WO2023247731A1 - Molécules de liaison à l'antigène bispécifiques ror1/ptk7 - Google Patents

Molécules de liaison à l'antigène bispécifiques ror1/ptk7 Download PDF

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WO2023247731A1
WO2023247731A1 PCT/EP2023/067045 EP2023067045W WO2023247731A1 WO 2023247731 A1 WO2023247731 A1 WO 2023247731A1 EP 2023067045 W EP2023067045 W EP 2023067045W WO 2023247731 A1 WO2023247731 A1 WO 2023247731A1
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seq
fusion protein
recombinant fusion
binding molecule
hfc
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PCT/EP2023/067045
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Paul Richard TRUMPER
Jennifer THOM
Andrei KAMENSKI
Graham John Cotton
Aiden MCCANN
Estelle MCLEAN
Greg PAPADAKOS
Mark WAPPETT
Caroline Jane BARELLE
Marina KOVALEVA
Andrew Justin Radcliffe Porter
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Almac Discovery Limited
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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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to bi-specific antigen binding molecules with specificity for both receptor tyrosine kinase-like orphan receptor 1 (ROR1) and inactive protein tyrosine kinase 7 (PTK7, CCK4) and associated fusion proteins and conjugates.
  • ROR1 receptor tyrosine kinase-like orphan receptor 1
  • PTK7, CCK4 inactive protein tyrosine kinase 7
  • the present invention relates to conjugated immunoglobulin-like shark variable novel antigen receptors (VNARs).
  • VNARs conjugated immunoglobulin-like shark variable novel antigen receptors
  • BACKGROUND Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a 937 amino acid glycosylated type I single pass transmembrane protein.
  • the extracellular region consists of three distinct domains composing an N-terminal immunoglobulin domain (Ig), followed by a cysteine rich fizzled domain (fz) which in turn is linked to the membrane proximal kringle domain (kr).
  • Ig N-terminal immunoglobulin domain
  • fz cysteine rich fizzled domain
  • kr membrane proximal kringle domain
  • the intracellular region of the protein contains a pseudo kinase domain followed by two Ser/Thr rich domains which are interspersed by a proline-rich region, and this same overall domain architecture is conserved in the closely related family member ROR2, with which it shares high sequence identity.
  • ROR1 is expressed during embryonic development, where it is prominently expressed in neural crest cells and in the necrotic and interdigital zones in the later stages of development. However, its expression is quickly silenced after birth, and is largely absent in normal adult tissue.
  • ROR1 expression has been observed at both the mRNA and protein level across a broad range of solid tumours and haematological malignancies including lung, endometrial, pancreatic, ovarian, colon, head and neck and prostate cancers, melanoma and renal cell carcinoma, breast cancer and chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (AML). Additionally, increased ROR1 expression is reported to correlate with poor clinical outcomes for a number of cancer indications including breast cancer, ovarian cancer, colorectal cancer, lung adenocarcinoma and CLL.
  • ROR1 Consistent with ROR1’s expression pattern and the link to poor clinical prognosis, a functional role for ROR1 in tumorigenesis and disease progression has been demonstrated for a number of different cancer indications.
  • ROR1 promotes epithelial-mesenchymal transition and metastasis in models of breast cancer and spheroid formation and tumour engraftment in models of ovarian cancer.
  • ROR1 is a transcript target of the NKX2-1/TTF-1 lineage survival factor oncogene in lung adenocarcinoma, where it sustains EGFR signalling and represses pro-apoptotic signalling and an EGF induced interaction between ROR1 and EGFR has been observed.
  • ROR1 has been noted from breast cancer gene expression database mining.
  • ROR1 has also been shown to act as a scaffold to sustain caveolae structures and by-pass signalling mechanism that confer resistance to EGFR tyrosine kinase inhibitors.
  • Signalling through an ROR1-HER3 complex modulates the Hippo-YAP pathway and promotes breast cancer bone metastasis and the protein can promote Met-driven tumorigenesis.
  • ROR1 expression is associated with chemotherapy resistance in breast cancer through activation of Hippo-YAP/TAZ and BMI1 pathways.
  • CLL CLL
  • ROR1 has been reported to hetero- oligomerise with ROR2 in response to Wnt5a to transduce signalling and enhance proliferation and migration.
  • ROR1 Given the functional role of ROR1 in cancer pathology and the general lack of expression on normal adult tissue, this oncofetal protein is an attractive target for cancer therapy.
  • Antibodies to ROR1 have been described in the literature WO2021097313 (4A5 kipps), WO2014031174 (UC961), WO2016187220 (Five Prime) WO2010124188 (2A2), WO2012075158 (R11, R12), WO2011054007 (Oxford Bio), WO2011079902 (Bioinvent) WO2017127664, WO2017127664 (NBE Therapeutics, SCRIPPS), WO2016094847 (Emergent), WO2017127499), and a humanised murine anti-ROR1 antibody, UC961, has entered clinical trials for relapsed or refractory chronic lymphocytic leukemia.
  • Chimeric antigen receptor T-cells targeting ROR1 have also been reported (Hudecek M et al, Clin. Cancer Res., 2013, 19, 3153-64) and preclinical primate studies with UC961 and with CAR-T cells targeting ROR1 showed no overt toxicity, which is consistent with the general lack of expression of the protein on adult tissue (Choi M et al, Clinical Lymphoma, myeloma & leukemia, 2015, S167; Berger C et al, Cancer Immunol. Res., 2015, 3, 206).
  • PTK7 (CCK4) is a protein comprising an extracellular domain with seven immunoglobulin like domains a transmembrane domain and a catalytically defective tyrosine kinase and is thus categorised as a pseudokinase.
  • Overexpression of PTK7 has been documented in multiple solid tumours and may be associated with a worse prognosis (for example epithelial ovarian cancer, esophageal adenocarcinoma).
  • RNA-seq data from the cancer genome atlas reports increased PTK7 mRNA levels in various cancer tissues including endometrial, head and neck, lung, ovary, cervical, prostate, breast, pancreatic, bladder, thyroid, and esophageal cancers when compared with the overall mean level from normal tissues (Shin WS et al Sci Reports, 2018, 8, 8519).
  • IHC staining of primary human tumours shows overexpression of PTK7 across a number of cancer indications including ovarian cancer, TNBC, NSCLC.
  • PTK7 expression is also enriched on tumour initiating cells (Damelin M et al, Sci Transl. Med., 2017, 9, 1-11.
  • PTK7 is also expressed on a number of normal human tissues including eosophagus, kidney, urinary bladder and lung. Although PTK7 does not play a role in the biology of mature cells it has been linked with regulating planar cell polarity (PCP) and canonical and non-canonical Wnt signaling during organ development through interactions with a non-canonical Wnt/PCP ligand, Wnt5A, .
  • PCP planar cell polarity
  • Wnt receptors such as Fz7 and LRP6 and intracellular Wnt signaling proteins such as Dvl and ⁇ -catenin (Peradziryi et al.2011) and ROR2 (Martinez et al.2015. J. Biol. Chem.209, 30562-72).
  • PTK7 signalling increases the proliferation, survival, migration, and invasion of cancer cells through activation of ERK, JNK, p38, and NF- ⁇ B signaling pathways, whereas it decreases apoptosis through suppression of caspase-9 and -10.
  • ADC antibody drug conjugate targeting PTK7 has entered clinical trials.
  • This ADC Cofetuxumab Pelidotin, consists of a humanised mouse anti-PTK7 monoclonal antibody (hu24) conjugated with an auristatin 0101 payload, via mc-valine-citrulline-PABC linker, using endogenous cysteine residues.
  • Single domain binding molecules can be derived from an array of proteins from distinct species.
  • the immunoglobulin isotope novel antigen receptor is a homodimeric heavy-chain complex originally found in the serum of the nurse shark (Ginglymostoma cirratum) and other sharks and ray species. IgNARs do not contain light chains and are distinct from the typical immunoglobulin structure. Each molecule consists of a single-variable domain (VNAR) and five constant domains (CNAR).
  • VNAR single-variable domain
  • CNAR constant domains
  • Type I has germline encoded cysteine residues in framework 2 and framework 4 and an even number of additional cysteines within CDR3. Crystal structure studies of a Type I IgNAR isolated against and in complex with lysozyme enabled the contribution of these cysteine residues to be determined. Both the framework 2 and 4 cysteines form disulphide bridges with those in CDR3 forming a tightly packed structure within which the CDR3 loop is held tightly down towards the HV2 region. To date Type I IgNARs have only been identified in nurse sharks – all other elasmobranchs, including members of the same order have only Type II or variations of this type.
  • Type II IgNAR are defined as having a cysteine residue in CDR1 and CDR3 which form intra-molecular disulphide bonds that hold these two regions in close proximity, resulting in a protruding CDR3 that is conducive to binding pockets or grooves.
  • Type I sequences typically have longer CDR3s than type II with an average of 21 and 15 residues respectively. This is believed to be due to a strong selective pressure for two or more cysteine residues in Type I CDR3 to associate with their framework 2 and 4 counterparts.
  • Studies into the accumulation of somatic mutations show that there are a greater number of mutations in CDR1 of type II than type I, whereas HV2 regions of Type I show greater sequence variation than Type II.
  • a third IgNAR type known as Type III has been identified in neonates. This member of the IgNAR family lacks diversity within CDR3 due to the germline fusion of the D1 and D2 regions (which form CDR3) with the V-gene. Almost all known clones have a CDR3 length of 15 residues with little or no sequence diversity.
  • Another structural type of VNAR, termed type (IIb or IV) has only two canonical cysteine residues (in framework 1 and framework 3b regions). So far, this type has been found primarily in dogfish sharks and was also isolated from semisynthetic V-NAR libraries derived from wobbegong sharks.
  • ROR1-specific antigen binding molecules including VNARs
  • VNARs are described in WO 2019/122447, hereby incorporated by reference in its entirety.
  • WO 2019/122447 describes the sequences of B1, P3A1 and P3A1 G1 identified below.
  • WO 2019/122445 describes ROR1/EGFR bi-specific binding molecules where the ROR1 binding molecules are as described in WO 2019/122447, however ROR1/PTK7 bi-specific binding molecules are not disclosed.
  • Conjugates of ROR1-specific antigen binding molecules, including VNARs, are described in WO 2020/254640, hereby incorporated by reference in its entirety.
  • WO 2020/254640 describes anthracycline (PNU) derivatives suitable for use in drug conjugates. Specifically, derivatives of PNU159682 are provided, which lack the C14 carbon and attached hydroxyl functionality, and in which an ethylenediamino (EDA) group forms part of a linker region between the C13 carbonyl of PNU159682 and a maleimide group.
  • EDA ethylenediamino
  • the same molecules may be described with EDA-PNU as the “warhead” such that the EDA group is not considered part of the linker region.
  • the linker comprises val-cit-PAB
  • the maleimide group may be replaced with any reactive group suitable for a conjugation reaction.
  • payloads are able to react with a free thiol group on another molecule.
  • the free thiol is on a protein a protein-drug conjugate (PDC) may be formed.
  • the anthracycline derivative PNU-159682 has been described as a metabolite of nemorubicin and has been reported to exhibit extremely high potency for in vitro cell killing in the pico- to femtomolar range with one ovarian (A2780) and one breast cancer (MCF7) cell line (WO2012/073217 A1).
  • Derivatives of PNU-159682 have also been described in WO2016/102679. Conjugation of PNU-159682 derivatives to antibodies is described in WO2009/099741, WO2016/127081 and WO2016/102679, Yu et al, Clin. Cancer Res 2015, 21, 3298 and Stefan et al, Mol. Cancer. Ther., 2017, 16,879.
  • Auristatin E (AE) and monomethylauristatin E (MMAE) are synthetic analogs of the dolastatins, a special group of linear pseudopeptides originally isolated from marine sources, some of which have very potent cytotoxic activity against tumour cells.
  • MMAE has the disadvantage of a comparatively high systemic toxicity.
  • MMAE is used in particular in conjunction with enzymatically cleavable valine citrulline linkers in the ADC setting for more targeted tumour therapy (see for example WO 2005/081711. After proteolytic cleavage, MMAE is preferably released intracellularly from corresponding ADCs.
  • Monomethylauristatin F is an auristatin derivative having a C- terminal phenylalanine moiety.
  • MMAF as well as various ester and amide derivatives thereof have been disclosed in WO 2005/081711. Further auristatin analogues with a C-terminal, amidically substituted phenylalanine unit are described in WO 01/18032.
  • WO 02/088172 and WO 2007/008603 which claim MMAF analogs which relate to side-chain modifications of phenylalanine, and in WO 2007/008848 those in which the carboxyl group of the phenylalanine is modified.
  • Auristatin conjugates linked via the C- terminus have been described in WO 2009/117531 and further conjugates are described in WO2013/087716.
  • ROR1-specific variant antigen binding molecules having improved properties and conjugates thereof to derivatives of PNU-159682 are described in PCT/EP2021/086667, filed on 17 December 2021, which is hereby incorporated by reference in its entirety.
  • PCT/EP2021/086667 does not disclose any bi- specifics comprising a ROR1-specific variant antigen binding molecule of PCT/EP2021/086667 and an PTK7-specific variant antigen binding molecule.
  • SUMMARY OF INVENTION The present invention generally relates to bi-specific antigen binding molecules.
  • the present invention relates to bi-specific molecules having the ability to bind to both ROR1 and PTK7.
  • the invention provides a PTK7-specific binding molecule as disclosed in relation to any of the following aspects of the invention.
  • the invention provides a bi-specific antigen binding molecule comprising: (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQW
  • the invention provides a bi-specific antigen binding molecule comprising: (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30) and DTSYGLYS (SEQ ID NO:207); FW1 is a framework region; FW2 is a framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from
  • the invention provides a recombinant fusion protein comprising a bi- specific antigen binding molecule according to the first or the second aspects of the invention.
  • a recombinant fusion protein dimer comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24),
  • the invention provides a recombinant fusion protein dimer comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30) and DTSYGLYS (SEQ ID NO:207); FW1 is a framework region
  • the invention provides a recombinant fusion protein dimer comprising (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a first PTK7-specific binding protein as disclosed in relation to any of the aspects of the invention and wherein the first antigen binding molecule is fused to a first fragment of an immunoglobulin Fc region engineered to dimerize with a second fragment of an immunoglobulin Fc region, and (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises a second PTK7-specific binding protein as disclosed in relation to any of the aspects of the invention fused to a second fragment of an immunoglobulin Fc region engineered to dimerize with the first fragment of an immunoglobulin Fc region.
  • the invention provides a ROR1-specific chimeric antigen receptor (CAR), comprising at least one bi-specific antigen binding molecule as defined by the first or second aspects of the invention, at least one recombinant fusion protein as defined by the third aspect of the invention, or at least one recombinant fusion protein dimer as defined by the fourth or fifth aspects of the invention, at least one PTK7 specific binding molecule as defined by the first configuration, or at least one recombinant fusion protein dimer as defined in the second or third configuration, fused or conjugated to at least one transmembrane region and at least one intracellular domain.
