcagatatctcaccttaccatatctttc cttattttcactgcatgcatttaatcactgtattacttaatgtttgattt gttattatgggcatttcaaataggcaagcattgaattgtaatgacaaaaa ggctattttatattaaggatatatgcatttgtatttcacacaccagagat gatattaaacactgattattttatgctgctgtttattaaaaatgtttact ataaaaaaaaaaaaaaaaaaa
ROR1 mhrprrrgtrppllallaalllaargaaaqetelsvsaelvptsswnis s 128 isoforml eInkdsyltldepmnnittslgqtaelhckvsgnppptirwfkndapvvq precursor eprrlsfrstiygsrlrirnldttdtgyfqcvatngkevvsstgvlfvkf
gppptaspgysdeyeedgfcqpyrgiacarfignrtvymeslhmqgeien predicted full
qitaaftmigtsshlsdkcsqfaipslchyafpycdetssvpkprdlcrd length eceilenvlcqteyifarsnpmilmrlklpncedlpqpespeaancirig protein ipmadpinknhkcynstgvdyrgtvsvtksgrqcqpwnsqyphthtftal sequence rfpelngghsycrnpgnqkeapwcftldenfksdlcdipacds kdskekn
kmeilyilvpsvaiplaiallffficvcrnnqks ssapvqrqpkhvrgqn vemsmlnaykpks kakelplsavrfmeelgecafgkiykghlylpgmdha qlvaiktlkdynnpqqwtefqqeaslmaelhhpnivcllgavtqeqpvcm 1 feyinqgdlheflimrsphsdvgcs sdedgtvkssldhgdflhiaiqia agmeyls shffvhkdlaarniligeqlhvkisdlglsreiysadyyrvqs ksllpirwmppeaimygkfs sdsdiws fgvvlweifs fglqpyygfsnqe viemvrkrqllpcsedcpprmyslmtecwneips rrprfkdihvrlrswe gls shts sttpsggnattqttslsaspvsnlsnprypnymfpsqgitpqg qiagfigppipqnqrfipingypippgyaafpaahyqptgpprviqhcpp pksrspssasgststghvtslpssgsnqeanipllphmsipnhpggmgit vfgnksqkpykids kqasllgdanihghtesmisael
ROR1 atgcaccggccgcgccgccgcgggacgcgcccgccgctcctggcgctgct 129 recombinant ggccgcgctgctgctggccgcacgcggggctgctgcccaagaaacagagc ectodomain, tgtcagtcagtgctgaattagtgcctacctcatcatggaacatctcaagt
gaactcaacaaagattcttacctgaccctcgatgaaccaatgaataacat nucleotide
caecaegtctctgggccagacagcagaactgcactgcaaagtctctggga sequence atccacctcccaccatccgctggttcaaaaatgatgctcctgtggtccag
gagccccggaggctctcctttcggtccaccatctatggctctcggctgcg gattagaaacctcgacaccacagacacaggctacttccagtgcgtggcaa caaacggcaaggaggtggtttcttccactggagtcttgtttgtcaagttt ggcccccctcccactgcaagtccaggatactcagatgagtatgaagaaga tggattctgtcagccatacagagggattgcatgtgcaagatttattggca accgcaccgtctatatggagtctttgcacatgcaaggggaaatagaaaat cagatcacagctgccttcactatgattggcacttccagtcacttatctga taagtgttctcagttcgccattccttccctgtgccactatgccttcccgt actgcgatgaaacttcatccgtcccaaagccccgtgacttgtgtcgcgat gaatgtgaaatcctggagaatgtcctgtgtcaaacagagtacatttttgc aagatcaaatcccatgattctgatgaggctgaaactgccaaactgtgaag atctcccccagccagagagcccagaagctgcgaactgtatccggattggc attcccatggcagatcctataaataaaaatcacaagtgttataacagcac aggtgtggactaccgggggaccgtcagtgtgaccaaatcagggcgccagt gccagccatggaactcccagtatccccacacacacactttcaccgccctt cgtttcccagagctgaatggaggccattcctactgccgcaacccagggaa tcaaaaggaagctccctggtgcttcaccttggatgaaaactttaagtctg atctgtgtgacatcccagcgtgcgattcaaaggattccaaggagaagaat aaactcgagctcaacgatatttttgaagcccaaaaaattgagtggcatga agattacaaggacgatgacgacaaagactataaggacgacgacgataagg attacaaggatgacgatgataagcaccatcatcatcaccatcaccaccac cactga
ROR1 mhrprrrgtrppllallaalllaargaaaqetelsvsaelvptsswnis s 130 recombinant elnkdsyltldepmnnittslgqtaelhckvsgnppptirwfkndapvvq
ectodomain, eprrlsfrstiygsrlrirnldttdtgyfqcvatngkevvsstgvlfvkf
amino acid gppptaspgysdeyeedgfcqpyrgiacarfignrtvymeslhmqgeien
qitaaftmigtsshlsdkcsqfaipslchyafpycdetssvpkprdlcrd sequence
eceilenvlcqteyifarsnpmilmrlklpncedlpqpespeaanci rig ipmadpinknhkcyns tgvdyrgtvsvtksgrqcqpwnsqyphthtftal rfpelngghsycrnpgnqkeapwcftldenfksdlcdipacds kdskekn klelndi feaqkiewhedykddddkdykddddkdykddddkhhhhhhhhh h
EXAMPLE 9: Phage Display to Improve Binding of Anti-ROR1 Humanized Molecules
[00299] Overall binding affinity of humanized anti-ROR1 bispecific molecules containing the L6 and H9 humanized variable domains was evaluated by SPR analysis (see Table 4, Example 2) and found to be below that of the parental rabbit variable domains. To improve the overall affinity, error-prone PCR was used for mutagenesis to create a library of variant humanized scFv molecules, which were then transferred to a phage display vector to produce a phage library. This library was then panned in the presence of a chaotropic agent to identify scFv variants with higher affinity binding to recombinant ROR1 ectodomain.
[00300] Methods
[00301] To clone the scFv for R1 1-L6H9 into the phagemid vector, the mammalian expression vector ROR101 (see Table 3) was digested with Age\ and Xho\, and the band corresponding to the scFv was gel purified (Qiagen Gel Extraction kit) following the manufacturer's protocol. The purified fragment was subsequently ligated into a phagemid vector backbone containing a gene 3 fragment that had previously been digested using the same enzymes, generating the construct R1 1-L6H9-g3.
[00302] Error-prone PCR was carried out to construct a random mutagenesis library of the R1 1-L6H9 scFv. First, a 1421 bp restriction fragment containing the scFv was generated by digesting phage-R1 1-L6H9 using Hind\\\ and EcoRI. This fragment was then gel purified and used as template in an error-prone PCR reaction using a commercial mutagenesis kit (GeneMorph II Random Mutagenesis Kit, Agilent Technologies) following the manufacturer's protocol. Approximately 2.5 ng of scFv, corresponding to 1.5 ng of the scFv target gene, was amplified using forward primer phage-F6 (5 -
ACTTTTATTCATGATCCCGTTAGTTGTACCGTTCGTGGCCCAGCCG (SEQ ID NO:364)) and reverse primer phage-R4 (3'-ATGGTGATGATGATGACTCGAG (SEQ ID NO:365)). The PCR library was then digested using Age\ and Xho\ and PCR purified (Qiagen PCR Purification kit) following the manufacturer's protocol. Approximately 2.7 ug of the digested PCR library was ligated into 6 ug of phagemid vector backbone in a 200 μΙ_ reaction containing 50 mM Tris-HCI (pH 7.5), 10 mM MgCI2, 1 mM ATP, 10 mM DTT, and 5000 U T4 ligase (NEB). The ligation reaction was incubated overnight at 16°C and PCR purified (Qiagen PCR Purification kit) following manufacturer's protocol, with a final elution volume of 70 μΙ_. The entire volume was transformed into electrocompetent E. coli SS320 cells that had previously been infected with M 13K07 helper phage, as described in Tonikian et al. (2007). The electroporated cells were rescued in 25 mL prewarmed SOC and incubated at 37°C with shaking at 200 rpm for one hour. The culture was then transferred to a 2-L baffled shake flask containing 500 mL 2YT media supplemented with 50 mg/L carbenicillin and 25 mg/L kanamycin and incubated overnight at 37°C with shaking at 200 rpm.
[00303] The phage library was rescued by centrifuging the overnight culture at 16,000 g for 10 minutes at 4°C. The supernatant was transferred to a fresh tube containing 1/5 volume of 20% PEG-8000 in 2.5M NaCI to precipitate the phage. After incubating for 5 minutes at room temperature, the culture was centrifuged at 16,000 g for 10 minutes at 4°C. The supernatant was discarded, and the phage pellet was resuspended in 10 mL of PBT (PBS, 0.05% Tween-20, 0.5% BSA) buffer. Insoluble matter was pelleted by centrifuging the suspension at 27,000 g for an additional 5 minutes at 4°C. The supernatant was transferred to a new tube, and the phage concentration was estimated by measuring OD268nm on a NanoDrop spectrophotometer.