  • CAR ROR1-specific chimeric antigen receptor
  • the present invention also provides a cell comprising a chimeric antigen receptor according to the sixth aspect, which cell is preferably an engineered T-cell.
  • a nucleic acid sequence comprising a polynucleotide sequence that encodes a PTK7-specific binding molecule of the first configuration of the invention, or a bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor according to the first, second, third, fourth, fifth or sixth aspects of the invention, the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • a vector comprising a nucleic acid sequence in accordance with the seventh aspect and a host cell comprising such a nucleic acid.
  • a method for preparing a PTK7-specific binding molecule of the first configuration of the invention, the recombinant fusion protein dimer of the second or third configuration, or a bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor, of the first, second, third, fourth, fifth or sixth aspect comprising cultivating or maintaining a host cell comprising the polynucleotide or vector described above under conditions such that said host cell produces the bi-specific antigen binding molecule, recombinant fusion protein or chimeric antigen receptor, optionally further comprising isolating the specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor.
  • a pharmaceutical composition comprising a PTK7-specific binding molecule of the first configuration of the invention, the recombinant fusion protein dimer of the second or third configuration, or the bi-specific antigen binding molecule, fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspects.
  • the pharmaceutical composition may contain a variety of pharmaceutically acceptable carriers.
  • Pharmaceutical compositions of the invention may be for administration by any suitable method known in the art, including but not limited to intravenous, intramuscular, oral, intraperitoneal, or topical administration.
  • the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule, or foam.
  • the bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspects, the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration may be for use in therapy. More specifically, the bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspects may be for use in the treatment of cancer.
  • the cancer is a ROR1-positive cancer type and/or an PTK7-positive cancer type.
  • the cancer is selected from the group comprising blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B- ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, renal cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, Head and Neck cancer, bladder cancer, oesophageal cancer, stomach cancer or liver cancer.
  • blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B- ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL),
  • Also provided herein is the use of a bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspects, the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration in the manufacture of a medicament for the treatment of a disease in a patient in need thereof.
  • the bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth, sixth aspects, the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration or pharmaceutical composition of the eighth aspect may be administered in a single dose.
  • single dose refers to a dosage regiment consisting of one dose. Alternatively, a multi- dose regiment may be used.
  • the advantages of the specific binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth, sixth aspects or pharmaceutical composition of the eighth aspect may be particularly apparent when administered in a single dose.
  • a method of treatment of a disease in a patient in need of treatment comprising administration to said patient of a therapeutically effective dosage of a bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspects or a pharmaceutical composition of the eighth aspect, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • the cancer is a ROR1-positive cancer type and/or a PTK7-positive cancer type.
  • the cancer is selected from the group comprising blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, renal cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, Head and Neck cancer, bladder cancer, oesophageal cancer, stomach cancer or liver cancer.
  • blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL),
  • Also provided herein is a method of assaying for the presence of a target analyte in a sample, comprising the addition of a detectably labelled bi-specific antigen binding molecule of the first aspect or second aspect, or a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration, to the sample and detecting the binding of the molecule to the target analyte.
  • a method of imaging a site of disease in a subject comprising administration of a detectably labelled bi-specific antigen binding molecule of the first aspect or second aspect, or a detectably labelled recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect to a subject or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • a method of diagnosis of a disease or medical condition in a subject comprising administration of a bi-specific antigen binding molecule of the first aspect or second aspect, or a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • a bi-specific antigen binding molecule comprising an antibody, antibody fragment or antigen-binding molecule that competes for binding to ROR1 with the ROR1-specific antigen binding molecule of the first or second aspect.
  • antigen binding proteins e.g., neutralizing antigen binding proteins or neutralizing antibodies
  • competition means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or functional fragment thereof) under test prevents or inhibits specific binding of a the antigen binding molecule defined herein (e.g., specific antigen binding molecule of the first aspect) to a common antigen (e.g., ROR1 in the case of the specific antigen binding molecule of the first or second aspect).
  • kits for diagnosing a subject suffering from cancer, or a pre-disposition thereto, or for providing a prognosis of the subject's condition comprising detection means for detecting the concentration of antigen present in a sample from a test subject, wherein the detection means comprises a bi-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, a chimeric antigen receptor of the sixth aspect or a nucleic acid sequence of the seventh aspect, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration, each being optionally derivatized, wherein presence of antigen in the sample suggests that the subject suffers from cancer.
  • the antigen comprises ROR1 protein, more preferably an extracellular domain thereof. More preferably, the kit is used to identify the presence or absence of ROR1-positive cells in the sample, or determine the concentration thereof in the sample.
  • the kit may also comprise a positive control and/or a negative control against which the assay is compared and/or a label which may be detected.
  • the present invention also provides a method for diagnosing a subject suffering from cancer, or a pre- disposition thereto, or for providing a prognosis of the subject's condition, the method comprising detecting the concentration of antigen present in a sample obtained from a subject, wherein the detection is achieved using a bi-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, a chimeric antigen receptor of the sixth aspect or a nucleic acid sequence of the seventh aspect, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration each being optionally derivatized, and wherein presence of antigen in the sample suggests that the subject suffers from cancer.
  • Also contemplated herein is a method of killing or inhibiting the growth of a cell expressing ROR1 and/or PTK7 in vitro or in a patient, which method comprises administering to the cell a pharmaceutically effective amount or dose of (i) bi-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, a nucleic acid sequence of the seventh aspect, or the CAR or cell according to the sixth aspect, or (ii) of a pharmaceutical composition of the eighth aspect, or (iii) the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • the cell expressing ROR1 and/or is a cancer cell. More preferably, the ROR1 is human ROR1 and/or the PTK7 is human PTK7.
  • the invention provides a bi-specific antigen binding molecule comprising an amino acid sequence represented by the formula (II): X-FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4-Y (II) wherein FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 is a ROR1-specific antigen binding molecule according to the first or second aspect X and Y are optional amino acid sequences wherein the ROR1-specific antigen binding molecule is conjugated to a second moiety and wherein the bi-specific antigen binding molecule further comprises a PTK7-specific antigen binding molecule.
  • the invention provides a target-binding molecule-drug conjugate, comprising (a) a PTK7-specific binding molecule of the first configuration of the invention, or a bi-specific antigen binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein of the third aspect or a recombinant fusion protein dimer of the fourth or fifth aspect or the second or third configuration, and (b) at least one toxin.
  • a target-binding molecule-drug conjugate comprising (a) a PTK7-specific binding molecule of the first configuration of the invention, or a bi-specific antigen binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein of the third aspect or a recombinant fusion protein dimer of the fourth or fifth aspect or the second or third configuration, and (b) at least one toxin.
  • DESCRIPTION OF FIGURES Figure 1 Design of B1 loop library: The sequence of B1 is shown with the “X
  • Figure 2 Cell surface binding of B1 VNAR loop variants (His 6 Myc tag) to A549 (ROR1 hi ) lung cancer cells by flow cytometry.
  • Figure 3 Cell surface binding of B1 VNAR loop variants (His6Myc tag) to A427 (ROR1 low ) lung cancer cells by flow cytometry.
  • Figure 4 Sequence and loop library design of P3A1 G1. CDR1 diversity results in 448 combinations, HV2 diversity results in 768 combinations and HV4 diversity results in 24 combinations.
  • Figure 5 Binding of P3A1G1 loop variants to human ROR1 by ELISA.
  • FIG. 6 Binding of P3A1G1 loop variants and parental P3A1G1 protein to human ROR1 by ELISA.
  • Figure 7 Linker mouse IgG and linker human IgG sequences used in VNAR IgG Fc fusion proteins. Engineered hIgG1 Fc fusion proteins incorporate an engineered cysteine substitution in the hIgG1 Fc sequence, for example at position S239C or S442C (EU numbering) to enable site specific labelling.
  • Figure 8 Cell surface binding of B1 loop variant – hFc fusion proteins to A549 (ROR1 hi ) lung cancer cells by flow cytometry.
  • Figure 9 Analysis of ROR1 bi-paratopic VNAR-hFc fusions by SDS-PAGE (4-12% Bis Tris gel, MOPS buffer, ⁇ 50mM DTT).
  • Figure 10 Cell surface binding of ROR1 bi-paratopic VNAR-hFc fusions to A549 (ROR1 hi ) and A427 (ROR1 low ) lung cancer cells by flow cytometry.
  • Figure 11 Structures of PNU-linker payloads MA-PEG-vc-PAB-EDA-PNU159682 and MA- PEG-va-EDA-PNU159682
  • Figure 12 Dose response showing binding of G3CP-hFc and G3CPG4-hFc PNU conjugates and the corresponding parental proteins to human ROR1 by ELISA
  • Figure 13 Potency of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in killing the ROR1 positive PA-1 cell-line and a PA-1 cell-line with ROR1 knockout
  • Figure 14 In vivo efficacy of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in the ROR1+ HBCx-28 patient-derived TNBC xenograft model.
  • FIG. 15 An alignment of the sequences for B1, B1 G4, B1V15, G3CP and G3CPG4. Points of variation within the CDRs and HV regions are emphasised in underline.
  • B1V15 SEQ ID NO: 115: is not a loop library variant of B1; they have identical CDR1, HV2, HV4 and CDR3 sequences.
  • Figure 16 UV analysis of B1-hFc, B1G4-hFc, G3CP-hFc and G3CPG4-hFc after incubation in PBS pH 7.4 buffer at 37°C for 96h.
  • Figure 17 Size exclusion analysis (SEC) of B1-hFc, B1G4-hFc, G3CP-hFc and G3CPG4-hFc after incubation in PBS pH 7.4 buffer at 37°C for 96h.
  • Figure 18 Potency of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in killing ROR1 low HEK293 cells and HEK293 cells stably transfected with human ROR1
  • Figure 19 In vivo efficacy of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in the ROR1+ HBCx-10 patient-derived TNBC xenograft model.
  • Figure 21 Potency of bi-paratopic G3CP-P3A1-hFc PNU and G3CPG4-P3A1-hFc PNU conjugates in killing the ROR1 positive PA-1 cell-line and a PA-1 cell-line with ROR1 knockout
  • Figure 22 In vivo efficacy of bi-paratopic G3CP-P3A1 hFc PNU and G3CPG4-P3A1-hFc PNU conjugates in the ROR1+ HBCx-28 patient-derived TNBC xenograft model.
  • Figure 23 Binding of VNAR clones to huPTK7 (ECD)-Fc, huPTK7 (5-7)-Fc or HSA by ELISA.
  • CCK4, hu24 and E06 are controls.
  • CCK4 – anti huPTK7 polyclonal antibody, hu24 is anti PTK7 antibody and E06 is an albumin-binding VNAR.
  • Figure 24 Binding of VNAR clones to huPTK7 (ECD)-Fc, huPTK7 (5-7)-Fc or cynoPTK7-Fc and mouse PTK7-Fc in ELISA.
  • CCK4, hu24 and E06 are controls.
  • hu24 is anti PTK7 antibody.
  • E06 is an albumin-binding VNAR used as a negative control
  • Figure 25 Cell surface binding of PTK7 mono- and bi-paratopic VNAR-hFc fusion molecules to NCI-H661 (PTK7 hi ) and AsPC1 (PTK7 low ) human cancer cells by flow cytometry
  • Figure 26 Cell surface binding of PTK7 mono- and bi-paratopic VNAR-hFc fusion molecules to NCI-H661 (PTK7 hi ) and AsPC1 (PTK7 low ) human cancer cells by flow cytometry.
  • Figure 27 Cell surface binding of PTK7 mono- and bi-paratopic VNAR-hFc-drug conjugate molecules to NCI-H661 (PTK7 hi ) and AsPC1 (PTK7 low ) human cancer cells by flow cytometry.
  • Figure 28 Cell surface binding of PTK7 mono- and bi-paratopic VNAR-hFc-drug conjugate molecules to NCI-H661 (PTK7 hi ) and AsPC1 (PTK7 low ) human cancer cells by flow cytometry.
  • Figure 29 Cell surface binding of PTK7 mono- and bi-paratopic VNAR-hFc-drug conjugate molecules to NCI-H661 (PTK7 hi ) and AsPC1 (PTK7 low ) human cancer cells by flow cytometry.
  • Figure 30 Potency of PTK7 mono- and bi-paratopic drug-conjugates in killing the PTK7 positive PA-1 cell-line and a PA-1 cell-line with PTK7 knockout
  • Figure 31 In vivo efficacy of PTK7 mono- and bi-paratopic drug conjugates in the OVXF_OV55 patient-derived ovarian xenograft model.
  • Figure 32 Binding of ROR1 x PTK7 hFc bi-specific proteins to ROR1 by ELISA.
  • FIG. 34 Size exclusion analysis (SEC) of G3CP-P2A7 hFc, G3CPG4-P2A7 hFc, P3A1-P2A7 hFc and B1-P2A7 hFc (top panel G 4 S linker, bottom panel (G 4 S) 3 linker) before and after incubation in PBS pH 7.4 buffer at 37°C for 96h
  • Figure 35 Size exclusion analysis (SEC) of G3CP-4D2 hFc, G3CPG4-4D2 hFc, P3A1-4A7 hFc and B1-P2A7 hFc (top panel G 4 S linker, bottom panel (G 4 S) 3 linker) before and after incubation in PBS pH 7.4 buffer at 37°C for 96h
  • Figure 35 Size exclusion analysis (SEC) of G3CP-4D2 hFc, G3CPG4-4D2 hFc, P3A1-4
  • Figure 37 Western Blot analysis of PDX models from different cancer indications for dual ROR1 and PTK7 expression
  • Figure 38 Further Western Blot analysis of PDX models from different cancer indications for dual ROR1 and PTK7 expression
  • Figure 39 Cell surface binding of ROR1 x PTK7 VNAR hFc MMAE conjugate with corresponding ROR1 and PTK7 VNAR-hFc monospecific MMAE control conjugates to PA-1 (ROR1 hi / PTK7 hi ) and SW403 (ROR1 neg / PTK7 low ) human cancer cells by flow cytometry.
  • Figure 40 Cell surface binding of ROR1 x PTK7 VNAR hFc MMAE conjugate with corresponding ROR1 and PTK7 VNAR-hFc monospecific MMAE control conjugates to PA-1 (ROR1 hi / PTK7 hi ) and SW403 (ROR1 neg / PTK7 low ) human cancer cells by flow cytometry.
  • Figure 41 Internalisation of ROR1 x PTK7 VNAR hFc MMAE conjugate with corresponding ROR1 and PTK7 VNAR-hFc monospecific MMAE control conjugates to HEK-293-ROR1 (ROR1 hi / PTK7 hi ).
  • Figure 42 Potency of ROR1 x PTK7 bispecific drug-conjugates in killing the ROR1 positive and PTK7 positive NCI-H1975 cells lung adenocarcinoma cells.
  • Figure 43 Cell surface binding of ROR1 x PTK7 VNAR hFc PNU conjugate with corresponding ROR1 and PTK7 VNAR-hFc monospecific PNU control conjugates to PA-1 (ROR1 hi / PTK7 hi ) and SW403 (ROR1 neg / PTK7 low ) human cancer cells by flow cytometry.
  • Figure 44 Cell surface binding of ROR1 x PTK7 VNAR hFc PNU conjugate with corresponding ROR1 and PTK7 VNAR-hFc monospecific PNU control conjugates to PA-1 (ROR1 hi / PTK7 hi ) and SW403 (ROR1 neg / PTK7 low ) human cancer cells by flow cytometry.
  • Figure 45 Potency of ROR1 x PTK7 bispecific PNU drug-conjugates in killing the PTK7 positive PA-1 cells, and normal human epidermal keratinocytes (NHEKAd).
  • the present invention generally relates to bi-specific antigen binding molecules.
  • the invention provides immunoglobulin-like shark variable novel antigen receptors (VNARs) specific for receptor tyrosine kinase-like orphan receptor 1 (ROR1) and associated fusion proteins, chimeric antigen receptors, conjugates, and nucleic acids, as well as accompanying methods.
  • VNARs immunoglobulin-like shark variable novel antigen receptors
  • ROR1-specifc VNAR domains are described herein as ROR1-specific antigen binding molecules.
  • the Novel or New antigen receptor (IgNAR) is an approximately 160 kDa homodimeric protein found in the sera of cartilaginous fish (Greenberg A. S., et al., Nature, 1995.374(6518): p.168-173, Dooley, H., et al, Mol.
  • Each molecule consists of a single N-terminal variable domain (VNAR) and five constant domains (CNAR).
  • VNAR N-terminal variable domain
  • CNAR constant domains
  • the IgNAR domains are members of the immunoglobulin-superfamily.
  • the VNAR is a tightly folded domain with structural and some sequence similarities to the immunoglobulin and T-cell receptor Variable domains and to cell adhesion molecules and is termed the VNAR by analogy to the N Variable terminal domain of the classical immunoglobulins and T Cell receptors.
  • the VNAR shares limited sequence homology to immunoglobulins, for example 25-30% similarity between VNAR and human light chain sequences.
  • Kovaleva M. et al Expert Opin. Biol. Ther.2014.14(10): p.1527-1539 and Zielonka S. et al mAbs 2015. 7(1): p.15-25 provided summaries of the structural characterization and generation of the VNARs, which are hereby incorporated by reference.
  • the VNAR does not appear to have evolved from a classical immunoglobulin antibody ancestor.
  • VNARs The distinct structural features of VNARs are the truncation of the sequences equivalent to the CDR2 loop present in conventional immunoglobulin variable domains and the lack of the hydrophobic VH/VL interface residues which would normally allow association with a light chain domain, which is not present in the IgNAR structure. Furthermore, unlike classical immunoglobulins some VNAR subtypes include extra cysteine residues in the CDR regions that are observed to form disulphide bridges in addition to the canonical Immunoglobulin superfamily bridge between the Cysteines in the Framework 1 and 3 regions N terminally adjacent to CDRs 1 and 3. To date, there are three defined types of shark IgNAR known as I, II and III.
  • Type I has germline encoded cysteine residues in framework 2 and framework 4 and an even number of additional cysteines within CDR3. Crystal structure studies of a Type I IgNAR isolated against and in complex with lysozyme enabled the contribution of these cysteine residues to be determined. Both the framework 2 and 4 cysteines form disulphide bridges with those in CDR3 forming a tightly packed structure within which the CDR3 loop is held tightly down towards the HV2 region. To date Type I IgNARs have only been identified in nurse sharks - all other elasmobranchs, including members of the same order have only Type II or variations of this type.
  • Type II IgNAR are defined as having a cysteine residue in CDR1 and CDR3 which form intramolecular disulphide bonds that hold these two regions in close proximity, resulting in a protruding CDR3 that is conducive to binding pockets or grooves.
  • Type I sequences typically have longer CDR3s than type II with an average of 21 and 15 residues respectively. This is believed to be due to a strong selective pressure for two or more cysteine residues in Type I CDR3 to associate with their framework 2 and 4 counterparts.
  • Studies into the accumulation of somatic mutations show that there are a greater number of mutations in CDR1 of type II than type I, whereas HV2 regions of Type I show greater sequence variation than Type II.
  • a third IgNAR type known as Type III has been identified in neonates. This member of the IgNAR family lacks diversity within CDR3 due to the germline fusion of the D1 and D2 regions (which form CDR3) with the V-gene. Almost all known clones have a CDR3 length of 15 residues with little or no sequence diversity.
  • Another structural type of VNAR, termed type (IIb or IV) has only two canonical cysteine residues (in framework 1 and framework 3b regions). So far, this type has been found primarily in dogfish sharks and was also isolated from semisynthetic V-NAR libraries derived from wobbegong sharks.
  • VNAR binding surface unlike the variable domains in other natural immunoglobulins, derives from four regions of diversity: CDR1, HV2, HV4 and CDR3, joined by intervening framework sequences in the order: FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4.
  • CDR1, HV2, HV4 and CDR3 joined by intervening framework sequences in the order: FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4.
  • the IgNAR shares some incidental features with the heavy chain only immunoglobulin (HCAb) found in camelidae (camels, dromedaries and llamas) Unlike the IgNAR the HCAb is clearly derived from the immunoglobulin family and shares significant sequence homology to standard immunogloblulins. Importantly one key distinction of VNARs is that the molecule has not had at any point in its evolution a partner light chain, unlike classical immunoglobulins or the HCAbs. Flajnik M.F. et al PLoS Biol 2011. 9(8): e1001120 and Zielonka S.
  • the present invention provides such agents in the form of the ROR1-specific antigen binding molecules described herein. Without being bound by theory, the presently-described ROR1-specific antigen binding molecules are thought to bind to both human and murine ROR1.
  • a number of variants including G3CP, G3CPG4, 1E5, 1B11, C3CP, 1G9, 1H8, G11CP, D9CP, 1B6, 1 F10, F2CP, B6CP, 1E1 and P3A1, P3A1G1 NAC6.S, P3A1G1 AE3.S, P3A1G1 NAC6, P3A1G1 AE3 and P3A1G1 NAG8 have been experimentally confirmed to bind to both hROR1 and mROR1. Furthermore, the ROR1-specific antigen binding molecules described herein may bind to deglycosylated forms of ROR1.
  • ROR1-specific antigen binding molecules may not bind to a number of linear peptides associated with anti-ROR1 antibodies described in the prior art.
  • the presently- described ROR1-specific antigen binding molecules are therefore thought to bind to distinct epitopes in the ROR1 sequence compared to these prior art anti-ROR1 antibodies. Binding of ROR1-specific antigen binding molecules to cancer cell lines, as well as internalisation, have been demonstrated. This confirms the potential for the use of such molecules in the treatment of cancers, specifically cancers which express ROR1.
  • Various forms of the ROR1-specific antigen binding molecules are described, including fusion proteins of several types. Fusion proteins including an immunoglobulin Fc region are described, as well as both homo and heterodimers.
  • VNAR molecules conjugated to a variety of moieties and payloads The application therefore discloses chemically conjugated VNARs. More specifically, ROR1- specific antigen binding molecules in several conjugated formats are provided. ROR1-specific antigen binding molecules described herein have been formatted in combination with an PTK7-specific binding molecule. The present invention therefore relates to bi-specific ROR1/PTK7 antigen binding molecules. In a first configuration, the invention provides a PTK7-specific binding molecule as disclosed as part of any of the following aspects of the invention. The invention therefore provides a mono-specific PTK7 binding molecule.
  • the mono-specific PTK7 binding molecule may comprise the amino acid sequence of any PTK7 specific binding molecule disclosed herein, including those disclosed in the context of bi- specific antigen binding molecules.
  • the mono-specific PTK7 binding molecule may comprise any CDR1 and/or CDR3 sequence of any PTK7 specific binding molecule disclosed herein, including those disclosed in the context of bi-specific antigen binding molecules.
  • the mono-specific PTK7 binding molecule may comprise any HV2 and/or HV4 sequence of any PTK7 specific binding molecule disclosed herein, including those disclosed in the context of bi-specific antigen binding molecules.
  • the mono-specific PTK7 binding molecule may comprise any FW1, FW2, FW3a, FW3b and/or FW4 sequence of any PTK7 specific binding molecule disclosed herein, including those disclosed in the context of bi-specific antigen binding molecules.
  • the mono-specific PTK7 binding molecule may have the characteristics of any PTK7 specific binding molecule disclosed herein, including those disclosed in the context of bi-specific antigen binding molecules.
  • the invention provides a bi-specific antigen binding molecule comprising: (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 12), YPWGAGAPRLVQWY (SEQ ID NO: 13), YPWGAGAPRQVQWY (SEQ ID NO:
  • CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO:11), YPWGAGAPCLVQWY (SEQ ID NO: 12), YPWGAGAPRLVQWY (SEQ ID NO:13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY
  • CDR1 may be a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1), DANYGLAA (SEQ ID NO: 5), GANYDLSA (SEQ ID NO: 2), GANYGLSA (SEQ ID NO: 3), and GANYDLAA (SEQ ID NO: 4).
  • the ROR1 specific antigen binding molecule may be defined as comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO:11), YPWGAGAPCLVQWY (SEQ ID NO: 12), YPWGAGAPRLVQWY (SEQ ID NO:13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO:
  • CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20) and YPWGAGAPWNVQWY (SEQ ID NO: 24), and/or CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1) and DANYGLAA (SEQ ID NO: 5).
  • CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23). In one embodiment of the ROR1-specific antigen binding molecule: CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23); CDR1 is a CDR sequence having an amino acid sequence according to GANYGLAA (SEQ ID NO: 1); HV2 is a hypervariable sequence having an amino acid sequence according to SSNQERISIS (SEQ ID NO: 6); and HV4 is a hypervariable sequence having an amino acid sequence according to NKRTM (SEQ ID NO: 8).
  • CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23);
  • CDR1 is a CDR sequence having an amino acid sequence according to DANYGLAA (SEQ ID NO: 5);
  • HV2 is a hypervariable sequence having an amino acid sequence according to SSNKERISIS (SEQ ID NO: 7); and
  • HV4 is a hypervariable sequence having an amino acid sequence according to NKGTM (SEQ ID NO: 9).
  • CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPWLVQWY (SEQ ID NO: 10);
  • CDR1 is a CDR sequence having an amino acid sequence according to DANYGLAA (SEQ ID NO: 5);
  • HV2 is a hypervariable sequence having an amino acid sequence according to SSNKERISIS (SEQ ID NO: 7); and
  • HV4 is a hypervariable sequence having an amino acid sequence according to NKGTM (SEQ ID NO: 9).
  • the ROR1-specific antigen binding molecule comprises an amino acid sequence selected from the group consisting of: ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50) referred to herein as G3CP; TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPWLVQWY DGAGTKVEIK (SEQ ID NO: 51) referred to herein as B1G4; ASVNQTPRTATKETGESLTINCVVTGANYDLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPSGAGAPRPVQWYDGAGTV;
  • the ROR1-specific antigen binding molecule may comprise an amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50).
  • the ROR1-specific antigen binding molecule may comprise an amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50) or a functional variant thereof having CDR1, HV2, HV4 and CDR2 sequences according to SEQ ID NO: 50 and having FW1, FW2, FW3a, FW3b and FW4 sequences having a combined sequence identity of at least 45% to the combined FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO: 50.
  • G3CP SEQ ID NO: 50
  • functional variants thereof include increased expression yields and hydrophilicity and increased ease of analysis, purification and monomericity in non-optimised aqueous buffer systems for these proteins. Without being bound by theory, these advantages may be particularly evident in VNAR-hFc fusion proteins comprising the G3CP sequence or functional variants thereof.
  • the G3CP sequence and functional variants thereof may therefore provide improved manufacturing and/or handling properties.
  • G3CP-hFc shows excellent in vivo efficacy in a patient-derived xenograft model of Triple Negative Breast Cancer (TNBC) when conjugated to a cytotoxic anthracycline (PNU) derivative.
  • TNBC Triple Negative Breast Cancer
  • PNU cytotoxic anthracycline
  • ROR1xPTK7 bi-specifics comprising G3CP have shorter retention time (RT) in size exclusion chromatography (SEC) compared to B1 comprising bi-specifics, indicating improved hydrophilicity of ROR1xPTK7 bi-specifics containing G3CP.
  • ROR1xPTK7 bi-specifics comprising G3CP may have reduced turbidity and reduced formation of high molecular weight (HMW) species compared to B1 comprising bi-specifics, indicating increased ease of analysis, purification and monomericity of ROR1xPTK7 bi-specifics comprising G3CP in non-optimised aqueous buffer systems for these proteins. Therefore, the G3CP sequence and functional variants thereof may therefore provide improved manufacturing and/or handling properties that are retained in ROR1xPTK7 bi-specific format.
  • HMW high molecular weight
  • ROR1 x PTK7 bi-specifics may also improve efficacy where ROR1 mono-specifics are refractory or have low specificity.
  • ROR1 mono-specifics are refractory or have low specificity.
  • disclosed herein are data showing not only co-expression of ROR1 and PTK7 in TNBC (the application also contains data showing excellent in vivo efficacy of a G3CP-hFc PNU conjugate in a patient-derived xenograft model of TNBC) but high expression of PTK7 alongside ROR1 expression in non-small cell lung cancer models and ovarian cancer models using western blotting.
  • the ROR1-specific antigen binding molecule may comprise an amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN (SEQ ID NO: 61).
  • the ROR1-specific antigen binding molecule may comprise an amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51).
  • SEQ ID NO: 51 Particular advantages associated with SEQ ID NO: 51 (“B1G4”) and functional variants thereof include increased expression yields and monomericity in aqueous buffer systems for fusion proteins comprising the B1G4 sequence or functional variants thereof, such as VNAR-hFc fusion proteins.
  • the B1G4 sequence and functional variants thereof may therefore provide fusion proteins with improved manufacturing and/or handling properties.
  • the ROR1-specific antigen binding molecule may comprise an amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51) or a functional variant thereof having CDR1, HV2, HV4 and CDR2 sequences according to SEQ ID NO: 51 and having FW1, FW2, FW3a, FW3b and FW4 sequences having a combined sequence identity of at least 45% to the combined FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO: 51.