[00304] Approximately 1x1013 phage particles were incubated in 1 mL of blocking buffer (SuperBlock, LifeTechnologies) for 30 minutes. Subtractive panning was then performed by pre-incubating the blocked phage library with streptavidin beads for 10 minutes. This step was repeated a total of 5 times to eliminate streptavidin-specific scFvs from the pool before incubating with antigen. The depleted phage library was next incubated with 50 nM biotinylated ROR1 extracellular domain for 1 hour. The phage-antigen complex was then incubated with streptavidin beads for 15 minutes. Panning was performed by washing the beads with PBST (PBS, 0.05% Tween-20) for 10 minutes. The washes were repeated a total of 4 times, followed by 2 washes in PBS for 5 minutes each. The bound phage were eluted from the beads by incubating in 1 mL glycine-HCI (pH 2.2) for 10 minutes. All incubations were performed on a rotator at room temperature.
[00305] Next, to prepare exponentially growing cells, 250 μί of an overnight XL1 -Blue culture was diluted into 25 mL 2YT containing 5 mg/L tetracycline and incubated at 37°C with shaking at 250 rpm for approximately 1 .5 hours, until the OD6oonm reached 0.5. To amplify the eluted phage, the 1 mL elution was mixed with approximately 5 mL of XL1 -Blue cells and incubated at 37°C for 30 minutes with gentle shaking. Approximately 2.5x1010 M13K07 helper phage were added to the infected cells and incubated at 37°C for 30 minutes with gentle shaking. Finally, the cells were added to 25 mL of 2YT medium containing 50 mg/L carbenicillin and 25 mg/L kanamycin in a 250-mL shake flask and incubated overnight at 30°C with shaking at 200 rpm. [00306] To purify the phage for the next round of panning, the culture was centrifuged at 27,000 g for 10 minutes at 4°C to pellet the cells. The supernatant was transferred to a new tube containing 1/5 volume of 20% PEG-8000 in 2.5M NaCI and incubated on ice for 30 minutes. The phage were then pelleted by centrifuging the tubes at 12,000 g for 10 minutes at 4°C. The phage pellet was resuspended in 1 mL PBS and centrifuged for an additional 2 minutes to remove remaining insoluble material. The supernatant was transferred to a new tube, and the phage concentration was estimated by measuring OD268nm on a NanoDrop.
[00307] Four additional rounds of panning were performed as described above, with a few exceptions. Approximately 5 x 1012 phage particles were used for each subsequent round of panning. The wash steps were performed as follows: two PBST washes for 12 minutes each, three washes in 0.5M MgCI2 for 12 minutes each, one PBST wash for 12 minutes, and two PBS washes for 5 minutes each. The stringency of selection was increased each round by adjusting the concentration of the MgCI2 washes to 1 M, 1.5M and 2M in rounds 3, 4, and 5 of panning, respectively.
[00308] After the last round of panning, a portion of the infected XL1-Blue cells were streaked onto 2YT agar plates containing 100 mg/L carbenicillin and 2% glucose and incubated overnight at 37°C. Colony PCR was then performed on selected colonies using primers phage-F8 (5'-CAGGCTTTACACTTTATGCTTCCG (SEQ ID NO:366)) and glll-RSP (3'-TGCCTTTAGCGTCAGACTGTAG (SEQ ID NO:367)). The scFv region was sequenced to identify mutations that were enriched during selection.
[00309] Results
[00310] Screening of phage libraries revealed consistent pressure for multiple mutations at particular positions in both the L6 light chain and L9 heavy chain. Key mutations observed are listed in Table 6:
Table 6. Key Mutations Selected in Phage Display Screen
Chain Mutation Location
Light R98W CDRL3
Heavy A25V FR1
Heavy S28T CDRH1
Heavy S40T FR2
Heavy K72E FR3
Heavy G113R CDRH3
[00311] Certain combinations of mutations were observed to correlate across multiple sequences in the presence of additional mutations, such as S40T G1 13R or A25V S28T G1 13R in the heavy chain. These mutations may result in improvements in binding or expression of humanized ROR1 scFv sequences.
[00312] References
[00313] Tonikian, R., Zhang, Y., Boone, C, & Sidhu, S. S. (2007). Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nature Protocols, 2(6), 1368-1386.
EXAMPLE 10. Preparation of Optimized Humanized R11 Sequences
[00314] Given that binding observed for the L6H9 humanized R1 1 scFv was below that observed for the rabbit LH scFv (ROR063) or the hemi-humanized L6H0 scFv (ROR091), additional humanized R1 1 frameworks were constructed and evaluated. One sequence (H10) consisted of rehumanization of the R1 1 CDRs to an alternate human framework (H10). A second sequence (H15) consisted of introducing the F103Y and T105A mutations into the H9 sequence at the start of the CDRH3 to revert these residues to the original human framework. Monospecific scFv-Fc L6H 10 (ROR1 12) and L6H15 constructs were transiently expressed in HEK293 cells, purified, and then examined for binding to ROR1 + Kasumi-2 cells using flow cytometry, following the protocol provided in Example 3. Binding results are shown in Figure 10. The humanized R1 1 construct with the L6H10 variable domain pair (ROR1 12) showed similar levels of binding saturation to the humanized construct with the existing L6H9 variable domain pair (ROR101). However, the construct with the L6H15 variable domain pair (ROR1 1 1) showed higher levels of binding saturation than the other two humanized constructs (L6H9, L6H10). However, binding saturation was not at the same level as a construct featuring the original rabbit binding domains (ROR063).
[00315] The light chain sequence was further examined for potential elements that could impair binding. A putative N-linked glycosylation site in CDRL3 (N94) was identified for mutagenesis, and a new humanized light chain construct was prepared with a N94A mutation (L7). A humanized scFv-Fc construct was prepared (ROR1 19) featuring this new light chain (L7) with the existing H15 humanized heavy chain, expressed, purified, and was similarly assessed for binding to Kasumi-2 cells by flow cytometry (Figure 1 1). Binding saturation of ROR1 19 to Kasumi-2 cells was comparable to the parental rabbit binding domains (data not shown) and the hemi-humanized construct ROR091 . This was a surprising result, as the N-linked glycosylation site is present in the parental rabbit light chain - it was unexpected that mutation of the N-linked glycosylation site would restore binding saturation to parental levels.
[00316] Overall expression levels and relative purity after protein A purification were also examined for these constructs (Table 7). Table 7. Expression Levels and Purity of Humanized R11 Sequences
Construct ID Rll Domains 1st Step Yield (ue/mL) % purity (SEC)
ROR101 L6H9 15.2 42.3
ROR112 L6H10 25.7 77.9
ROR111 L6H15 15.2 43.8
ROR119 L7H15 18.2 72.1
[00317] Expression levels for these new humanized R1 1 constructs featuring the H10, H15 and L7 variable domains were comparable or higher than the previous best humanized molecule based on L6H9 (ROR101). A noticeable increase in purity (>30%) after protein A purification was observed from either switching heavy chains to the H10 framework or switching light chains to the L7 framework. As a result, the L7H10 and L7H15 combinations were chosen for further optimization based on higher expression, higher purity, and improved binding in contrast to the original L6H9 combination.
[00318] Next, the effects of introducing mutations identified from phage display from random mutagenesis of the L6 and H9 sequences was examined in the context of the L7H15 and L7H10 scFv sequences. Examples are shown in Table 8.
Table 8. Examples of Additional Humanized Sequences Tested
Construct ID Original Light Chain Mutations Original Heavy Chain
Light Chain Heavy Chain Mutations
ROR181 L7 n/a H10 A25V S28T G113R
ROR154 L7 n/a H15 S40T G113R
ROR179 L7 R98W H15 S40T G113R
[00319] Constructs were transiently expressed in human HEK293 cells and purified. New constructs were evaluated for binding to recombinant ROR1 by surface plasmon resonance, following the protocol used in Example 2. Binding data for new constructs is shown in Table 9:
Table 9. Binding results to monomeric human ROR1 ECD by SPR.
Molecule Database Molecule D (by SPR) Ka (1/Ms) Kd (1/s)
Name Type (nM)
R11 Rabbit mAb ROR066 mAb 117 1.3E+5 1.5E-2
R11 VLVH scFv-Fc ROR063 scFv-Fc 8 4.4E+5 3.3E-3
R11 L6H9 scFv-Fc ROR101 scFv-Fc 48 4.3E+5 2.1 E-2 R11 L7H15 x CD3 scFv- ROR133 scFv-Fc-scFv 21 2.7E+5 5.7E-3 Fc-scFv
R11 L7H16 x CD3 scFv- ROR154 scFv-Fc-scFv 5 2.6E+5 1.3E-3 Fc-scFv
R11 L8H16 x CD3 scFv- ROR179 scFv-Fc-scFv 2 2.7E+5 4.5E-4 Fc-scFv
R11 L7H10 x CD3 scFv- ROR134 scFv-Fc-scFv 47 2.7E+5 1.3E-2 Fc-scFv
R11 L7H17 x CD3 scFv- ROR181 scFv-Fc-scFv 2 2.3E+5 5.4E-4 Fc-scFv
[00320] Constructs featuring the L7H10 and L7H15 variable domains had comparable dissociation constants (47 nM, 21 nM) for binding to monomeric ROR1 monomer as the L6/H9 variable domain pair (48 nM). Inclusion of the mutations identified by phage display in constructs ROR154, ROR179, and ROR181 led to a noticeable improvement in binding affinity, with a reduction of the dissociation constant to 2-5 nM. These dissociation constants are lower or comparable to that observed with the original rabbit scFv (ROR063), and appear to be due to a 4-to-10 fold reduction in the observed off rate.