  • the ROR1-specific antigen binding molecule comprises an amino acid sequence selected from the group consisting of: TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) referred to herein as G3CP G4; ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSL RISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK (SEQ ID NO: 72) referred to herein as G3CP V15; TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAG
  • the ROR1-specific antigen binding molecule may comprise an amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71).
  • the ROR1-specific antigen binding molecule may comprise an amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) or a functional variant thereof having CDR1, HV2, HV4 and CDR2 sequences according to SEQ ID NO: 71 and having FW1, FW2, FW3a, FW3b and FW4 sequences having a combined sequence identity of at least 45% to the combined FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO: 71.
  • G3CP G4 SEQ ID NO: 71
  • functional variants thereof include increased expression yields and hydrophilicity and increased ease of analysis, purification and monomericity in non-optimised aqueous buffer systems for these proteins. Without being bound by theory, these advantages may be particularly evident in VNAR-hFc fusion proteins comprising the G3CP G4 sequence or functional variants thereof.
  • the G3CP G4 sequence and functional variants thereof may therefore provide improved manufacturing and/or handling properties.
  • G3CPG4-hFc shows excellent in vivo efficacy in a patient-derived xenograft model of Triple Negative Breast Cancer (TNBC) when conjugated to a cytotoxic anthracycline (PNU) derivative.
  • TNBC Triple Negative Breast Cancer
  • PNU cytotoxic anthracycline
  • ROR1xPTK7 bi-specifics comprising G3CP G4 have shorter retention time (RT) in size exclusion chromatography (SEC) compared to B1 comprising bi-specifics, indicating improved hydrophilicity of ROR1xPTK7 bi-specifics containing G3CP G4.
  • ROR1 x PTK7 bi-specifics including but not limited to those comprising the G3CP G4 sequence, may also improve efficacy where ROR1 mono-specifics are refractory or have low specificity.
  • RNA showing not only co-expression of ROR1 and PTK7 in TNBC the application also contains data showing excellent in vivo efficacy of a G3CPG4-hFc PNU conjugate in a patient-derived xenograft model of TNBC) but high expression of PTK7 alongside ROR1 expression in non-small cell lung cancer models and ovarian cancer models using western blotting.
  • the sequences of G3CP and G3CPG4 have in common two single amino acid changes relative to the sequence of B1. These are both within CDR3 and are the substitution: 1. Of a W residue with a Y residue, and 2. Of an L residue with a N residue.
  • G3CPG4 has a further single amino acid change in each of CDR1, HV2 and HV4 relative to B1 (which also appear in B1 G4) and changes to humanise the framework regions (some of which also appear in B1V15, SEQ ID NO: 115, as shown in Figure 15 - B1V15 has the same CDR1, HV2, HV4 and CDR3 sequences as B1 i.e. it is not a loop library variant; the changes to B1V15 relative to B1 are in the framework regions only).
  • any improvements over B1 shown by both G3CP and G3CPG4 which are not shown by B1 G4 or B1 V15 are thought to result from one or both of the two mutations they share in CDR3. Accordingly, advantages of G3CP and G3CPG4 are thought to derive from a CDR3 comprising the sequence YPWGAGAPYNVQWY (SEQ ID NO: 23). Without being bound by theory, the surprising advantages associated with YPWGAGAPYNVQWY (SEQ ID NO: 23) may represent a synergistic effect of both the W to Y and the L to N substitutions.
  • the surprising advantages may derive primarily from the W to Y substitution thus being shared by YPWGAGAPYLVQWY (SEQ ID NO: 20).1B6, which has the L to N mutation and a CDR3 sequence of YPWGAGAPWNVQWY (SEQ ID NO: 24), has a lower elution volume than B1, therefore the L to N mutation in CDR3 does lead to improved manufacturing and/or handling properties.
  • the PTK7-specific antigen binding molecule may be any molecule which binds to PTK7.
  • the PTK7-specific antigen binding molecule may be selected from the group comprising an immunoglobulin, an immunoglobulin Fab region, an Fv, a single chain Fv (scFv), a diabody, a triabody, a tetrabody, a VNAR domain, a single domain antibody (sdAb), a VH domain, or a scaffold protein (affibodies, centyrins, darpins etc.).
  • the PTK7-specific antigen binding molecule is a VNAR domain.
  • the PTK7 specific antigen binding molecule may derive from or compete with hu24.
  • the PTK7-specific antigen binding molecule may be an immunoglobulin, an immunoglobulin Fab region, an Fv, a single chain Fv (scFv), a diabody, a triabody, a tetrabody, a VNAR domain, a single domain antibody (sdAb), a VH domain, or a scaffold protein (affibodies, centyrins, darpins etc.) that is derived from or competes with hu24.
  • the PTK7-specific antigen binding molecule may comprise the CDRs of hu24.
  • the PTK7-specific antigen binding molecule may comprise the VH and/or VL domains of hu24
  • the PTK7 specific antigen binding molecule may comprise an antibody or antibody fragment.
  • hu24 a humanised mouse IgG targeting PTK7 Ig domains 1-4 with an affinity of 1.2 nM for the full PTK7 extracellular domain [WO2015168019].
  • the Fab HC may be fused to a fragment of an immunoglobulin Fc region.
  • the Fab LC and the Fab HC are associated via a disulphide bond.
  • the PTK7-specific antigen binding molecule may comprise SEQ ID NO: 548 and SEQ ID NO: 549 wherein SEQ ID NO: 548 is be fused to a fragment of an immunoglobulin Fc region and wherein SEQ ID NO: 548 and SEQ ID NO: 549 are associated via a disulphide bond.
  • the PTK7-specific antigen binding molecule may comprise the sequence of any PTK7- specific antigen binding molecule disclosed herein comprising: (i) at least 85% identity thereto, and/or (ii) one, two, or three amino acid substitutions relative thereto.
  • Said sequence identity is at least about 85% sequence identity and may therefore be 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% identity.
  • Preferably said sequence identity is at least 90% or at least 95%.
  • the one or more amino acid substitution may be a conservative amino acid substitution.
  • the PTK7-specific antigen binding molecule selectively interacts with PTK7 protein with an affinity constant of approximately 1 to 2,000 nM or 2 to 2,000 nM, preferably 1 to 200 nM, even more preferably 1 to 20 nM.
  • the affinity constant may be around 2 nM.
  • An affinity constant may be measured as described elsewhere herein for ROR1-specific antigen binding molecule for instance by surface plasmon resonance (SPR) or by Bio-layer interferometry (BLI).
  • the ROR1-specific antigen binding molecule and PTK7-specific antigen binding molecule may be combined in any order to form the bi-specific antigen binding molecule of the first aspect, i.e., the ROR1-specific antigen binding molecule may be N-terminal to the PTK7-specific antigen binding molecule or vice versa, or when the bi-specific antigen binding molecule is formed by Knobs-into-holes for example, the ROR1-specific antigen binding molecule may be on one arm while the PTK7-specific antigen binding molecule may be on the other arm or vice versa.
  • Preferred linkers include [G4S]3, [G4S]5, and G4S.
  • a preferred linker is [G4S]3.
  • a preferred linker is G4S.
  • a preferred linker is [G4S]5.
  • Other linkers may include, but are not limited to PGVQPSPGGGGS (SEQ ID NO: 89) (Wobbe-G4S), PGVQPAPGGGGS (SEQ ID NO: 90) (Wobbe-G4S GM). It will be appreciated that different combinations of different linkers can be combined within the same construct.
  • ROR1 binders may the same ROR1 specific antigen binding molecule or two different ROR1 specific antigen binding molecules.
  • the PTK7 binder may be any PTK7 specific antigen binding molecule disclosed herein. Where two PTK7 binders are present, they may the same PTK7 specific antigen binding molecule or two different PTK7 specific antigen binding molecules.
  • linkers between domains are preferentially, but not limited to (G4S)X, where X is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, PGVQPSPGGGGS (SEQ ID NO: 89) (Wobbe-G4S), PGVQPAPGGGGS (SEQ ID NO: 90) (Wobbe-G4S GM) and wherein different combinations of different linkers can be combined within the same construct.
  • PGVQPSPGGGGS SEQ ID NO: 89
  • PGVQPAPGGGGS SEQ ID NO: 90
  • additional C-terminal (or N-terminal) tag sequences may or may not be present.
  • the invention provides a bi-specific antigen binding molecule comprising: (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30) and DTSYGLYS (SEQ ID NO:207); FW1 is a framework region; FW2 is a framework region; HV2 is
  • the ROR1 specific antigen binding molecule may be fused to a first fragment of an immunoglobulin Fc region and the PTK7 specific binding molecule may be fused to a second fragment of an immunoglobulin Fc region and the first fragment of an immunoglobulin Fc region and the second fragment of an immunoglobulin Fc region may be engineered to dimerise.
  • first fragment and second fragment are interchangeable.
  • the ROR1-specific antigen binding molecule comprises an amino acid sequence selected from the group consisting of: TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFS LRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (SEQ ID NO: 206) referred to herein as P3A1; TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTL TISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 77) referred to herein as P3A1 G1 AE3; TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFT LTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEI
  • Framework region FW1 is preferably from 20 to 28 amino acids in length, more preferably from 22 to 26 amino acids in length, still more preferably from 23 to 25 amino acids in length. In certain preferred embodiments, FW1 is 26 amino acids in length. In other preferred embodiments, FW1 is 25 amino acids in length. In still other preferred embodiments, FW1 is 24 amino acids in length. In alternative definitions, CDR region CDR1 is preferably from 7 to 11 amino acids in length, more preferably from 8 to 10 amino acids in length.
  • CDR1 is 9 amino acids in length. In other preferred embodiments, CDR1 is 8 amino acids in length.
  • Framework region FW2 is preferably from 6 to 14 amino acids in length, more preferably from 8 to 12 amino acids in length. In certain preferred embodiments, FW2 is 12 amino acids in length. In other preferred embodiments, FW2 is 10 amino acids in length. In other preferred embodiments, FW2 is 9 amino acids in length. In other preferred embodiments, FW2 is 8 amino acids in length.
  • Hypervariable sequence HV2 is preferably from 4 to 11 amino acids in length, more preferably from 5 to 10 amino acids in length. In certain preferred embodiments, HV2 is 10 amino acids in length.
  • HV2 is 9 amino acids in length. In other preferred embodiments, HV2 is 6 amino acids in length.
  • Framework region FW3a is preferably from 6 to 10 amino acids in length, more preferably from 7 to 9 amino acids in length. In certain preferred embodiments, FW3a is 8 amino acids in length. In certain preferred embodiments, FW3a is 7 amino acids in length.
  • Hypervariable sequence HV4 is preferably from 3 to 7 amino acids in length, more preferably from 4 to 6 amino acids in length. In certain preferred embodiments, HV4 is 5 amino acids in length. In other preferred embodiments, HV4 is 4 amino acids in length.
  • Framework region FW3b is preferably from 17 to 24 amino acids in length, more preferably from 18 to 23 amino acids in length, still more preferably from 19 to 22 amino acids in length. In certain preferred embodiments, FW3b is 21 amino acids in length. In other preferred embodiments, FW3b is 20 amino acids in length. In alternative definitions, CDR region CDR3 is preferably from 8 to 21 amino acids in length, more preferably from 9 to 20 amino acids in length, still more preferably from 10 to 19 amino acids in length. In certain preferred embodiments, CDR3 is 17 amino acids in length. In other preferred embodiments, CDR3 is 14 amino acids in length. In still other preferred embodiments, CDR3 is 12 amino acids in length. In yet other preferred embodiments, CDR3 is 10 amino acids in length.
  • FW1 is a framework region of from 20 to 28 amino acids
  • FW2 is a framework region of from 6 to 14 amino acids
  • FW3a is a framework region of from 6 to 10 amino acids
  • FW3b is a framework region of from 17 to 24 amino acids
  • FW4 is a framework region of from 7 to 14 amino acids.
  • FW1 has an amino acid sequence selected from the group consisting of: ASVNQTPRTATKETGESLTINCVVT (SEQ ID NO: 40), TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 41) and ASVTQSPRSASKETGESLTITCRVT (SEQ ID NO: 42), or a functional variant of any thereof with a sequence identity of at least 45%;
  • FW2 has an amino acid sequence according to TYWYRKNPG (SEQ ID NO: 43), or a functional variant of any thereof with a sequence identity of at least 45%;
  • FW3a has an amino acid sequence selected from the group consisting of: GRYVESV (SEQ ID NO: 44) and GRYSESV (SEQ ID NO: 45), or a functional variant of any thereof with a sequence identity of at least 45%;
  • FW3b has an amino acid sequence selected from the group consisting of: SFSLRIKDLTVADSATYYCKA (SEQ ID NO: 40), TRVDQSP
  • humanised sequences of the invention include, but are not limited to: B1G4 G3CP G4 G3CP V15 1H8 G4 1H8 V15 C3CP G4 C3CPV15 P3A1 G1 AE3 P3A1 G1 AE3.S P3A1 G1 NAC6 P3A1 G1 NAC6.S P3A1 G1 NAG8 P3A1 G1 NAG8.S P3A1 G1 AF7.S It will be appreciated by the skilled person that the humanised ROR1-specific antigen binding molecules described herein may be further humanised, for instance by substituting further FW region amino acids with amino acids of DPK-9.
  • the ROR1-specific antigen binding molecule may also be conjugated to a detectable label, dye, toxin, drug, pro-drug, radionuclide or biologically active molecule.
  • the ROR1-specific antigen binding molecule does not bind to receptor tyrosine kinase-like orphan receptor 2 (ROR2). More preferably, the ROR1-specific antigen binding molecule binds to both human ROR1 and murine ROR1 (mROR1). Yet more preferably, the ROR1-specific antigen binding molecule binds to deglycosylated ROR1.
  • ROR1-specific antigen binding molecules of the invention may not bind to a linear peptide sequence selected from: YMESLHMQGEIENQI (SEQ ID NO: 91) CQPWNSQYPHTHTFTALRFP (SEQ ID NO: 92) RSTIYGSRLRIRNLDTTDTGYFQ (SEQ ID NO: 93) QCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYE (SEQ ID NO: 94)
  • the ROR1-specific antigen binding molecule selectively interacts with ROR1 protein with an affinity constant of approximately 0.01 to 50 nM, preferably 0.1 to 30 nM, even more preferably 0.1 to 10 nM.
  • An affinity constant may be measured by Bio-layer interferometry (BLI). For monomers the interaction is 1:1.
  • VNAR-hFc format the inventors have used two approaches. One where the ROR1 is immobilized and thus a bi-valent VNAR-hFc binds with an apparent KD as the avidity effect comes into play. The other approach is in a 1:1 format whereby the VNAR-hFc is immobilized and ROR1 is flowed across the surface thus giving the K D for ‘true’ 1:1 binding.
  • affinity constants refer to those measured by Bio-layer interferometry (BLI) using the 1:1 binding format.
  • G3CP and G3CP G4 are within the 0.1 – 10 nM range.
  • P3A1 G1 loop variants examples have KD values of 5.0 nM (AE3), 13.8 nM (NAC6) and 12.2 nM (NAG8).
  • the antigen binding molecule specifically binds ROR1 with an affinity determined by SPR, (e.g., under SPR conditions disclosed herein).
  • the antigen binding molecule may specifically bind PTK7 with an affinity determined by SPR, (e.g., under SPR conditions disclosed herein).
  • the SPR is carried out at 37 o C.
  • the SPR is carried out at physiological pH, such as about pH7 or at pH7.6 (e.g., using Hepes buffered saline at pH7.6 (also referred to as HBS-EP)).
  • the SPR is carried out at a physiological salt level, e.g., 150mM NaCl.
  • the SPR is carried out at a detergent level of no greater than 0.05% by volume, e.g., in the presence of P20 (polysorbate 20; e.g., Tween-20 TM ) at 0.05% and EDTA at 3mM.
  • the SPR is carried out at 25 o C or 37 o C in a buffer at pH7.6, 150mM NaCl, 0.05% detergent (e.g., P20) and 3mM EDTA.
  • the buffer can contain 10mM Hepes.
  • the SPR is carried out at 25 o C or 37 o C in HBS-EP.
  • HBS-EP is available from Teknova Inc (California; catalogue number H8022).
  • the affinity of the antibody or fragment is determined using SPR by 1.
  • Coupling target antigen e.g. ROR1 or PTK7
  • a biosensor chip e.g., GLM chip
  • target antigen may be coupled indirectly to biosensor chip via an initial anti-tag IgG capture step (e.g. appropriate anti-Fc IgG) 2. Passing the test ROR1 x PTK7 bispecific over the chip’s capture surface at 1024nM, 256nM, 64nM, 16nM, 4nM with 0nM (i.e. buffer alone); 3. Determining the affinity of binding of test ROR1 x PTK7 bispecific to target antigen using surface plasmon resonance, e.g., under an SPR condition discussed above (e.g., at 25 o C in physiological buffer).
  • surface plasmon resonance e.g., under an SPR condition discussed above (e.g., at 25 o C in physiological buffer).
  • test ROR1 x PTK7 bispecific may be coupled to biosensor chip directly (e.g. primary amine coupling) or indirectly via an initial anti-tag IgG capture step (e.g. anti-hFc IgG) and passing the target antigen (e.g. ROR1 or PTK7 ) over the chip’s capture surface.
  • initial anti-tag IgG capture step e.g. anti-hFc IgG
  • target antigen e.g. ROR1 or PTK7
  • Regeneration of the capture surface can be carried out with 10mM glycine at pH1.7. This removes the captured antibody and allows the surface to be used for another interaction.
  • the binding data can be fitted to 1:1 model inherent using standard techniques, e.g., using a model inherent to the ProteOn XPR36 TM analysis software.
  • BLI methods are used to determine affinity using Octet BLI system (Sartorius). Unless otherwise stated, BLI was used to determine affinity of the PTK7 monomers, PTK7 mono-specific hFc fusions and the ROR1 x PTK7 all binding measurements disclosed herein.
  • ROR1 or PTK7 ligand is attached to biosensors by standard amine coupling (ARG2 biosensors) or by affinity capture (for example using anti-human Fc capture with AHC biosensors, or anti-His capture using HIS1K biosensors).
  • the ROR1-specific antigen binding molecule is preferably capable of mediating killing of ROR1-expressing tumour cells or is capable of inhibiting cancer cell proliferation.
  • the ROR1-specific antigen binding molecule may also be capable of being endocytosed upon binding to ROR1. In other embodiments, the ROR1-specific antigen binding molecule may not be endocytosed upon binding to ROR1.
  • the ROR1-specific antigen binding molecule may comprise any of the sequences set out below, each of which is disclosed in WO 2019/122447, hereby incorporated by reference in its entirety.
  • the ROR1-specific antigen binding molecules comprises any one of the sequences set out below.
  • B1 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 113)
  • D3 ASVNQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFS LRIKDLTVADSATYYCKAQSGMAISTGSGHGYNWYDGAGTVLTVN (SEQ ID NO: 457)
  • BA11 TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTL TISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK SEQ ID NO:
  • the ROR1 binding molecules disclosed in WO 2019/122447 may for example be fused to one or more biologically active proteins, such as hFc, optionally via a linker. Also disclosed herein is a binding molecule 2V which is disclosed in WO 2019/122447 but acts as a negative control VNAR from a na ⁇ ve library with no known target.
  • the invention provides a recombinant fusion protein comprising a bi- specific antigen binding molecule according to the first or the second aspects of the invention.
  • the ROR1 specific antigen binding molecule and/or the PTK7 specific antigen binding molecule is fused to one or more biologically active proteins.
  • the specific antigen binding molecule may be fused to one or more biologically active proteins via one or more linker domains.
  • Preferred linkers include but are not limited to [G4S]x, where x is 1, 2, 3, 4, 5, or 6. Particular preferred linkers are G4S (SEQ ID NO: 222), [G4S]3 (SEQ ID NO: 86) and [G4S]5 (SEQ ID NO: 87) .
  • Other preferred linkers include the sequences PGVQPSP (SEQ ID NO: 88), PGVQPSPGGGGS (SEQ ID NO: 89) and PGVQPAPGGGGS (SEQ ID NO: 90).
  • linkers may be particularly useful when recombinant fusion proteins are expressed in different expression systems that differ in glycosylation patterns, such as CHO and insect, and those that do not glycosylate expressed proteins (e.g. E. coli).
  • Any recombinant fusion protein sequence disclosed herein comprising a [G 4 S] 3 linker may alternatively possess any other linker sequence disclosed herein.
  • the fusion proteins of the invention can be constructed in any order, i.e., with the ROR1-specific antigen binding molecule at the N-terminus, C-terminus, or at neither terminus (e.g. in the middle of a longer amino acid sequence).
  • the PTK7-specific antigen binding molecule may be at the N-terminus, C-terminus, or at neither terminus (e.g. in the middle of a longer amino acid sequence).
  • Preferred biologically active proteins include, but are not limited to an immunoglobulin, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobulin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv)2, a diabody, a triabody, a tetrabody, a bispecific t-cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, or a scaffold protein (affibodies, centyrins, darpins etc.).
  • a particularly preferred biologically active protein is an immunoglobulin Fc region.
  • Other preferred fusion proteins include VNAR-VNAR and VNAR-VNAR-VNAR.
  • the at least one biologically active protein is an immunoglobulin Fc region.
  • the recombinant fusion protein may comprise a ROR1 specific antigen binding molecule fused to an immunoglobulin Fc region or a fragment thereof.
  • the immunoglobulin Fc region or fragment thereof may be fused to the ROR1 specific antigen binding molecule via a linker.
  • the immunoglobulin Fc region or fragment thereof and/or the linker may be fused to the C-terminus of the ROR1 specific antigen binding molecule.
  • the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 223..
  • G3CP-hFc ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 186) G3CPG4-hFc TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWY
  • the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 186, SEQ ID NO: 187 or SEQ ID NO: 183.
  • the at least one biologically active protein is an immunoglobulin Fc region further modified to comprise an S to C mutation.
  • the S to C mutation may be at position S239 (EU numbering). Therefore, the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, or SEQ ID NO: 182 or SEQ ID NO: 224.
  • G3CP-hFc(S239C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKT HTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 178) G3CPG4-hFc(S239C) TRVDQSPSSLSASVG
  • the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 178, SEQ ID NO: 179 or SEQ ID NO: 180.
  • the S to C mutation may be at position S442 (EU numbering). Therefore, the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, or SEQ ID NO: 229 or SEQ ID NO: 230..
  • G3CP-hFc (S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 225) G3CPG4-hFc (S442C) TRVDQSPSSLSAS
  • the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 225, SEQ ID NO: 226 or SEQ ID NO: 227.
  • the S to C mutation may be at both position S239 and S442 (EU numbering). Therefore, the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, or SEQ ID NO: 235 or SEQ ID NO: 236.
  • G3CP-hFc (S239C & S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKT HTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 231) G3CPG4-hFc (S239C & S44
  • the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 231, SEQ ID NO: 232 or SEQ ID NO: 233.
  • the recombinant fusion protein may comprise an PTK7 specific antigen binding molecule fused to an immunoglobulin Fc region or a fragment thereof.
  • the immunoglobulin Fc region or fragment thereof may be fused to the PTK7 specific antigen binding molecule via a linker.
  • the immunoglobulin Fc region or fragment thereof and/or the linker may be fused to the C-terminus of the PTK7 specific antigen binding molecule.
  • the PTK7 specific antigen binding molecule fused to an immunoglobulin Fc region may therefore comprise a sequence according to any one of SEQ ID Nos 473 to 484.
  • the PTK7 specific antigen binding molecule fused to an immunoglobulin Fc region may therefore comprise a sequence according to any one of SEQ ID Nos 473 to 475.
  • the PTK7 specific antigen binding molecule fused to an immunoglobulin Fc region may therefore comprise a sequence according to any one of SEQ ID Nos 476 to 478.
  • the PTK7 specific antigen binding molecule fused to an immunoglobulin Fc region may therefore comprise a sequence according to any one of SEQ ID Nos 479 to 481.
  • the PTK7 specific antigen binding molecule fused to an immunoglobulin Fc region may therefore comprise a sequence according to any one of SEQ ID Nos 482 to 484.
  • P2A7 hFc (SEQ ID NO: 473) TRVDQTPRTATKETGESLTINCVLTDTNYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFS LRIKDLTVADSATYYCKAAYIERNGFLTWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 4D2 hFc (SEQ ID NO: 474) ASVNQTPRTATKETGESLTINCVVTGAIC
  • the recombinant fusion protein may comprise one or more of the following SEQ ID Nos 408 to 431 and 485 to 496.
  • G3CP G 4 S-hFc ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 408) G3CPG4 G4S-hFc TRVDQSPSSLSASVGDRVTITCVLTDANYGLAA
  • the fragment of an immunoglobulin Fc region is an Fc heavy chain.
  • one or more residues of fusion protein comprises one or more amino acid substitution suitable for conjugation.
  • the one or more residues suitable for conjugation may be residues of the fragment of the immunoglobulin Fc region.
  • Any part of the fusion protein of the invention may be engineered to enable conjugation.
  • an immunoglobulin Fc region it may be engineered to include a cysteine residue as a conjugation site.
  • Preferred introduced cysteine residues include, but are not limited to S252C and S473C (Kabat numbering), which correspond to S239C and S442C in EU numbering, respectively.
  • any of the fusion proteins disclosed herein may comprise the S239C point mutation. In some embodiments, any of the fusion proteins disclosed herein may comprise the S442C point mutation. In some embodiments, any of the fusion proteins disclosed herein may comprise both S239C and S442C point mutations. It is explicitly contemplated herein that sequence of any of the fusion proteins disclosed herein may be modified to include an S239C and/or S442C point mutation.
  • recombinant fusions comprising multiple VNAR domains are provided. Accordingly, the recombinant fusions of the invention may be dimers, trimers or higher order multimers of VNARs.
  • each VNAR may be the same or different.
  • Recombinant fusions of the invention include, but are not limited to, bi-specific or tri-specific molecules in which each VNAR domain binds to a different antigen, or to different epitopes on a single antigen (bi-paratopic binders).
  • bi-paratopic binders The term “bi-paratopic” as used herein is intended to encompass molecules that bind to multiple epitopes on a given antigen.
  • recombinant fusions which include a ROR1- specific antigen binding molecule of the first aspect and a humanised VNAR domain.
  • Humanised VNAR domains may be referred to as soloMERs and include but are not limited to the VNAR BA11, which is a humanised VNAR that binds with high affinity to human serum albumin.
  • Examples of bi-paratopic and multivalent fusion proteins include, but are not limited to:
  • the specific binding molecules or recombinant fusions of the invention may be expressed with N- or C-terminal tags to assist with purification. Examples include but are not limited to His6 and/or Myc.
  • the N- or C-terminal tag may be further engineered to include additional cysteine residues to serve as conjugation points. It will therefore be appreciated that reference to specific binding molecules or recombinant fusions in all aspects of the invention is also intended to encompass such molecules with a variety of N- or C-terminal tags, which tags may also include additional cysteines for conjugation. Additional recombinant fusions are listed below. It will be appreciated that not every combination of linker and VNAR or fusion partner is listed below.
  • Monovalent-BA11 fusions Dimeric biparatopic BA11 fusions BA11-G3CP G3CP-P3A1G1 AE3-BA11 G3CP-BA11 P3A1G1 AE3-G3CP-BA11 BA11-G3CPG4 G3CP-BA11-P3A1G1 AE3 G3CPG4-BA1 P3A1G1 AE3-BA11-G3CP P3A1G1 AE3-BA11 G3CPG4-P3A1G1 AE3-BA11 BA11-P3A1G1 AE3 P3A1G1 AE3-G3CPG4-BA11 G3CPG4-BA11-P3A1G1 AE3 Divalent-BA11 fusions P3A1G1 AE3-BA11-G3CPG4 P3A1G1 AE3-P3A1G1 AE3-BA11 B1G
  • the WobbeCys-G 4 S sequence also contains a single cysteine residue to facilitate site-selective bioconjugation of payloads to the proteins, in this linker, using thiol mediated chemical coupling strategies.
  • the use of this linker sequence for bioconjugation is advantageous as reoxidation and capping of the reduced cysteine is minimal, leading to high yielding conversion of the protein to the corresponding conjugate in bioconjugation reactions.
  • bi-paratopic fusion protein can also be made by fusing any of the ROR1 specific antigen binding molecules disclosed herein to any of the protein tyrosine kinase 7 (PTK7) specific antigen binding molecule disclosed herein.
  • the bi-paratopic-fusion protein may have the “beads-on-a-string” format.
  • the bi-paratopic fusion protein may comprise any of the linkers disclosed herein.
  • the bi-paratopic fusion protein may comprise the WobbeCys-G4S linker disclosed herein.
  • the bi-paratopic fusion protein may have a sequence selected from the group consisting of SEQ ID NO: 497 to 499.
  • C-terminal tags include, but are not limited to, tags that contain poly-Histidine sequences to facilitate purification (such as His6), contain c-Myc sequences (such as EQKLISEEDL (SEQ ID NO: 112)) to enable detection and / or contain Cysteine residues to enable labelling and bioconjugation using thiol reactive payloads and probes and combinations thereof.