[00321] In conclusion, inclusion of mutations determined by phage display resulted in successful affinity maturation of humanized R1 1 scFv constructs to a higher affinity than that of the original rabbit R1 1 scFv construct.
EXAMPLE 11 : Isoelectric Point Engineering of ROR1 Bispecific Constructs
[00322] Bispecific molecules with humanized R1 1 scFv and humanized anti-CD3 scFv components were predicted to have relatively high isoelectric points (8.9-9.2). Protein therapeutics such as monoclonal antibodies with high isoelectric points (>9.0) are highly positively charged at neutral pH values, such as that seen in serum, and have been shown to have reduced serum elimination half-lives in vivo owing to higher rates of non-specific clearance. Reduction of isoelectric point values has been shown in the literature to improve serum elimination half-lives of monoclonal antibodies and other proteins.
[00323] To test if the isoelectric point of molecules featuring humanized R1 1 scFvs could be successfully reduced, framework mutations were introduced into the L7 light chain (K42Q S60D Q79E P80A) and H16 heavy chain (K43Q). Constructs featuring the framework mutations (L8/H19) were transiently expressed and purified, then analyzed by analytical capillary isoelectric focusing chromatography to determine pi values. Comparison of multiple constructs is shown in Table 10: Table 10. pi Values of anti-ROR1 x anti-CD3 Constructs
Construct Rll scFv Anti-CD3 scFv Observed pi
ROR154 L7 H 16 DRA222 9.1
ROR185 L7 H 16 TSC394 E86D F87Y 9.2
ROR182 L9 H 18 DRA222 8.0
ROR192 L9 H 18 TSC394 E86D F87Y 8.4
[00324] In conclusion, inclusion of the framework mutations to lower the net charge of the molecules resulted in a successful reduction of observed pi by 0.8 to 1.1 pH units.
EXAMPLE 12: Serum Stability of Bispecific anti-ROR1 x anti-CD3 Molecules
[00325] Resistance to serum proteases is a key attribute of protein therapeutics and can be correlated with overall pharmacokinetics. Molecules with higher thermodynamic stability are also frequently more resistant to proteolysis, which can improve stability in human serum. This can in turn improve overall serum pharmacokinetics and the overall exposure of a therapeutic.
[00326] Serum stability of bispecific anti-ROR1 x anti-CD3 molecules (humanized R1 1 L7H15) with two different anti-CD3 scFv molecules with differing levels of thermodynamic stability (DRA222, TSC394 F87Y) was evaluated to determine if the stability of the C- terminal anti-CD3 scFv would impact the stability of a bispecific anti-ROR1 x anti-CD3 molecule.
[00327] Method: Human serum donated by a random healthy donor was collected in a Red/Grey Vacutanor (BD# 367988 ), and was prepared according to vendor suggested protocol. Test articles were spiked into 50 μΙ_ serum at a concentration of 1 μΜ in sterile PCR tubes, and were incubated in a humidified 37°C tissue culture incubator for up to 21 days. Specific time points were 21 , 14, 7, 3 and 0 days. Samples in were incubated in a reverse chronological order starting as "assay day 21 ", and all samples were assessed simultaneously using a chromium release RTCC assay at the end of incubation on
"experiment day 0" following the protocol listed above in Example 1. EC50 values were fit from titration curves conducted with samples at each time points and were normalized against the EC50 value measured for each construct at day 0.
[00328] Plotting the EC50 values over time showed a dramatic difference for the observed serum stability of ROR133 vs ROR193 (Figure 12), with a 2.5 fold loss in observed EC50 over 21 days for ROR133 but minimal change in EC5o for ROR193. This demonstrates that a change in thermodynamic stability of one component of a bispecific molecule can have a noticeable impact on serum stability. EXAMPLE 13: Impacts of Improved Thermodynamic Stability on Protein Expression and Quality
[00329] Previously, it has also been shown that improvements in thermodynamic stability can result in improvements in protein expression and overall protein quality, as measured by the production of high molecular weight aggregates during protein production.
[00330] To test whether or not the presence of a more stable anti-CD3 scFv translated into improved protein expression or protein quality for anti-ROR1 x anti-CD3 bispecific
molecules, one of the stabilized anti-CD3 domains (TSC394 E86D F463Y, or TSC394DY for short) was compared to DRA222 in the context of five different pairs of anti-ROR1 x anti- CD3 bispecific molecules, each featuring the same anti-ROR1 scFv (Table 1 1).
Table 11. Relative Expression and Protein Quality
CD3 % binding Expression % SEC (post- % HMW reduction
BD pair Construct ID domain (ug/mL) improvement ProA) aggregate aggregate
A ROR134 DRA222 26 78 22
ROR189 TSC394DY 37 42% 89 11 50%
B ROR154 DRA222 16 91 9
ROR185 TSC394DY 26 63% 94 6 33%
C ROR179 DRA222 14 83 17
ROR186 TSC394DY 27 93% 88 12 29%
D ROR181 DRA222 25 81 19
ROR191 TSC394DY 33 32% 90 10 47%
E ROR182 DRA222 16 90 10
ROR192 TSC394DY 21 31% 92 8 20%
[00331] With each molecule pair, a higher titer of overall protein expression was seen - from 31 % to 93% higher - with the construct featuring the stabilized anti-CD3 scFv (TSC394DY). Also, within each molecule pair, the construct featuring the stabilized anti-CD3 scFv had a lower level of high molecular weight aggregates after protein A purification (anywhere from a 20% to 50% reduction in aggregate levels). This confirms that inclusion of a stabilized anti- CD3 scFv can result in improved protein expression and improved protein quality of anti- ROR1 x anti-CD3 bispecific molecules when compared to the original anti-CD3 scFv.
EXAMPLE 14: Inhibition of growth of MDA-MB-231 xenografts
[00332] To compare the effectiveness of different bispecific molecules directed against ROR1 at inhibiting tumor growth, the ROR1-directed bispecific molecules ROR182 and ROR192 were tested for the ability to inhibit subcutaneous tumor outgrowth of MDA-MB-231 tumors co-implanted with human T-cells in NOD/SCID mice. The day prior to tumor challenge, frozen Leukopak peripheral blood mononuclear cells were thawed and human T- cells were isolated using Pan T Cell Isolation Kit II (Miltenyi Biotech, San Diego, CA).
Purified T-cells were held overnight in complete RPMI media at 37°C. Female NOD/SCID mice were co-implanted in the right flank with a 100μΙ_ mixture containing 2 million MDA-MB- 231 XV-3 triple negative breast tumor cells mixed with or without 1 million human T-cells in 50% high content matrigel on the day zero. Due to the tumor challenge containing matrigel, a measurable mass was present from day 0; however, in the absence of tumor growth, the matrigel mass will naturally resolve over time. Mice were treated with 3 μg anti-ROR1 molecules intravenously on day 0, 4 and 8. Mice were monitored for tumor outgrowth by taking perpendicular measurements with digital calipers. Tumor volume is calculated as (length x width2)/2. Tumor volume and body weight were captured throughout the duration of the study. Significant differences in tumor growth were determined using SAS/JMP software version 10.02 (SAS Institute) and RM ANOVA Add-In with Tukey HSD multiple comparison test.
Table 12. Experimental design
Figure imgf000184_0001
[00333] Treatment with ROR182 and ROR192 proteins significantly inhibited the outgrowth of MDA-MB-231 XV-3 tumors when administered on day 0, 4 and 8 post tumor challenge (Figure 13). No statistical difference was seen between growth inhibition from treatment with the ROR182 and ROR192 molecules in the 3 μg dose groups (Table 13). No treatment-related weight loss was observed with any of the treatment groups (Figure 14).
Table 13. Statistical analysis of the effects of ROR182 and ROR192 proteins on tumor volume
Figure imgf000184_0002
[00334] These results demonstrate that ROR1 -targeted molecules are capable of preventing ROR1 + tumor outgrowth in the presence of human T-cells.
EXAMPLE 15: Mapping of R11 binding epitope on ROR1 ectodomain
[00335] To identify the binding epitope for the R1 1 antibody (ROR066), a series of linear, cyclic, constrained or discontinuous constrained peptides (CLIPS approach) was synthesized corresponding to the ROR1 ectodomain sequence (residues 30-406) and analyzed for binding to the rabbit mAb ROR066 by ELISA (Pepscan Presto BV, Lelystad, Netherlands).
Synthesis of Peptides
[00336] To reconstruct epitopes of the target molecule a library of peptides was synthesized based on the ROR1 ectodomain sequence (residues 30-406). An amino functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with Nhydroxybenzotriazole (HOBt) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA). Standard Fmoc-peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS liquid handling stations (Perkin Elmer).