  • Preferential C-terminal tags include but are not limited to: QASGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 98) QACGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 99) QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) AAAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 100) ACAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 101) QASGAHHHHHH (SEQ ID NO: 102) QACGAHHHHHH (SEQ ID NO: 103) QACKAHHHHHH (SEQ ID NO: 104) AAAHHHHHH (SEQ ID NO: 105) ACAHHHHHH (SEQ ID NO: 106) QASGA (SEQ ID NO: 107) QACGA (SEQ ID NO: 108) QACKA (SEQ ID NO: 109) ACA (SEQ ID NO: 110) SAPSA (SEQ ID NO: 111) Wherein: G3CP is ASVNQTPRTATKET
  • recombinant fusion protein disclosed herein which does refer to the presence an PTK7 specific binding molecule can be modified to include an PTK7 specific binding molecule.
  • Any PTK7 specific binding molecules disclosed herein are explicitly contemplated as combined with any recombinant fusion protein disclosed herein which does refer to the presence an PTK7 specific binding molecule in any configuration.
  • recombinant fusions are provided which include a ROR1- specific antigen binding molecule and a recombinant toxin. Examples of recombinant toxins include but are not limited to Pseudomonas exotoxin PE38 and diphtheria toxin.
  • recombinant fusions which include a ROR1- specific antigen binding molecule and a recombinant CD3 binding protein.
  • ROR1 and CD3 binding agents include but are not limited to: B1G4–[WGM]–CD3 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELAR PGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTA YMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGSDIQLTQSPAI MSASPGEKVTMTCRASSSVSYMN
  • UCL OKT3 sequence (WO2019008379) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFK DRVTITADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYCLDYWGQGTMVTVSSVEGGSGGSGGSG GSGGVDDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPFTFGQGTKVEIK (SEQ ID NO: 130) Harpoon ID20 (WO2016187594) DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQ
  • the Fc regions disclosed herein may be engineered to reduce Fc ⁇ R binding.
  • the fragment may be a first fragment of an immunoglobulin Fc region which is engineered to dimerize with a second fragment of an immunoglobulin Fc region.
  • the ROR1 specific antigen binding molecule may be fused to a first fragment of an immunoglobulin Fc region and the PTK7 specific binding molecule may be fused to a second fragment of an immunoglobulin Fc region.
  • an immunoglobulin Fc region that is “engineered to dimerise” may comprise at least one amino acid substitution.
  • the at least one amino acid substitution promotes and/or makes more energetically favourable, an interaction and/or association with a second fragment of an immunoglobulin Fc region, which thus promotes dimerization and/or makes dimerization more energetically favourable.
  • Such recombinant fusion proteins may have particular utility in the preparation of bi-specific and/or bi-paratopic binders.
  • Methods for generating Fc based bi-specific and / or bi-paratopic binders, through pairing of two distinct Fc heavy chains that are engineered to dimerize, are known in the art. These methods enable an Fc region to be assembled from two different heavy chains, each fused to a target binding domain or sequence with different binding characteristics.
  • the target binding domains or sequences can be directed to different targets to generate multi-specific binders and/or to different regions or epitopes on the same target to generate bi-paratopic binding proteins. Multiple binding domains or sequences can be fused to the Fc sequences to create multi-specific or multi-paratopic binders or both multi-specific multi-paratopic binders within the same protein.
  • Methods to generate these asymmetric bispecific and/or bi-paratopic binders through heterodimerisation of two different Fc heavy chains, or fragments thereof include but are not limited to: Knobs-into-holes (Y-T), Knobs-into-holes (CW-CSAV), CH3 charge pair, Fab-arm exchange, SEED technology, BEAT technology, , HA-TF, ZW1 approach, Biclonic approach, EW-RVT and Triomab See for example, Brinkman & Kontermann, (2017) mAbs, 9:2, 182-212; Klein et al (2012) mAbs 4:6, 653–663; Wang et al (2019) Antibodies, 8, 43; and Dietrich et al (2020) BBA - Proteins and Proteomics 1868140250; each of which is incorporated herein by reference in its entirety.
  • the first fragment of an immunoglobulin Fc region is engineered to dimerize with the second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs-into-holes (Y-T), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab-arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT and Triomab.
  • Knobs-into-holes (Y-T) may comprise a T366Y substitution in a first CH3 domain and a Y407T substitution in a second CH3 domain.
  • Knobs-into-holes may comprise one or more (preferably all) of the following substitutions in a first CH3 domain: S354C, T366W.
  • Knobs-into-holes may comprise one or more (preferably all) of the following substitutions in a second CH3 domain: Y349C, T366S, L368A, Y407V.
  • Knobs-into-holes may comprise a disulphide bond in CH3.
  • CH3 charge pairing may comprise one or more (preferably all) of the following substitutions in a first CH3 domain: K392D, K409D.
  • CH3 charge pairing may comprise one or more (preferably all) of the following substitutions in a second CH3 domain: E356K, D399K.
  • Fab-arm exchange may comprise a K409R substitution in a first CH3 domain and a F405L substitution in a second CH3 domain.
  • Fab arm exchange and DuoBody capture the same Fc changes.
  • DuoBody technology may therefore comprise a K409R substitution in a first CH3 domain and a F405L substitution in a second CH3 domain.
  • SEED technology may incorporate known substitutions and/or result in an IgG/A chimera.
  • SEED strand-exchange engineered domain
  • HA-TF may comprise one or more (preferably all) of the following substitutions in a first CH3 domain: S364H, F405A.
  • HA-TF may comprise one or more (preferably all) of the following substitutions in a second CH3 domain: Y349T, T394F.
  • ZW1 approach may comprise one or more (preferably all) of the following substitutions in a first CH3 domain: T350V, L351Y, F405A, Y407V.
  • ZW1 approach may comprise one or more (preferably all) of the following substitutions in a second CH3 domain: T350V, T366L, K392L, T394W.
  • Biclonic approach may comprise one or more (preferably all) of the following substitutions in a first CH3 domain: 366K (+351K).
  • Biclonic approach may comprise one or more (preferably all) of the following substitutions in a second CH3 domain: 351D or E or D at 349, 368, 349, or 349 + 355.
  • EW-RVT may comprise one or more (preferably all) of the following substitutions in a first CH3 domain: K360E, K409W.
  • EW-RVT may comprise one or more (preferably all) of the following substitutions in a second CH3 domain: Q347R, D399V, F405T.
  • EW-RVT may comprise a disulphide bond in CH3.
  • a disulphide bridge may be supported by the further incorporation of Y349C to a first CH3 domain and S354C to a second CH3 domain.
  • Triomabs may be formed by fusing a mouse hybridoma with a rat hybridoma, resulting in production of a bispecific, assymmetric hybrid IgG molecule.
  • one or more residues of the fragment of the immunoglobulin Fc region comprises one or more amino acid substitution suitable for heterodimerization with a second fragment of an immunoglobulin Fc region comprising one or more corresponding amino acid substitution. In one embodiment, one or more residues of the fragment of the immunoglobulin Fc region comprises one or more amino acid substitution suitable for knobs-in-holes (KIH) dimerization with a second fragment of an immunoglobulin Fc region comprising one or more corresponding amino acid substitution.
  • KH knobs-in-holes
  • the one or more corresponding amino acid substitution may be one or more corresponding amino acid substitution suitable for knobs-in-holes (KIH) dimerization with the first fragment of an immunoglobulin Fc region.
  • the one or more amino acid substitution is selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.
  • the one or more amino acid substitution is selected from the group consisting of T366Y and Y407T.
  • the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 146 or SEQ ID NO: 147.
  • G3CP hFc(S239C+Y407T) SEQ ID NO: 146 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKT HTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK G3CPG4 hFc(S239C+Y407T)
  • P3A1 hFc(S239C+T366Y) SEQ ID NO: 148 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFS LRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHT CPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCS VMHEALHNHYTQKSLSPGK
  • the recombinant fusion protein may comprise a sequence according to SEQ ID NO
  • P2A7 hFc (Y407T+S239C) (SEQ ID NO: 500) TRVDQTPRTATKETGESLTINCVLTDTNYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFS LRIKDLTVADSATYYCKAAYIERNGFLTWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTC PPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 4D2 hFc (Y407T+S239C) (
  • P2A7 hFc (Y407T+S239C+S442C) (SEQ ID NO: 506) TRVDQTPRTATKETGESLTINCVLTDTNYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFS LRIKDLTVADSATYYCKAAYIERNGFLTWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTC PPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLCLSPGK 4D2 hFc (Y407T+S
  • the bi-paratopic dimer may comprise one of SEQ ID NOs 146, 147, 194, 195, 196 and 193 comprising the Y407T point mutation.
  • the bi-paratopic dimer may comprise one of SEQ ID NOs 148, 191, 192, 197, 198 and 199 comprising the T366Y point mutation.
  • the bi-paratopic dimer may comprise SEQ ID NO: 146 and SEQ ID NO: 148 or SEQ ID NO: 147 and SEQ ID NO: 148.
  • any of the recombinant fusion proteins disclosed herein may be associated with any of the linkers and payloads disclosed herein, Any of the bi-paratopic dimers disclosed herein may be associated with any of the linkers and payloads disclosed herein, Conjugation may be by any one or more S239C or S442C residue in the bi-paratopic dimer.
  • the bi-paratopic dimer may be associated with the linker and payload vc-MMAE.
  • the bi-paratopic dimer may comprise G3CP hFc(S239C+Y407T) (SEQ ID NO: 146) and P3A1 hFc(S239C+T366Y) (SEQ ID NO: 148), conjugated to vc-PAB-EDA-PNU
  • the bi-paratopic dimer may be associated with the linker and payload vc-PAB-EDA-PNU.
  • the bi- paratopic dimer may comprise G3CP hFc(S239C+Y407T) (SEQ ID NO: 146) and P3A1 hFc(S239C+T366Y) (SEQ ID NO: 148), conjugated to vc-PAB-EDA-PNU, or G3CPG4 hFc(S239C+Y407T) (SEQ ID NO: 147) and P3A1 hFc(S239C+T366Y) (SEQ ID NO: 148), conjugated to vc-PAB-EDA-PNU which have been shown to be highly efficacious in vivo.
  • SEQ ID Nos: 146, 147, 194, 195, 196, 148, 191, 192, 193, 197, 198 and 199 include an S239C mutation, for use in conjugation reactions.
  • the recombinant fusion protein is not conjugated (for example to an anthracycline (PNU) derivative) the S239C mutation is not needed and position 239 may be an S rather than a C.
  • PNU anthracycline
  • the recombinant fusion protein or bi-paratopic dimer may comprise a sequence according to any one of SEQ ID Nos: 146, 147, 194, 195, 196, 148, 191, 191, 192, 193, 197, 198 and 199 except that each sequence does not include an S239C mutation.
  • the recombinant fusion protein may be a bi-paratopic dimer comprising any one or any two of SEQ ID NO:550 to 561.
  • the bi-paratopic dimer may comprise one of SEQ ID NOs 556 to 561 comprising the Y407T point mutation.
  • the bi-paratopic dimer may comprise one of SEQ ID NOs 550 to 555 comprising the T366Y point mutation.
  • the bi-paratopic dimer may comprise SEQ ID NO: 556 and SEQ ID NO: 555 or SEQ ID NO: 557 and SEQ ID NO: 555. Any of the recombinant fusion proteins disclosed herein may be associated with any of the linkers and payloads disclosed herein, Any of the bi-paratopic dimers disclosed herein may be associated with any of the linkers and payloads disclosed herein, Conjugation may be by any one or more S239C or S442C residue in the bi-paratopic dimer.
  • the bi-paratopic dimer may be associated with the linker and payload vc-MMAE.
  • the bi-paratopic dimer may comprise G3CP hFc(S2442C+Y407T) (SEQ ID NO: 556) and P3A1 hFc(S442C+T366Y) (SEQ ID NO: 555), conjugated to vc-PAB-EDA-PNU
  • the bi-paratopic dimer may be associated with the linker and payload vc-PAB-EDA-PNU.
  • the bi- paratopic dimer may comprise G3CP hFc(S442C+Y407T) (SEQ ID NO: 556) and P3A1 hFc(S442C+T366Y) (SEQ ID NO: 555), conjugated to vc-PAB-EDA-PNU, or G3CPG4 hFc(S442C+Y407T) (SEQ ID NO: 557) and P3A1 hFc(S442C+T366Y) (SEQ ID NO: 555), conjugated to vc-PAB-EDA-PNU which have been shown to be highly efficacious in vivo.
  • bi-paratopic dimers corresponding to those described above comprising both a S442C and S239C.
  • the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 165 or SEQ ID NO: 166.
  • 1H8 hFc (Y407T) SEQ ID NO: 200 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQGNVFSCS VMHEALHNHYTQKSLSLSPGK 1H8 G4 hFc (Y407T) SEQ ID NO: 201 TRVD
  • P2A7 hFc Y407T (SEQ ID NO: 518) TRVDQTPRTATKETGESLTINCVLTDTNYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFS LRIKDLTVADSATYYCKAAYIERNGFLTWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 4D2 hFc Y407T (SEQ ID NO: 519) ASVNQTP
  • the recombinant fusion protein may comprise one or more of the following SEQ ID Nos; 297 to 332, 408 to 431, 485 to 496, 524 to 547 and 562 to 573.
  • the invention provides a recombinant fusion protein dimer comprising (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 12), YPWGAGAPRLV
  • the second fragment of an immunoglobulin Fc region selected from the group consisting of an Fc heavy chain, a CH2 region and a CH3 region. In one embodiment, the second fragment of an immunoglobulin Fc region is an Fc heavy chain. In one embodiment, the second fragment of an immunoglobulin Fc region is engineered to dimerize with the second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs-into-holes (Y-T), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab-arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT and Triomab.
  • the one or more amino acid substitution is selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.
  • the skilled person knows which amino acid substitutions represent a “knob” and which amino acid substitutions represent a “hole” and therefore which mutation is suitable for KIH dimerization with a corresponding mutation.
  • T366Y is a knob variant
  • Y407T is a hole variant.
  • the first fragment of the immunoglobulin Fc region comprises T366Y
  • the second fragment of the immunoglobulin Fc region may comprise Y407T, and vice versa.
  • the PTK7 specific antigen binding molecule may comprise any PTK7 specific antigen binding molecule disclosed herein.
  • the PTK7 specific antigen binding molecule may for instance comprise P2A2, P2A7, P2B1, P2B12, P2C6, P2C7, P2F8, P2G3, P2H9, 4A12, 4C7, 4E5, 4H3, 4D2, E02, PB4, PC2 or a derivative thereof.
  • the first recombinant fusion protein may comprise a ROR1 specific antigen binding molecule, such as G3CP, 1H8 or G3CPG4, fused to an Fc heavy chain, optionally via a [G4S]x linker.
  • the first fragment of an immunoglobulin Fc region may be a first Fc heavy chain.
  • the second fragment of an immunoglobulin Fc region may be a second Fc heavy chain.
  • One or more residues of the first Fc heavy chain may comprise one or more amino acid substitution suitable for knobs-in-holes (KIH) dimerization with the second Fc heavy chain comprising one or more corresponding amino acid substitutions suitable for knobs-in-holes (KIH) dimerization with the first Fc heavy chain.