[00337] Synthesis of structural mimics was done using Pepscan's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows for the synthesis of structured peptides in single loops, double loops, triple loops, sheet-like folds, helix-like folds and combinations thereof. CLIPS templates were coupled to cysteine residues introduced into the peptide sequences. The side-chains of multiple cysteines in the peptide sequences were coupled to one or two CLIPS templates. For example, a 0.5 mM solution of the P2 CLIPS (2,6-bis(bromomethyl)pyridine) was dissolved in ammonium bicarbonate (20 mM, pH 7.8)/acetonitrile (1 :3(v/v)), and this solution was added onto the peptide arrays. The CLIPS template was used to bind to side-chains of two cysteines present in the solid-phase bound peptides of the peptide-arrays (455 well plate with 3 μΙ wells). The peptide arrays were gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrays were washed extensively with excess of H20 and sonicated in buffer containing 1 % SDS/0.1 % beta-mercaptoethanol in PBS (pH 7.2) at 70°C for 30 minutes, followed by sonication in H20 for another 45 minutes. The T3 CLIPS carrying peptides were made in a similar way but using three cysteines. Table 14. Peptide library design.
Figure imgf000186_0001
ELISA Screening
[00338] The binding of antibody to each of the synthesized peptides was tested in a PEPSCAN-based ELISA. The peptide arrays were incubated with primary antibody solution (ROR066 at 7 μg/ml in PBST buffer, overnight at 4°C) after blocking with serum and ovalbumin. After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (SBA) for one hour at 25°C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μΙ/ml of 3 percent H202 were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD) camera and an image processing system.
Synthesis Quality Control
[00339] To verify the quality of the synthesized peptides, a separate set of positive and negative control peptides was synthesized in parallel. These were screened with antibody 57.9 (ref. Posthumus et al., J. Virology, 1990, 64:3304-3309).
Results [00340] When tested under high stringency conditions antibody ROR066 did not bind any peptide present on the arrays. When tested under lower stringency conditions the antibody bound peptides only from set 5 (combinatorial mimics of discontinuous epitopes). ROR066 repeatedly bound peptides containing residues corresponding to stretches 259-273
(LCQTEYIFARSNPMI (residues 259-273 of SEQ ID NO: 128)) and 389-403
(PACDSKDSKEKNKME (residues 389-403 of SEQ ID NO: 128)) of the ROR1 ectodomain, near the junction of the Frizzled and Kringle domains.
[00341] In conclusion, the R1 1 antibody is predicted to bind a discontinuous, conformational epitope near the junction of the frizzled and kringle domains.
Literature References
[00342] Timmerman et al. (2007). Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS™ technology. J. Mol. Recognit. 20:283-99.
[00343] Slootstra et al. (1996). Structural aspects of antibody-antigen interaction revealed through small random peptide libraries, Molecular Diversity] : 87-96.
EXAMPLE 16: In vitro cytotoxicity assays with codon-optimized sequences
[00344] In order to compare the potency of various codon-optimized, anti-ROR1 molecules, PC-3 cells which were stably transfected with GFP were cultured in 96 well plates (BD Biocoat #356640) for 2 days, in the presence of human donor T-cells and anti-ROR1 molecules. ROR1 molecules were added to wells to have final concentrations of 125, 41 .7, 13.9, 4.6, 1 .5, 0.51 , or 0.17 pM, or none, in one experiment. In the other experiment, final concentrations were two-fold higher: 250, 83.3, 27.8, 9.3, 3.1 , 1 .0, or 0.34 pM, or none. Fresh or frozen T-cells from a healthy donor were added at a ratio of 75,000 T-cells to 15,000 PC-3(GFP) cells per well. Culture media during the assay was RPMI-1640 media plus 10% FBS, supplemented with NEAA, sodium pyruvate, Glutamax, 20 mM HEPES (all from Life Technologies), and 55 uM B-mercaptoethanol.
[00345] After 2 days culture at 37°C in 5% C02, media was aspirated from wells and 100 ul of 0.4% NP-40 was added to each well. Fluorescent signal from GFP in PC-3 cells adherent in wells was detected by a Spectramax plate reader. Data were processed to express percent maximum growth for each sample according to the equation: (sample RFU from well with drug added) divided by (sample RFU from well with no drug added). These data were converted to percent specific mortality for each sample by subtracting each value from 100%. The data were fit to a 4-parameter logistic curve and graphed as concentration vs. % specific mortality using GraphPad PRISM® software. [00346] Codon-optimized variants of anti-ROR1 molecules did not vary appreciably in their ability to induce RTCC in this fluorolysis assay, whether observed after 2 or 3 days of coculture of T-cells with target cells. EC50 values for ROR238, ROR242, ROR243, and ROR244 molecules varied from 7.4 to 10.9 pM after 2 days' exposure using fresh primary T- cells and PC3(GFP) target cells (Figure 15A). For ROR241 , ROR250, ROR251 , ROR252, and ROR253 molecules, EC50 values varied from 4.2 to 6.0 pM after 2 days' exposure using frozen primary T-cells and PC3(GFP) target cells (Figure 15B).
[00347] In conclusion, these results demonstrate that the codon-optimized anti-ROR1 molecules tested induce equivalent levels of target-dependent T-cell cytotoxicity.
EXAMPLE 17: On-cell binding assays with codon-optimized sequences
Binding of bispecific ROR1 -bindinq molecules to RORK+) cell line
[00348] Binding characteristics of codon-optimized bispecific anti-ROR1 molecules
(ROR238, ROR239, ROR241 , ROR242, ROR243, ROR244, ROR246, ROR247, ROR248, ROR249, ROR250, ROR251 , ROR252, ROR253) to ROR1 + tumor cells were compared with those of the parental bispecific anti-ROR1 molecules (ROR185 and ROR192). Binding studies were performed by standard flow cytometry-based staining procedures used on ROR1 (+) Kasumi-2 B-cell precursor leukemia cell line. All labeling and washes were performed in U-bottom 96-well plates in saline buffer with 0.1 % BSA and 2mM EDTA.
Kasumi-2 cells were plated at 200,000 cells per well and incubated with a range of 0.012 nM to 200 nM concentrations of test molecules in 50 μΙ volume/well, for 30 minutes on ice. Cells were washed three times then incubated for another 30 min on ice with fluorescently-labeled secondary polyclonal antibody, F(ab')2 goat anti-human IgG, with minimum cross reactivity to other species (Jackson ImmunoResearch Laboratories) and 7-AAD. The cells were then washed twice and the samples acquired in a BD LSRII flow cytometer. The sample files were analyzed using FlowJo software; the mean fluorescence intensity (MFI) of the live population of Kasumi-2 cells in each well was calculated after gating on live cells (forward vs side scatter, 7-AAD" cells) and excluding doublets.
[00349] In these assays, the codon-optimized bispecific anti-ROR1 molecules (ROR238, ROR239, ROR241 , ROR242, ROR243, ROR244, ROR246, ROR247, ROR248, ROR249, ROR250, ROR251 , ROR252, ROR253) showed comparable affinities for binding to Kasumi- 2 cells as the the parental bispecific anti-ROR1 molecules (ROR185 and ROR192) (Figure 16A, Figure 17A, Figure 18A). Binding of bispecific ROR1 -bindinq molecules to CD3(+) T-cell line
[00350] Binding characteristics of codon-optimized bispecific anti-ROR1 molecules
(ROR238, ROR239, ROR241 , ROR242, ROR243, ROR244, ROR246, ROR247, ROR248, ROR249, ROR250, ROR251 , ROR252, ROR253) to CD3(+) T-cells were compared with those of the parental bispecific anti-ROR1 molecules (ROR185 and ROR192). Binding studies were performed by standard flow cytometry-based staining procedures used on CD3(+) Jurkat-60 cell line. Jurkat is a human T lymphocyte cell line (TIB- 152, ATCC). Jurkat- 60 is a TCRH| sub-clone of the Jurkat. All labeling and washes were performed in U-bottom 96-well plates in saline buffer with 3% BSA and 2 mM EDTA. Jurkat-60 cells were plated at 200,000 cells per well and incubated with a range of either 0.012 nM to 200 nM or 0.006 nM to 100 nM concentrations of test molecules in 50 μΙ volume/well, for 30 minutes on ice. Cells were washed three times then incubated for another 30 min on ice with fluorescently-labeled secondary polyclonal antibody, F(ab')2 goat anti-human IgG , with minimum cross reactivity to other species (Jackson I mmunoResearch Laboratories) and 7-AAD. The cells were then washed twice and the samples acquired in a BD LSRI I flow cytometer. The sample files were analyzed using FlowJo software; the mean fluorescence intensity (MFI) of the live population of Kasumi-2 cells in each well was calculated after gating on live cells (forward vs side scatter, 7-AAD" cells) and excluding doublets.
[00351] In these assays, the codon-optimized bispecific anti-ROR1 molecules (ROR238, ROR239, ROR241 , ROR242, ROR243, ROR244, ROR246, ROR247, ROR248, ROR249, ROR250, ROR251 , ROR252, ROR253) showed comparable affinities for binding to Jurkat- 60 cells as the the parental bispecific anti-ROR1 molecules (ROR185 and ROR192) (Figure 16B, Figure 17B, Figure 18B).
Table 15. SEQ ID NO Summary Table
Figure imgf000189_0001
Variant Rll LCDR3 (268)
Rll VH-VL scFv 17 (18)
Rll VL-VH scFv 20 (21)
Humanized Rll VL (L2 ) 22 (23)
Humanized Rll VL (L6) 24 (25)
Humanized Rll VH (H9) 26 (27)
Hemi- Rll VL-VH scFv (L2H0) 28(29) humanized
Hemi- Rll VL-VH scFv (L6H0) 30(31) humanized
Humanized Rll VL-VH scFv (L2H9) 32 (33)
Humanized Rll VL-VH scFv (L6H9) 34 (35)
Rabbit R12 VH 36(37)
Rabbit R12 VL 38(39)
Rabbit R12 HCDR1 40(41)
Rabbit R12 HCDR2 42 (43)
Rabbit R12 HCDR3 44(45)
Rabbit R12 LCDR1 46(47)
Rabbit R12 LCDR2 48(49)
Rabbit R12 LCDR3 50(51)
R12 VH-VL scFv 52 (53)
Humanized R12 VL (LI) 54 (55)
Humanized R12 VL (L2 ) 56(57)
Humanized R12 VH (HI) 58(59)
Humanized R12 VH (H2 ) 60 (61)
Humanized R12 VH-VL scFv (H1L1) 62 (63)
Humanized R12 VH-VL scFv (H1L2) 64 (65)
Humanized R12 VH-VL scFv (H2L1) 66 (67)
Humanized R12 VH-VL scFv (H2L2) 68 (69)
ROR015 Chimeric Rll VH-VL scFv-Fc 70(71)
ROR063 Chimeric Rll VL-VH scFv-Fc 72 (73)
ROR016 Chimeric R12 VH-VL scFv-Fc 74(75)
ROR073 Hemi- Rll VL-VH scFv-Fc (L2H0) 76(77) humanized
ROR091 Hemi- Rll VL-VH scFv-Fc (L6H0) 78(79) humanized
ROR081 Humanized Rll VL-VH scFv-Fc (L2H9) 80(81)
ROR101 Humanized Rll VL-VH scFv-Fc (L6H9) 82 (83)
ROR033 Humanized R12 VH-VL scFv-Fc (H1L1) 84(85)
ROR034 Humanized R12 VH-VL scFv-Fc (H1L2) 86(87)
ROR035 Humanized R12 VH-VL scFv-Fc (H2L1) 88(89)
ROR036 Humanized R12 VH-VL scFv-Fc (H2L2) 90(91)
ROR064 Rabbit Rll Heavy chain 92 (93)
ROR065 Rabbit Rll Light chain 94 (95)
ROR067 Rabbit R12 Heavy chain 96(97)
ROR068 Rabbit R12 Light chain 98(99)
ROR020 Chimeric RllxCD3 scFv-Fc-scFv Rll HL x CD3 100(101)
ROR070 Chimeric RllxCD3 scFv-Fc-scFv Rll LH x CD3 102 (103)
ROR084 Humanized RllxCD3 scFv-Fc-scFv Rll LH (L2H9) 104 (105) x CD3
ROR021 Chimeric R12xCD3 scFv-Fc-scFv R12 HL x CD3 106(107)
ROR050 Humanized R12xCD3 scFv-Fc-scFv R12 HL (H1L2) 108 (109) x CD3
Cris7 and HCDR1 (110) DRA222 (anti- CD3) (Rabat)
Cris7 and HCDR2 (111) DRA222 (anti- CD3) (Rabat) Cris7 and HCDR3 (112) DRA222 (anti- CD3) (Rabat)
Cris7 and LCDR1 (113) DRA222 (anti- CD3) (Rabat)
Cris7 and LCDR2 (114) DRA222 (anti- CD3) (Rabat)
Cris7 and LCDR3 (115) DRA222 (anti- CD3) (Rabat)
Cris7 and HCDR1 (269) DRA222 (anti- CD3) (IMGT)
Cris7 and HCDR2 (270) DRA222 (anti- CD3) (IMGT)
Cris7 and HCDR3 (271) DRA222 (anti- CD3) (IMGT)
Cris7 and LCDR1 (272) DRA222 (anti- CD3) (IMGT)
Cris7 and LCDR2 (273) DRA222 (anti- CD3) (IMGT)
Cris7 and LCDR3 (274) DRA222 (anti- CD3) (IMGT)
I2C (anti- HCDR1 (275) CD3) (Rabat)
I2C (anti- HCDR2 (276) CD3) (Rabat)
I2C (anti- HCDR3 (277) CD3) (Rabat)
I2C (anti- LCDR1 (278) CD3) (Rabat)
I2C (anti- LCDR2 (279) CD3) (Rabat)
I2C (anti- LCDR3 (280) CD3) (Rabat)
I2C (anti- HCDR1 (281) CD3) (IMGT)
I2C (anti- HCDR2 (282) CD3) (IMGT)
I2C (anti- HCDR3 (283) CD3) (IMGT)
I2C (anti- LCDR1 (284) CD3) (IMGT)
I2C (anti- LCDR2 (285) CD3) (IMGT)
I2C (anti- LCDR3 (286) CD3) (IMGT)
HuM291 (anti- HCDR1 (287) CD3) (Rabat)
HuM291 (anti- HCDR2 (288) CD3) (Rabat)
HuM291 (anti- HCDR3 (289) CD3) (Rabat) HuM291 (anti- LCDR1 (290)
CD3) (Rabat)
HuM291 (anti- LCDR2 (291)
CD3) (Rabat)
HuM291 (anti- LCDR3 (292)
CD3) (Rabat)
HuM291 (anti- HCDR1 (293)
CD3) (IMGT)
HuM291 (anti- HCDR2 (294)
CD3) (IMGT)
HuM291 (anti- HCDR3 (295)
CD3) (IMGT)
HuM291 (anti- LCDR1 (296)
CD3) (IMGT)
HuM291 (anti- LCDR2 (297)
CD3) (IMGT)
HuM291 (anti- LCDR3 (298)
CD3) (IMGT)
Humanized Rll VH (H10) 209(210)
Humanized Rll VH(H15) (H9 F224Y T226A) 211 (212)
ROR112 Humanized Rll VL-VH scFv-Fc (L6H10) 213(214)
ROR134 Humanized RllxCD3 scFv-Fc- 215(216) scFv (L7H10) x (DRA222 )
ROR111 Humanized Rll VL-VH scFv-Fc (L6H15) 217 (218)
Humanized Rll VL (L7 ) (L6 N94A) 219 (220)
ROR119 Humanized Rll VL-VH scFv-Fc 221 (222)
(L7H15) (L6H15 N94A)
ROR133 Humanized RllxCD3 scFv-Fc- 223 (224) scFv (L7H15) x (DRA222 )
ROR193 Humanized RllxCD3 scFv-Fc- 225 (226) scFv (L7H15) x (TSC394 F463Y)
ROR183 Humanized RllxCD3 scFv-Fc- 227 (228) scFv (L7H15) x (TSC394 E86D
F87Y)
ROR189 Humanized RllxCD3 scFv-Fc- 229 (230) scFv (L7H10) x (TSC394 E86D
F87Y)
Humanized Rll VH (H16) (H15 S40T G113R) 231 (232)
ROR154 Humanized RllxCD3 scFv-Fc- 233(234) scFv (L7H16) x (DRA222 )
ROR185 Humanized RllxCD3 scFv-Fc- 235(236) scFv (L7H15) x (TSC394 E86D
F87Y)
ROR185 Humanized RllxCD3 scFv-Fc- (299) variant scFv (L7H15) x (TSC394 E86D
F87Y)
Humanized Rll VL (L8 ) (L7 R98W) 237 (238)
ROR179 Humanized RllxCD3 scFv-Fc- 239(240) scFv (L8H16) x (DRA222 )
ROR186 Humanized RllxCD3 scFv-Fc- 241(242) scFv (L8H16) x (TSC394 E86D
F87Y)
Humanized Rll VH (H17) (H10 A25V S28T 243(244)
G113R)
ROR181 Humanized RllxCD3 scFv-Fc- 245(246) scFv(L7H17) x(DRA222)
ROR191 Humanized RllxCD3 scFv-Fc- 247 (248) scFv(L7H17) x(TSC394 E86D
F87Y) Humanized Rll VL, pi variant (L9) (L7 249(250)
K42Q S60D Q79E P80A)
Humanized Rll VH, pi variant (H18) (H17 251 (252)
K43Q)
ROR182 Humanized RllxCD3 scFv-Fc- 253(254) scFv(L9H18) x(DRA222)
ROR192 Humanized RllxCD3 scFv-Fc- 255(256) scFv(L9H18) x(TSC394 E86D
F87Y)
ROR192 Humanized RllxCD3 scFv-Fc- (300) variant scFv(L9H18) x(TSC394 E86D
F87Y)
ROR238 Humanized, RllxCD3 scFv-Fc-scFv (parent 304 (305) codon molecule is ROR192)
optimized
ROR239 Humanized, RllxCD3 scFv-Fc-scFv (parent 306(307) codon molecule is ROR192)
optimized
ROR2 1 Humanized, RllxCD3 scFv-Fc-scFv (parent 308 (309) codon molecule is ROR185)
optimized
ROR2 2 Humanized, RllxCD3 scFv-Fc-scFv (parent 310 (311) codon molecule is ROR192)
optimized
ROR243 Humanized, RllxCD3 scFv-Fc-scFv (parent 312 (313) codon molecule is ROR192)
optimized
ROR244 Humanized, RllxCD3 scFv-Fc-scFv (parent 314 (315) codon molecule is ROR192)
optimized
ROR246 Humanized, RllxCD3 scFv-Fc-scFv (parent 316 (317) codon molecule is ROR192)
optimized
ROR247 Humanized, RllxCD3 scFv-Fc-scFv (parent 318 (319) codon molecule is ROR192)
optimized
ROR248 Humanized, RllxCD3 scFv-Fc-scFv (parent 320 (321) codon molecule is ROR192)
optimized
ROR249 Humanized, RllxCD3 scFv-Fc-scFv (parent 322 (323) codon molecule is ROR192)
optimized
ROR250 Humanized, RllxCD3 scFv-Fc-scFv (parent 324 (325) codon molecule is ROR185)
optimized
ROR251 Humanized, RllxCD3 scFv-Fc-scFv (parent 326 (327) codon molecule is ROR185)
optimized
ROR252 Humanized, RllxCD3 scFv-Fc-scFv (parent 328 (329) codon molecule is ROR185)
optimized
ROR253 Humanized, RllxCD3 scFv-Fc-scFv (parent 330 (331) codon molecule is ROR185)
optimized
Humanized HuRll Light chain 332 (333) mAb
Humanized HuRll Heavy chain 334 (335) mAb
Humanized HuRll Light chain, pi variant 336 (337) mAb Humanized HuRll Heavy chain, pi variant 338 (339) mAb
TSC455 CD3 TSC394 F87Y scFv (257)
TSC456 CD3 TSC394 E86D F87Y scFv (258)
TSC455 CD3 TSC455 and TSC456 variable (259) and heavy domain
TSC456
VH
TSC455 CD3 TSC455 variable light (260) VL domain
TSC456 CD3 TSC456 variable light (261) VL domain
DRA222 CD3 DRA222 CD3 scFv (262)
DRA222 CD3 DRA222 variable heavy (263) VH domain
DRA222 CD3 DRA222 variable light (264) VL domain

Claims

CLAIMS:
1. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein
(a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12 or a sequence that differs from SEQ ID NO: 12 by at least one amino acid substitution;
(b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14 or a sequence that differs from SEQ ID NO: 14 by at least one amino acid substitution;
(c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268, or a sequence that differs from SEQ ID NO: 16, SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268 by at least one amino acid substitution;
(d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:6 or SEQ ID
NO:266 or a sequence that differs from SEQ ID NO:6 or SEQ ID NO:266 by at least one amino acid substitution;
(e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:8 or a sequence that differs from SEQ ID NO:8 by at least one amino acid substitution; and
(f) the HCDR3 has an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID
NO:267 or a sequence that differs from SEQ ID NO: 10 or SEQ ID NO:267 by at least one amino acid substitution.
2. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein
(a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO: 12;
(b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO: 14;