  • the one or more amino acid substitution may be selected from the group consisting of T366Y and Y407T.
  • the first Fc heavy chain may comprise a S239C and/or a S442C mutation.
  • the second Fc heavy chain may comprise a S239C and/or a S442C mutation.
  • the second recombinant fusion protein may comprise a PTK7 specific antigen binding molecule, such as P2A7, 4D2, or E02, fused to an Fc heavy chain, optionally via a [G 4 S] x linker.
  • the first fragment of an immunoglobulin Fc region may be a first Fc heavy chain.
  • the second fragment of an immunoglobulin Fc region may be a second Fc heavy chain.
  • One or more residues of the second Fc heavy chain may comprise one or more amino acid substitution suitable for knobs-in-holes (KIH) dimerization with the first Fc heavy chain comprising one or more corresponding amino acid substitutions suitable for knobs- in-holes (KIH) dimerization with the second Fc heavy chain.
  • the one or more amino acid substitution may be selected from the group consisting of T366Y and Y407T.
  • the first Fc heavy chain may comprise a S239C and/or a S442C mutation.
  • the second Fc heavy chain may comprise a S239C and/or a S442C mutation.
  • the recombinant fusion protein dimer may be selected from the group consisting of: G3CP-hFc (S239C) and P2A7-hFc (S239C), G3CP-hFc (S239C) and 4D2-hFc (S239C), and G3CP-hFc (S239C) and E02-hFc (S239C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G 4 S] 3 linker or a G 4 S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution and the hFc region fused to the PTK7-specific binding molecule comprises
  • the recombinant fusion protein dimer may be selected from the group consisting of: G3CPG4-hFc (S239C) and P2A7-hFc (S239C), G3CPG4-hFc (S239C) and 4D2-hFc (S239C), and G3CPG4-hFc (S239C) and E02-hFc (S239C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution and the hFc region fused to the PTK7
  • the recombinant fusion protein dimer may be selected from the group consisting of: 1H8-hFc (S239C) and P2A7-hFc (S239C), 1H8-hFc (S239C) and 4D2-hFc (S239C), and 1H8-hFc (S239C) and E02-hFc (S239C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution and the hFc region fused to the PTK7-specific binding molecule comprises
  • the first recombinant fusion protein comprises G3CP-hFc (S442C), 1H8-hFc (S442C) or G3CPG4-hFc (S442C), and (b) the second recombinant fusion protein comprises P2A7-hFc (S442C), 4D2-hFc (S442C) or E02- hFc (S442C).
  • the recombinant fusion protein dimer may be selected from the group consisting of: G3CP-hFc (S442C) and P2A7-hFc (S442C), G3CP-hFc (S442C) and 4D2-hFc (S442C), and G3CP-hFc (S442C) and E02-hFc (S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution and the hFc region fused to the PTK7-specific binding molecule comprises
  • the recombinant fusion protein dimer may be selected from the group consisting of: G3CPG4-hFc (S442C) and P2A7-hFc (S442C), G3CPG4-hFc (S442C) and 4D2-hFc (S442C), and G3CPG4-hFc (S442C) and E02-hFc (S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution and the hFc region fused to the PTK7
  • the recombinant fusion protein dimer may be selected from the group consisting of: 1H8-hFc (S442C) and P2A7-hFc (S442C), 1H8-hFc (S442C) and 4D2-hFc (S442C), and 1H8-hFc (S442C) and E02-hFc (S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution and the hFc region fused to the PTK7-specific binding molecule comprises
  • the first recombinant fusion protein comprises G3CP-hFc (S239C & S442C), 1H8-hFc (S239C & S442C) or G3CPG4-hFc (S239C & S442C), and (b) the second recombinant fusion protein comprises P2A7-hFc (S239C & S442C), 4D2-hFc (S239C & S442C), or E02-hFc (S239C & S442C).
  • the recombinant fusion protein dimer may be selected from the group consisting of: G3CP-hFc (S239C & S442C) and P2A7-hFc (S239C & S442C), G3CP-hFc (S239C & S442C) and 4D2-hFc (S239C & S442C), and G3CP-hFc (S239C & S442C) and E02-hFc (S239C & S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the R
  • the recombinant fusion protein dimer may be selected from the group consisting of: G3CPG4-hFc (S239C & S442C) and P2A7-hFc (S239C & S442C), G3CPG4-hFc (S239C & S442C) and 4D2-hFc (S239C & S442C), and G3CPG4-hFc (S239C & S442C) and E02-hFc (S239C & S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc
  • the recombinant fusion protein dimer may be selected from the group consisting of: 1H8-hFc (S239C & S442C) and P2A7-hFc (S239C & S442C), 1H8-hFc (S239C & S442C) and 4D2-hFc (S239C & S442C), and 1H8-hFc (S239C & S442C) and E02-hFc (S239C & S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the R
  • the recombinant fusion protein dimers described above may comprise any other ROR1- specific binding molecule described herein in place of G3CP, 1H8 or G3CPG4.
  • the recombinant fusion protein dimers described above may comprise any other PTK7- specific binding molecule described herein in place of P2A7, 4D2, or E02. Any of the recombinant fusion protein dimers disclosed herein may be associated with any of the linkers and payloads disclosed herein, Conjugation may be by any one or more S239C or S442C residue in the recombinant fusion protein dimer.
  • conjugation is by a S239C residue in a first hFc region of the recombinant fusion protein dimer
  • conjugation will also be by a S239C residue in a second hFc region of the recombinant fusion protein dimer.
  • conjugation will also be by a S442C residue in a first hFc region of the recombinant fusion protein dimer.
  • conjugation is by a S239C and a S442C residue in a first hFc region of the recombinant fusion protein dimer
  • conjugation will also be by a S239C and a S442C residue in a second hFc region of the recombinant fusion protein dimer.
  • the invention provides a recombinant fusion protein dimer comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30) and DTSYGLYS (SEQ ID NO:207); FW1 is a framework region
  • the first recombinant fusion protein comprises P3A1
  • the second recombinant fusion protein comprises P2A7, 4D2, or E02.
  • the first recombinant fusion protein comprises P3A1-hFc
  • the second recombinant fusion protein comprises P2A7-hFc, 4D2-hFc, or E02-hFc.
  • the first recombinant fusion protein comprises P3A1-hFc (S239C), and (b) the second recombinant fusion protein comprises P2A7-hFc (S239C), 4D2-hFc (S239C), or E02- hFc (S239C).
  • the first recombinant fusion protein comprises P3A1-hFc (S239C)
  • the second recombinant fusion protein comprises P2A7-hFc (S239C), 4D2-hFc (S239C), or E02- hFc (S239C).
  • the recombinant fusion protein dimer may be selected from the group consisting of: P3A1-hFc (S239C) and P2A7-hFc (S239C), P3A1-hFc (S239C) and 4D2-hFc (S239C), and P3A1-hFc (S239C) and E02-hFc (S239C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution and the hFc region fused to the PTK7-specific binding
  • the first recombinant fusion protein comprises P3A1-hFc (S442C), and (b) the second recombinant fusion protein comprises P2A7-hFc (S442C), 4D2-hFc (S442C), or E02- hFc (S442C).
  • the first recombinant fusion protein comprises P3A1-hFc (S442C)
  • the second recombinant fusion protein comprises P2A7-hFc (S442C), 4D2-hFc (S442C), or E02- hFc (S442C).
  • the recombinant fusion protein dimer may be selected from the group consisting of: P3A1-hFc (S442C) and P2A7-hFc (S442C), P3A1-hFc (S442C) and 4D2-hFc (S442C), and P3A1-hFc (S442C) and E02-hFc (S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G 4 S] 3 linker or a G 4 S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution and the hFc region fused to the PTK7-specific binding
  • the first recombinant fusion protein comprises P3A1-hFc (S239C & S442C)
  • the second recombinant fusion protein comprises P2A7-hFc (S239C & S442C), 4D2-hFc (S239C & S442C), or E02-hFc (S239C & S442C).
  • the first recombinant fusion protein comprises P3A1-hFc (S239C & S442C)
  • the second recombinant fusion protein comprises P2A7-hFc (S239C & S442C), 4D2-hFc (S239C & S442C), or E02-hFc (S239C & S442C).
  • the recombinant fusion protein dimer may be selected from the group consisting of: P3A1-hFc (S239C & S442C) and P2A7-hFc (S239C & S442C), P3A1-hFc (S239C & S442C) and 4D2-hFc (S239C & S442C), and P3A1-hFc (S239C & S442C) and E02-hFc (S239C & S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises a Y407T substitution, or (b) the hFc region fused
  • the first recombinant fusion protein comprises any one of SEQ ID NO: 148, 167, 188, 189, 191, 192,, 197 to 199, 203 to 205, 259 to 264, 302 to 307, 315 to 320, 327 to 332, 550 to 555, and 568 to 573 and the second recombinant fusion protein comprises any one of SEQ ID NOs: 500 to 508, 518 to 520, 524 to 526, 533 to 536, 540 to 544 .
  • the first recombinant fusion protein comprises any one of SEQ ID NO: 146, 147, 165, 166, 190, 193 to 196, 200 to 202, 253 to 258, 297 to 301, 308 to 314, 321 to 326, and 556 to 567 and the second recombinant fusion protein comprises any one of SEQ ID NOs: 509 to 517, 521 to 523, 527 to 532, 537 to 539, and 545 to 547.
  • the first recombinant fusion protein comprises any one of SEQ ID NO: 148, 191, 192, 197 to 199, and 302 to 307 and the second recombinant fusion protein comprises any one of SEQ ID NOs: 500 to 502, and 524 to 526.
  • the first recombinant fusion protein comprises any one of SEQ ID NO: 146, 147, 193 to 196, 297 to 301, and 308 and the second recombinant fusion protein comprises any one of SEQ ID NOs: 509 to 511, and 527 to 529.
  • the first recombinant fusion protein comprises any one of SEQ ID NO: 327 to 332, and 550 to 555 and the second recombinant fusion protein comprises any one of SEQ ID NOs: 503 to 505, and 534 to 536.
  • the first recombinant fusion protein comprises any one of SEQ ID NO: 321 to 326, and 556 to 561 and the second recombinant fusion protein comprises any one of SEQ ID NOs: 512 to 514, and 537 to 539.
  • the first recombinant fusion protein comprises any one of SEQ ID NO: 259 to 264, and 315 to 320 and the second recombinant fusion protein comprises any one of SEQ ID NOs: 506 to 508, and 533, 540 and 541.
  • the first recombinant fusion protein comprises any one of SEQ ID NO: 253 to 258, and 309 to 314 and the second recombinant fusion protein comprises any one of SEQ ID NOs: 515 to 517, and 530 to 532.
  • the recombinant fusion protein dimers described above may comprise any other ROR1- specific binding molecule described herein in place of P3A1. In alternatives, the recombinant fusion protein dimers described above may comprise any other PTK7- specific binding molecule described herein in place of P2A7, 4D2, or E02.
  • the invention provides a recombinant fusion protein dimer comprising (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a first PTK7-specific binding protein as disclosed in relation to any of the aspects of the invention and wherein the first antigen binding molecule is fused to a first fragment of an immunoglobulin Fc region engineered to dimerize with a second fragment of an immunoglobulin Fc region, and (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises a second PTK7-specific binding protein as disclosed in relation to any of the aspects of the invention fused to a second fragment of an immunoglobulin Fc region engineered to dimerize with the first fragment of an immunoglobulin Fc region.
  • the first PTK7-specific binding protein will comprise a different sequence to the second PTK7-specific binding protein.
  • the recombinant fusion protein dimer of the second or third configuration may be a bi-paratopic recombinant fusion protein dimer.
  • the recombinant fusion protein dimer of the second or third configuration may be a bi-paratopic fusion protein dimer comprising P2A7-hFc and 4D2 -hFc, wherein the first and second fragment of an immunoglobulin Fc region are engineered to dimerise, for example by knobs-in-holes (KIH) dimerization.
  • the first and/or second recombinant fusion protein may comprise a PTK7 specific antigen binding molecule, such as P2A7, 4D2, or E02, fused to an Fc heavy chain, optionally via a [G4S]x linker.
  • the first fragment of an immunoglobulin Fc region may be a first Fc heavy chain.
  • the second fragment of an immunoglobulin Fc region may be a second Fc heavy chain.
  • One or more residues of the second Fc heavy chain may comprise one or more amino acid substitution suitable for knobs-in-holes (KIH) dimerization with the first Fc heavy chain comprising one or more corresponding amino acid substitutions suitable for knobs-in-holes (KIH) dimerization with the second Fc heavy chain.
  • the one or more amino acid substitution may be selected from the group consisting of T366Y and Y407T.
  • the first Fc heavy chain may comprise a S239C and/or a S442C mutation.
  • the second Fc heavy chain may comprise a S239C and/or a S442C mutation.
  • (a) the first recombinant fusion protein comprises P2A7
  • (b) the second recombinant fusion protein comprises 4D2.
  • (a) the first recombinant fusion protein comprises P2A7-hFc
  • the second recombinant fusion protein comprises 4D2-hFc.
  • the first recombinant fusion protein comprises P2A7-hFc (S239C), and (b) the second recombinant fusion protein comprises 4D2-hFc (S239C).
  • the first recombinant fusion protein comprises P2A7-hFc (S442C)
  • the second recombinant fusion protein comprises 4D2-hFc (S442C).
  • the first recombinant fusion protein comprises P2A7-hFc (S239C & S442C)
  • the second recombinant fusion protein comprises 4D2-hFc (S239C & S442C).
  • the first recombinant fusion protein comprises P2A7-hFc (T366Y), and (b) the second recombinant fusion protein comprises 4D2-hFc (Y407T).
  • the first recombinant fusion protein comprises P2A7-hFc (S239C & T366Y)
  • the second recombinant fusion protein comprises 4D2-hFc (S239C & Y407T).
  • the first recombinant fusion protein comprises P2A7-hFc (S442C & T366Y), and (b) the second recombinant fusion protein comprises 4D2-hFc (S442C & Y407T).
  • the first recombinant fusion protein comprises P2A7-hFc (S239C & S442C & T366Y)
  • the second recombinant fusion protein comprises 4D2-hFc (S239C & S442C & Y407T).
  • the first recombinant fusion protein comprises P2A7-hFc (Y407T), and (b) the second recombinant fusion protein comprises 4D2-hFc (T366Y).
  • the first recombinant fusion protein comprises P2A7-hFc (S239C & Y407T), and (b) the second recombinant fusion protein comprises 4D2-hFc (S239C & T366Y).
  • the first recombinant fusion protein comprises P2A7-hFc (S442C & Y407T), and (b) the second recombinant fusion protein comprises 4D2-hFc (S442C & T366Y).