(c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268;
(d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:266;
(e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:8; and
(f) the HCDR3 has an amino acid sequence set forth in SEQ ID NO:267.
3. The ROR1 -binding domain of claim 1 or 2, wherein the ROR1 -binding domain
(i) binds an epitope near the junction of the frizzled and kringle domains of human ROR1 ; or
(ii) binds residues in stretches 259-273 (LCQTEYIFARSNPMI (residues 259-273 of SEQ ID NO: 128)) and 389-403 (PACDSKDSKEKNKME (residues 389-403 of SEQ ID
NO: 128)) of the human ROR1 ectodomain.
4. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein
(a) the LCDR1 has an amino acid sequence set forth in SEQ ID NO:47 or a sequence that differs from SEQ ID NO:47 by at least one amino acid substitution;
(b) the LCDR2 has an amino acid sequence set forth in SEQ ID NO:49 or a sequence that differs from SEQ ID NO:49 by at least one amino acid substitution;
(c) the LCDR3 has an amino acid sequence set forth in SEQ ID NO:51 , or a sequence that differs from SEQ ID NO:51 by at least one amino acid substitution;
(d) the HCDR1 has an amino acid sequence set forth in SEQ ID NO:41 or a sequence that differs from SEQ ID NO:41 by at least one amino acid substitution;
(e) the HCDR2 has an amino acid sequence set forth in SEQ ID NO:43 or a sequence that differs from SEQ ID NO:43 by at least one amino acid substitution; and
(f) the HCDR3 has an amino acid sequence set forth in SEQ ID NO:45 or a sequence that differs from SEQ ID NO:45 by at least one amino acid substitution.
5. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises:
(i) an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250;
(ii) an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252; (iii) an immunoglobulin light chain comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:333 or SEQ ID NO:337; or
(iv) an immunoglobulin heavy chain comprising an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least
99% identical to the amino acid sequence set forth in SEQ ID NO:335 or SEQ ID NO:339.
6. The ROR1 -binding domain of claim 5, wherein
(i) the immunoglobulin light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220,
SEQ ID NO:238, or SEQ ID NO:250;
(ii) the immunoglobulin heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252;
(iii) the immunoglobulin light chain comprises an amino acid sequence set forth in
SEQ ID NO:333 or SEQ ID NO:337; or
(iv) the immunoglobulin light chain comprises an amino acid sequence set forth in SEQ ID NO:335 or SEQ ID NO:339.
7. The ROR1 -binding domain of claim 5 or 6, wherein
(i) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:23 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27;
(ii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27;
(iii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:59;
(iv) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:61 ;
(v) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:59;
(vi) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:61 ;
(vii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:210;
(viii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:210;
(ix) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:212;
(x) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:212;
(xi) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232;
(xii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:232;
(xiii) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:244;
(xiv) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:250 and the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:252;
(xv) the immunoglobulin light chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:333 and the
immunoglobulin heavy chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:335; or
(xvi) the immunoglobulin light chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:337 and the
immunoglobulin heavy chain comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:339.
8. The ROR1 -binding domain of any one of claims 5-7, wherein
(i) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27;
(ii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27;
(iii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59;
(iv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 ;
(v) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59;
(vi) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61 ;
(vii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:210;
(viii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:210;
(ix) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:25 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:212; (x) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:212;
(xi) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232;
(xii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:238 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:232;
(xiii) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:220 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:244;
(xiv) the immunoglobulin light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:250 and the immunoglobulin heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:252;
(xv) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:333 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:335; or
(xvi) the immunoglobulin light chain comprises the amino acid sequence set forth in SEQ ID NO:337 and the immunoglobulin heavy chain comprises the amino acid sequence set forth in SEQ ID NO:339.
9. The ROR1 -binding domain of any one of claims 1 -8, wherein at least one of the light chain variable region or the heavy chain variable region is humanized.
10. The ROR1 -binding domain of any one of claims 1 -9, wherein the ROR1 -binding domain is a single chain variable fragment (scFv).
1 1 . The ROR1 -binding domain of claim 10, wherein the light chain variable region of said scFv is carboxy-terminal to the heavy chain variable region of said scFv.
12. The ROR1 -binding domain of claim 10, wherein the light chain variable region of said scFv is amino-terminal to the heavy chain variable region of said scFv.
13. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding domain that specifically binds to human ROR1 , wherein said ROR1 -binding domain comprises SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69.
14. The ROR1 -binding domain of any one of claims 1 -13, wherein said binding domain is conjugated to a drug or a toxin.
15. The ROR1 -binding domain of any one of claims 1 -5, wherein the ROR1 -binding domain binds to human ROR1 with a dissociation constant that is lower than the dissociation constant of a ROR1 -binding domain comprising SEQ ID NO:73.
16. The ROR1 -binding domain of any one of claims 1 -5, wherein the ROR1 -binding domain has a reduced isoelectric point compared to the isoelectric point of a ROR1-binding domain comprising an immunoglobulin light chain variable region of SEQ ID NO:220 and an immunoglobulin heavy chain variable region of SEQ ID NO:232.
17. A ROR1 -binding polypeptide comprising the ROR1 -binding domain of any one of claims 1 -12 and an immunoglobulin constant region.
18. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide that specifically binds to human ROR1 , wherein said ROR1 -binding polypeptide comprises SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91 .
19. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising:
(i) the ROR1 -binding domain of any one of claims 1 -13, and
(ii) a second binding domain.
20. The ROR1 -binding polypeptide of claim 19, wherein said ROR1 -binding polypeptide comprises, in order from amino-terminus to carboxyl-terminus, (i) the ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker, and (v) the second binding domain.
21 . The ROR1 -binding polypeptide of claim 19 or 20, wherein
(i) the ROR1 -binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3; and (ii) the second binding domain comprises (a) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (b) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3.
22. The ROR1 -binding polypeptide of claim 20, wherein the carboxyl-terminus linker comprises or consists of SEQ ID NO:265, SEQ ID NO:301 , SEQ ID NO:302, or SEQ ID NO:303.
23. The ROR1 -binding polypeptide of any one of claims 20-22, wherein the
immunoglobulin constant region comprises immunoglobulin CH2 and CH3 domains of lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2 or IgD.
24. The ROR1 -binding polypeptide of claim 20, wherein the immunoglobulin constant region comprises a human lgG1 CH2 domain comprising the substitutions L234A, L235A, G237A, and K322A, according to the EU numbering system.
25. The ROR1 -binding polypeptide of any one of claims 20-24, wherein the ROR1 - binding polypeptide does not exhibit or exhibits minimal antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.
26. The ROR1 -binding polypeptide of any one of claims 19-25, wherein the second binding domain specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex.
27. The ROR1 -binding polypeptide of claim 26, wherein the second binding domain specifically binds CD3 and wherein the second binding domain comprises an
immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region;
wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is
(a) at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:260; or
(b) at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:261 ; and wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequence in SEQ ID NO:259.
28. The ROR1 -binding polypeptide of claim 26, wherein said ROR1 -binding polypeptide induces redirected T-cell cytotoxicity (RTCC).
29. The ROR1 -binding polypeptide of claim 26, wherein said ROR1 -binding polypeptide induces T-cell activation or T-cell proliferation.
30. The ROR1 -binding polypeptide of claim 26, wherein said ROR1 -binding polypeptide induces T-cell-dependent lysis of ROR1 -expressing cells.
31 . The ROR1 -binding polypeptide of claim 26, wherein said ROR1 -binding polypeptide when bound to a CD3 protein on a T cell does not induce or induces a minimally detectable cytokine release from said T cell.
32. The ROR1 -binding polypeptide of any one of claims 19-31 , wherein the second binding domain competes for binding to CD3z with a monoclonal antibody selected from CRIS-7, HuM291 and I2C.
33. The ROR1 -binding polypeptide of any one of claims 19-32, wherein the second binding domain is a single chain variable fragment (scFv).
34. The ROR1 -binding polypeptide of any one of claims 19-33, wherein the second binding domain comprises an immunoglobulin light chain variable region and an
immunoglobulin heavy chain variable region derived from a monoclonal antibody selected from CRIS-7, HuM291 and I2C.
35. The ROR1 -binding polypeptide of any one of claims 19-34, wherein the second binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein (a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 10, 1 1 1 and 1 12, respectively; or
(b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 272, 273 and 274, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 269, 270 and 271 , respectively.
36. The ROR1 -binding polypeptide of any one of claims 19-34, wherein the second binding domain comprises: (i) an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein
(a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 278, 279 and 280, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 275, 276 and 277, respectively; or
(b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in
SEQ ID NOs: 284, 285 and 286, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 281 , 282 and 283, respectively.
37. The ROR1 -binding polypeptide of any one of claims 19-34, wherein the second binding domain comprises: (i) an immunoglobulin light chain variable region comprising
LCDR1 , LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein
(a) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 290, 291 and 292, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 287, 288 and 289, respectively; or
(b) the LCDR1 , LCDR2 and LCDR3 has the amino acid sequences set forth in SEQ ID NOs: 296, 297 and 298, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 293, 294 and 295, respectively.
38. The ROR1 -binding polypeptide of any one of claims 19-37, wherein the second binding domain is a humanized binding domain.
39. The ROR1 -binding polypeptide of any one of claims 19-38, wherein the ROR1 - binding domain competes for binding to human ROR1 with a single chain variable fragment (scFv) having the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO:21 or SEQ ID NO:53.
40. The ROR1 -binding polypeptide of any one of claims 19-39, wherein the ROR1 - binding domain is a humanized binding domain.
41 . The ROR1 -binding polypeptide of any one of claims 19-40, wherein the ROR1 - binding domain is a single chain variable fragment (scFv).
42. The ROR1 -binding polypeptide of any one of claims 19-41 , wherein the ROR1 - binding domain comprises SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69.
43. The ROR1 -binding domain of claim 1 or 4, wherein the at least one amino acid substitution is a conservative amino acid substitution.
44. The ROR1 -binding domain of claim 1 or 4, wherein the at least one amino acid substitution is a non-conservative amino acid substitution.
45. The ROR1 -binding polypeptide of any one of claims 19-41 , wherein the light chain variable region of the ROR1 -binding domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:39, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; and the heavy chain variable region of the ROR1 -binding domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:2; SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252.
46. The ROR1 -binding polypeptide of any one of claims 19-41 and 45, wherein the light chain variable region of the ROR1 -binding domain comprises the amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:220, SEQ ID NO:238, or SEQ ID NO:250; and the heavy chain variable region of the ROR1 - binding domain comprises an amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:61 , SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:232, SEQ ID NO:244, or SEQ ID NO:252.
47. The ROR1 -binding polypeptide of any one of claims 19-41 , 45, and 46, wherein the ROR1 -binding domain is an scFv and comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO:21 , SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67 or SEQ ID NO:69.
48. The ROR1 -binding polypeptide of any one of claims 19-41 and 45-47, wherein said ROR1 -binding polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:71 , SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81 , SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91 , SEQ ID NO:214, SEQ ID NO:218, or SEQ ID NO:222.
49. The ROR1 -binding polypeptide of any one of claims 19-41 and 45-48, wherein said ROR1 -binding polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321 , SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, or SEQ ID NO:331 .
50. The ROR1 -binding polypeptide of any one of claims 19-41 and 45-49, wherein said ROR1 -binding polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321 , SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, or SEQ ID NO:331 .
51 . A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising in order from amino-terminus to carboxyl-terminus (i) a ROR1 -binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker, and (v) a second binding domain, wherein said ROR1 -binding polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID N0:31 1 , SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321 , SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, or SEQ ID NO:331 .
52. The ROR1 -binding polypeptide of any one of claims 19-41 and 45-50, wherein the ROR1 -binding domain is an scFv comprising an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; and wherein said light chain variable region and said heavy chain variable region are joined by an amino acid sequence comprising (Gly4Ser)n, wherein n=1 -5 (SEQ ID NO: 127).
53. The ROR1 -binding polypeptide of any one of claims 19-41 and 45-50, wherein the second binding domain is an scFv comprising an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region; and wherein said light chain variable region and said heavy chain variable region are joined by an amino acid sequence comprising (Gly4Ser)n, wherein n=1 -5 (SEQ ID NO: 127).
54. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding protein that is a dimer of two identical polypeptides, wherein each polypeptide is the ROR1 -binding polypeptide of any one of claims 19-41 and 45-53.
55. The ROR1 -binding polypeptide of any one of claims 19-42 and 45-53, wherein said ROR1 -binding polypeptide further comprises an immunoglobulin heterodimerization domain.
56. The ROR1 -binding polypeptide of claim 55, wherein the immunoglobulin
heterodimerization domain comprises an immunoglobulin CH1 domain or an immunoglobulin CL domain.
57. The ROR1 -binding polypeptide of any one of claims 19-42 and 45-56, wherein said ROR1 -binding polypeptide is a heterodimeric ROR1 -binding protein comprising (i) a first polypeptide chain comprising, in order from amino-terminus to carboxyl-terminus or from carboxyl-terminus to amino-terminus, (a) a ROR1 -binding domain that specifically binds human ROR1 , (b) a first hinge region, (c) a first immunoglobulin constant region, and (d) a first immunoglobulin heterodimerization domain; and (ii) a second polypeptide chain comprising, in order from amino-terminus to carboxyl-terminus or from carboxyl-terminus to amino-terminus, (a') a second hinge region, (b') a second immunoglobulin constant region, and (c') a second immunoglobulin heterodimerization domain that is different from the first immunoglobulin heterodimerization domain of the first single chain polypeptide, wherein the first and second immunoglobulin heterodimerization domains associate with each other to form a heterodimer.
58. The ROR1 -binding polypeptide of claim 57, wherein the first immunoglobulin heterodimerization domain comprises an immunoglobulin CH1 domain and the second immunoglobulin heterodimerization domain comprises an immunoglobulin CL domain, or wherein the first immunoglobulin heterodimerization domain comprises an immunoglobulin CL domain and the second immunoglobulin heterodimerization domain comprises an immunoglobulin CH1 domain.
59. The ROR1 -binding polypeptide of claim 58, wherein at least one of the first and second immunoglobulin constant regions comprises immunoglobulin CH2 and CH3 domains of lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD or any combination thereof; an immunoglobulin CH3 domain of lgG 1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgD, IgE, IgM or any combination thereof; or immunoglobulin CH3 and CH4 domains of IgE, IgM or a combination thereof.
60. The ROR1 -binding polypeptide of claim 57, wherein said second polypeptide chain further comprises a second binding domain.
61 . The ROR1 -binding polypeptide of claim 60, wherein the second binding domain is amino-terminal to the second hinge region.
62. The ROR1 -binding polypeptide of any one of claims 19-42 and 45-53, wherein said ROR1 -binding polypeptide is a bispecific single chain antibody molecule comprising a ROR1 binding domain and a CD3 binding domain, wherein the binding domains are arranged in the order VH ROR1 -VL ROR1 -VH CD3-VL CD3; VL ROR1 -VH ROR1 -VH CD3-VL CD3; VH CD3-VL CD3-VH ROR1 -VL ROR1 ; or VH CD3-VL CD3-VL ROR1 -VH ROR1 .
63. An isolated nucleic acid molecule encoding the ROR1 -binding polypeptide of any one of claims 19-42 and 45-53 or a portion of said ROR1 -binding polypeptide.
64. The isolated nucleic acid molecule of claim 63, wherein the nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:209, SEQ ID N0:21 1 , SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221 , SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231 , SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241 , SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251 , SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, or SEQ ID NO:338.
65. An expression vector comprising a nucleic acid segment encoding the ROR1 -binding polypeptide of any one of claims 19-42 and 45-53, wherein the nucleic acid segment is operatively linked to regulatory sequences suitable for expression of the nucleic acid segment in a host cell.
66. The expression vector of claim 65, wherein the nucleic acid segment comprises a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:209, SEQ ID NO:21 1 , SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221 , SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231 , SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241 , SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251 , SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, or SEQ ID NO:338.
67. A recombinant host cell comprising the expression vector of claim 65 or 66.
68. An expression vector comprising first and second expression units, wherein the first and second expression units respectively comprise first and second nucleic acid segments encoding the first and second polypeptide chains of the heterodimeric ROR1 -binding protein of any one of claims 57-61 , and
wherein the first and second nucleic acid segments are operably linked to regulatory sequences suitable for expression of the nucleic acid segments in a host cell.
69. A recombinant host cell comprising the expression vector of claim 68.
70. A method for producing a ROR1 -binding polypeptide, the method comprising
culturing a recombinant host cell comprising the expression vector of claim 65 or 66 under conditions whereby the nucleic acid segment is expressed, thereby producing the ROR1 -binding polypeptide.
71 . The method of claim 70, further comprising recovering the ROR1 -binding polypeptide.
72. A method for producing a heterodimeric ROR1 -binding protein, the method comprising
culturing a recombinant host cell comprising first and second expression units, wherein the first and second expression units respectively comprise first and second nucleic acid segments encoding the first and second polypeptide chains of a heterodimeric ROR1 - binding protein as set forth in any one of claims 57-61 , wherein the first and second nucleic acid segments are operably linked to regulatory sequences suitable for expression of the nucleic acid segments in a host cell, and
wherein said culturing is under conditions whereby the first and second nucleic acid segments are expressed and the encoded polypeptide chains are produced as the heterodimeric ROR1 -binding protein.
73. The method of claim 72, further comprising recovering the heterodimeric ROR1 - binding protein.
74. A pharmaceutical composition comprising the ROR1 -binding polypeptide of any one of claims 19-42, 45-53, and 55-62 or the ROR1 -binding protein of claim 54 and a pharmaceutically acceptable carrier, diluent, or excipient.
75. The pharmaceutical composition of claim 74 formulated in a dosage form selected from the group consisting of: an oral unit dosage form, an intravenous unit dosage form, an intranasal unit dosage form, a suppository unit dosage form, an intradermal unit dosage form, an intramuscular unit dosage form, an intraperitoneal unit dosage form, a
subcutaneous unit dosage form, an epidural unit dosage form, a sublingual unit dosage form, and an intracerebral unit dosage form.
76. The pharmaceutical composition of claim 74, formulated as an oral unit dosage form selected from the group consisting of: tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained-release formulations, aerosols, and sprays.
77. A method for inducing redirected T-cell cytotoxicity (RTCC) against a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 -expressing cell with the ROR1 -binding polypeptide of any one of claims 19-42, 45-53, and 55-62 or the ROR1 -binding protein of claim 54, wherein the second binding domain specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; and wherein said contacting is under conditions whereby RTCC against the ROR1 -expressing cell is induced.
78. A method for inducing redirected T-cell cytotoxicity (RTCC) against a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising:
contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and
a second binding domain that specifically binds a T-cell, CD3, CD3z or a T- cell receptor (TCR) complex or a component of a T-cell receptor complex;
wherein said contacting is under conditions whereby RTCC against the ROR1 - expressing cell is induced.
79. A method for inducing T-cell dependent lysis of a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising: contacting said ROR1 - expressing cell with the ROR1 -binding polypeptide of any one of claims 19-42, 45-53, and 55-62 or the ROR1 -binding protein of claim 54, wherein the second binding domain specifically binds a T-cell, CD3, CD3z or a T-cell receptor (TCR) complex or a component of a T-cell receptor complex; and wherein said contacting is under conditions whereby T-cell dependent lysis of the ROR1 -expressing cell is induced.
80. A method for inducing T-cell dependent lysis of a cell expressing receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising:
contacting said ROR1 -expressing cell with a ROR1 -binding polypeptide comprising a first binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 and
a second binding domain that specifically binds a T-cell, CD3, CD3z or a T- cell receptor (TCR) complex or a component of a T-cell receptor complex;
wherein said contacting is under conditions whereby T-cell dependent lysis of the ROR1 -expressing cell is induced.
81 . A method for treating a disorder in a subject, wherein said disorder is characterized by expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising
administering to the subject a therapeutically effective amount of the ROR1 -binding polypeptide of any one of claims 19-42, 45-53, and 55-62 or the ROR1 -binding protein of claim 54.
82. A method for treating a disorder in a subject, wherein said disorder is characterized by expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1), the method comprising
administering to the subject a therapeutically effective amount of a ROR1 -binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 .
83. The method of claim 81 or 82, wherein the disorder is a cancer.
84. The method of claim 83, wherein the cancer is breast cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), acute lymphoblastic leukemia (ALL), melanoma, adrenal cancer, bladder cancer or prostate cancer.
85. The method of claim 84, wherein the breast cancer is triple negative breast cancer (TNBC).
86. The receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide of any one of claims 19-42, 45-53, and 55-62 or the ROR1 -binding protein of claim 54 for the manufacture of a medicament for treatment of a disorder in a subject, wherein said disorder is characterized by expression of ROR1 .
87. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 for the manufacture of a medicament for treatment of a disorder in a subject, wherein said disorder is characterized by expression of ROR1 .
88. The receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide of any one of claims 19-42, 45-53, and 55-62 or the ROR1 -binding protein of claim 54 for use in treating a disorder in a subject, wherein said disorder is characterized by expression of ROR1 .
89. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising a ROR1 binding domain that specifically binds an epitope near the junction of the frizzled and kringle domains of human ROR1 for use in treating a disorder in a subject, wherein said disorder is characterized by expression of ROR1 .
90. The ROR1 -binding polypeptide of any one of claims 86-89, wherein the disorder is a cancer.
91 . The ROR1 -binding polypeptide of claim 90, wherein the cancer is breast cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), acute lymphoblastic leukemia (ALL), melanoma, adrenal cancer, bladder cancer or prostate cancer.
92. The ROR1 -binding polypeptide of claim 91 , wherein the breast cancer is triple negative breast cancer (TNBC).
93. An isolated nucleic acid molecule encoding the ROR1 -binding domain of any one of claims 1 -16 or a portion of said ROR1 -binding domain.
94. The isolated nucleic acid molecule of claim 93, wherein the nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:209, SEQ ID N0:21 1 , SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221 , SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231 , SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241 , SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251 , SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, or SEQ ID NO:338.
95. A receptor tyrosine kinase-like orphan receptor 1 (RORI)-binding polypeptide comprising a ROR1-binding domain and a CD3-binding domain;
wherein said ROR1 -binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein
(a) the LCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 12;
(b) the LCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 14;
(c) the LCDR3 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO: 1 16, SEQ ID NO: 1 17, SEQ ID NO: 1 18, or SEQ ID NO:268;
(d) the HCDR1 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:266;
(e) the HCDR2 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:8; and
(f) the HCDR3 of said ROR1 -binding domain has an amino acid sequence set forth in SEQ ID NO:267; and
wherein said CD3-binding domain comprises: an immunoglobulin light chain variable region comprising LCDR1 , LCDR2, and LCDR3, and an immunoglobulin heavy chain variable region comprising HCDR1 , HCDR2, and HCDR3, wherein
(a') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 1 13, 1 14 and 1 15, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 1 10, 1 1 1 and 1 12, respectively;
(b') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 272, 273 and 274, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 269, 270 and 271 , respectively;
(c') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 278, 279 and 280, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 275, 276 and 277, respectively;
(d') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 284, 285 and 286, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 281 , 282 and 283, respectively;
(e') the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 290, 291 and 292, respectively, and the HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 287, 288 and 289, respectively; or
(Γ) the LCDR1 , LCDR2 and LCDR3 of said CD3-binding domain have the amino acid sequences set forth in SEQ ID NOs: 296, 297 and 298, respectively, and the
HCDR1 , HCDR2, and HCDR3 has the amino acid sequences set forth in SEQ ID NOs: 293, 294 and 295, respectively.
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