  • the first recombinant fusion protein comprises P2A7-hFc (S239C & S442C & Y407T), and (b) the second recombinant fusion protein comprises 4D2-hFc (S239C & S442C & T366Y).
  • the recombinant fusion protein dimers described above may comprise any other PTK7- specific binding molecule described herein in place of P2A7 and/or 4D2.
  • the invention provides a recombinant fusion protein dimer comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule, and (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises a protein tyrosine kinase 7 (PTK7) specific antigen binding molecule.
  • ROR1 receptor tyrosine kinase-like orphan receptor 1
  • the ROR1 specific antigen binding molecule may comprise the amino acid sequence of any ROR1 specific binding molecule, for example any of those disclosed herein.
  • the ROR1 specific antigen binding molecule may comprise any CDR1 and/or CDR3 sequence of any ROR1 specific antigen binding molecule disclosed herein.
  • the ROR1 specific antigen binding molecule may comprise any HV2 and/or HV4 sequence of any ROR1 specific antigen binding molecule disclosed herein.
  • the ROR1 specific antigen binding molecule may comprise any FW1, FW2, FW3a, FW3b and/or FW4 sequence of any ROR1 specific antigen binding molecule disclosed herein.
  • the ROR1 specific antigen binding molecule may have the characteristics of any ROR1 specific antigen binding molecule disclosed herein.
  • the PTK7 specific antigen binding molecule may comprise the amino acid sequence of any PTK7 specific binding molecule, for example any of those disclosed herein.
  • the PTK7 specific antigen binding molecule may comprise any CDR1 and/or CDR3 sequence of any PTK7 specific antigen binding molecule disclosed herein.
  • the PTK7 specific antigen binding molecule may comprise any HV2 and/or HV4 sequence of any PTK7 specific antigen binding molecule disclosed herein.
  • the PTK7 specific antigen binding molecule may comprise any FW1, FW2, FW3a, FW3b and/or FW4 sequence of any PTK7 specific antigen binding molecule disclosed herein.
  • the PTK7 specific antigen binding molecule may have the characteristics of any PTK7 specific antigen binding molecule disclosed herein.
  • the invention provides a ROR1-specific chimeric antigen receptor (CAR), comprising at least one bi-specific antigen binding molecule as defined by the first or second aspects of the invention, at least one recombinant fusion protein as defined by the third aspect of the invention, or at least one recombinant fusion protein dimer as defined by the fourth or fifth aspects of the invention, at least one PTK7 specific binding molecule as defined by the first configuration, or at least one recombinant fusion protein dimer as defined in the second or third configurations, fused or conjugated to at least one transmembrane region and at least one intracellular domain.
  • CAR ROR1-specific chimeric antigen receptor
  • the present invention also provides a cell comprising a chimeric antigen receptor according to the sixth aspect, which cell is preferably an engineered T-cell.
  • a nucleic acid sequence comprising a polynucleotide sequence that encodes a PTK7-specific binding molecule of the first configuration of the invention, or a bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor according to the first, second, third, fourth, fifth or sixth aspects of the invention, the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • a vector comprising a nucleic acid sequence in accordance with the seventh aspect and a host cell comprising such a nucleic acid.
  • a method for preparing a PTK7-specific binding molecule of the first configuration of the invention, the recombinant fusion protein dimer of the second or third configuration, or a bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor, of the first, second, third, fourth, fifth, or sixth aspect comprising cultivating or maintaining a host cell comprising the polynucleotide or vector described above under conditions such that said host cell produces the bi-specific antigen binding molecule, recombinant fusion protein or chimeric antigen receptor, optionally further comprising isolating the specific antigen binding molecule, recombinant fusion protein or chimeric antigen receptor.
  • a pharmaceutical composition comprising a PTK7-specific binding molecule of the first configuration of the invention, the recombinant fusion protein dimer of the second or third configuration, or the bi-specific antigen binding molecule, fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspects.
  • the pharmaceutical composition may contain a variety of pharmaceutically acceptable carriers.
  • Pharmaceutical compositions of the invention may be for administration by any suitable method known in the art, including but not limited to intravenous, intramuscular, oral, intraperitoneal, or topical administration.
  • the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule, or foam.
  • the bi-specific antigen binding molecule, recombinant fusion protein or chimeric antigen receptor of the first, second, third, fourth, fifth, or sixth aspects may be for use in therapy. More specifically, the bi- specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth, or sixth aspects, the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration may be for use in the treatment of cancer.
  • the cancer is a ROR1-positive cancer type and/or an PTK7-positive cancer type.
  • the cancer is selected from the group comprising blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, renal cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, Head and Neck cancer, bladder cancer, oesophageal cancer, stomach cancer or liver cancer.
  • blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL),
  • the cancer may be selected from endometrial cancer and uterine carcinosarcoma. More preferably, the cancer is selected from the group comprising triple negative breast cancer (TNBC), breast adenocarcinoma, ovarian cancer, sarcoma, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung carcinoma, small cell lung carcinoma and pleuromesothelioma, Also provided herein is the use of a bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth, or sixth aspects, the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration in the manufacture of a medicament for the treatment of a disease in a patient in need thereof.
  • TNBC triple negative breast cancer
  • TNBC triple negative breast cancer
  • breast adenocarcinoma ovarian cancer
  • a method of treatment of a disease in a patient in need of treatment comprising administration to said patient of a therapeutically effective dosage of a bi-specific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth, or sixth aspects or a pharmaceutical composition of the sixth aspect, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • the cancer is a ROR1-positive cancer type and/or an PTK7-positive cancer type.
  • the cancer is selected from the group comprising blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, renal cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, Head and Neck cancer, bladder cancer, oesophageal cancer, stomach cancer or liver cancer.
  • blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL),
  • the cancer may be selected from endometrial cancer and uterine carcinosarcoma. More preferably, the cancer is selected from the group comprising triple negative breast cancer (TNBC), breast adenocarcinoma, ovarian cancer, sarcoma, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung carcinoma, small cell lung carcinoma and pleural mesothelioma.
  • TNBC triple negative breast cancer
  • breast adenocarcinoma ovarian cancer
  • sarcoma lung adenocarcinoma
  • lung squamous cell carcinoma large cell lung carcinoma
  • small cell lung carcinoma small cell lung carcinoma
  • Also provided herein is a method of assaying for the presence of a target analyte in a sample, comprising the addition of a detectably labelled bi-specific antigen binding molecule, recombinant fusion protein, or recombinant fusion protein dimer, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration to the sample and detecting the binding of the molecule to the target analyte.
  • a method of imaging a site of disease in a subject comprising administration of a detectably labelled bi-specific antigen binding molecule, recombinant fusion protein, or recombinant fusion protein dimer to a subject or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • a method of diagnosis of a disease or medical condition in a subject comprising administration of a bi-specific antigen binding molecule, recombinant fusion protein, or recombinant fusion protein dimer, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • an antibody, antibody fragment, antigen-binding molecule, bi-specific antigen binding molecule, recombinant fusion protein, or recombinant fusion protein dimer that competes for binding to ROR1 and/or PTK7 with the bi-specific antigen binding molecule, recombinant fusion protein, or recombinant fusion protein dimer.
  • antigen binding proteins e.g., neutralizing antigen binding proteins or neutralizing antibodies
  • competition means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or functional fragment thereof) under test prevents or inhibits specific binding of a the antigen binding molecule defined herein (e.g., specific antigen binding molecule of the first aspect) to a common antigen (e.g., ROR1 in the case of the specific antigen binding molecule of the first or second aspect).
  • kits for diagnosing a subject suffering from cancer, or a pre-disposition thereto, or for providing a prognosis of the subject's condition comprising detection means for detecting the concentration of one or more antigen present in a sample from a test subject, wherein the detection means comprises a bi-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein or recombinant fusion protein dimer of the third, fourth or fifth aspect, a chimeric antigen receptor of the sixth aspect or a nucleic acid sequence of the seventh aspect, or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration, each being optionally derivatized, wherein presence of antigen in the sample suggests that the subject suffers from cancer.
  • the one or more antigen comprises ROR1 protein, more preferably an extracellular domain thereof.
  • the one or more antigen comprises PTK7 protein. More preferably the one or more antigen comprises ROR1 protein and PTK7 protein. More preferably, the kit is used to identify the presence or absence of ROR1-positive cells and/or PTK7-positive cells in the sample, or determine the concentration thereof in the sample.
  • the kit may also comprise a positive control and/or a negative control against which the assay is compared and/or a label which may be detected.
  • the present invention also provides a method for diagnosing a subject suffering from cancer, or a pre- disposition thereto, or for providing a prognosis of the subject's condition, the method comprising detecting the concentration of antigen present in a sample obtained from a subject, wherein the detection is achieved using a bi-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein or recombinant fusion protein dimer of the third, fourth or fifth aspect, a chimeric antigen receptor of the sixth aspect or a nucleic acid sequence of the seventh aspect or the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration, each being optionally derivatized, and wherein presence of antigen in the sample suggests that the subject suffers from cancer.
  • Also contemplated herein is a method of killing or inhibiting the growth of a cell expressing ROR1 and/or PTK7 in vitro or in a patient, which method comprises administering to the cell a pharmaceutically effective amount or dose of (i) bi-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein or recombinant fusion protein dimer of the third, fourth or fifth aspect, a nucleic acid sequence of the sixth aspect, or the CAR or cell according the seventh aspect, or (ii) of a pharmaceutical composition of the eighth aspect, or (iii) the PTK7 specific binding molecule of the first configuration, or the recombinant fusion protein dimer of the second or third configuration.
  • the cell expressing ROR1 and/or PTK7 is a cancer cell. More preferably, the ROR1 is human ROR1 and/or the PTK7 is human PTK7.
  • the invention provides a bi-specific antigen binding molecule comprising an amino acid sequence represented by the formula (II): X-FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4-Y (II) wherein FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 is a ROR1-specific antigen binding molecule according to the first or second aspect X and Y are optional amino acid sequences wherein the ROR1-specific antigen binding molecule is conjugated to a second moiety and wherein the bi-specific antigen binding molecule further comprises an PTK7-specific antigen binding molecule.
  • the “ROR1-specific antigen binding molecule according to the first or second aspect” refers to any ROR1-specific antigen binding molecule described in connection with the bi-specific antigen binding molecules of the first and/or second aspects of the invention (which comprise a ROR1- specific antigen binding molecule).
  • the ROR1-specific antigen binding molecule may be as described anywhere herein.
  • the PTK7-specific antigen binding molecule may be an PTK7-specific antigen binding molecule according to the first or second aspect.
  • the “PTK7-specific antigen binding molecule according to the first or second aspect” refers to any PTK7-specific antigen binding molecule described in connection with the bi-specific antigen binding molecules of the first and/or second aspects of the invention (which comprise an PTK7-specific antigen binding molecule).
  • the PTK7-specific antigen binding molecule may be as described anywhere herein.
  • the bi-specific antigen binding molecule according to this aspect of the invention may additionally be conjugated to a third, fourth or fifth moiety. Conjugation of further moieties is also contemplated. In some cases, a third, fourth or fifth moiety may be conjugated to the second moiety.
  • any of the moieties according to this aspect of the invention may have additional moieties conjugated thereto.
  • Description of preferred features of the second moiety as set out below apply to the third, fourth, fifth or higher order moiety mutatis mutandis.
  • X or Y are individually either absent or selected from the group comprising an immunoglobulin, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobulin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv)2, a diabody, a triabody, a tetrabody, a bispecific t-cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, a scaffold protein (affibodies, centyrins, darpins etc.), or a toxin including but not limited to Pseudomonas exotoxin PE38, diphtheria toxin.
  • a toxin including but not limited to Pseudomonas
  • the conjugation is via a cysteine residue in the amino acid sequence of the specific antigen binding molecule.
  • the cysteine residue may be anywhere in the sequence, including in optional sequences X or Y (if present).
  • the conjugation may be via a thiol, aminoxy or hydrazinyl moiety incorporated at the N-terminus or C- terminus of the amino acid sequence of the specific antigen binding molecule.
  • the second moiety is selected from the group comprising detectable label, dye, toxin, drug, pro-drug, radionuclide or biologically active molecule.
  • the second moiety is at least one toxin selected from the group comprising: • Auristatins, • anthracyclines, preferably PNU-derived anthracyclines • maytansinoids, • calicheamicins, • amanitin derivatives, preferably ⁇ -amanitin derivatives • tubulysins • duocarmycins • radioisotopes for example alpha-emitting radionuclide, such as 227 Th or 225 Ac • liposomes comprising a toxic payload, • protein toxins • taxanes, • pyrrolbenzodiazepines and dimers thereof • indolinobenzodiazepine pseudodimers • spliceosome inhibitors • CDK11 inhibitors • nicotinamide phosphoribosyltransferase inhibitors (NAMPTi) • Pyridinobenzodiazepines and dimers thereof • Cyclopropapyrroloindole (CPI), cyclopropabenzin
  • the second moiety is a VNAR domain, which may be the same or different to the specific antigen binding molecule according to this aspect. Accordingly, dimers, trimers or higher order multimers of VNAR domains linked by chemical conjugation are explicitly contemplated herein. In such embodiments, each individual VNAR domain may have the same antigen specificity as the other VNAR domains, or they may be different.
  • the bi-specific antigen binding molecule may comprise, for example, bi- paratopic specific antigen binding molecules as described in relation to the first to fifth aspects fused to further biologically active molecules (including but not limited to molecules for half-life extension, for example BA11) and then further conjugated to a second moiety, including but not limited to cytotoxic payloads
  • the bi-specific antigen binding molecule may comprise a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule. This may be a ROR1- specific antigen binding molecule of the first or second aspect of the invention.
  • the bi-specific antigen binding molecule of the ninth aspect may be for use in therapy. More specifically, the bi-specific antigen binding molecule of the ninth aspect may be for use in the treatment of cancer.
  • the cancer is a ROR1-positive cancer type and/or an PTK7-positive cancer type.
  • the cancer is selected from the group comprising blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, renal cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, Head and Neck cancer, bladder cancer, oesophageal cancer, stomach cancer or liver cancer.
  • blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL),
  • a bi-specific antigen binding molecule of the ninth aspect in the manufacture of a medicament for the treatment of a disease in a patient in need thereof.
  • Pharmaceutical compositions comprising the bi-specific antigen binding molecule of the ninth aspect are also provided.
  • the pharmaceutical composition may contain a variety of pharmaceutically acceptable carriers.
  • a method of treatment of a disease in a patient in need of treatment comprising administration to said patient of a therapeutically effective dosage of a bi-specific antigen binding molecule of the ninth aspect or a pharmaceutical composition comprising a bi-specific antigen binding molecule of the ninth aspect.
  • the cancer is a ROR1-positive cancer type and/or an PTK7-positive cancer type.
  • the cancer is selected from the group comprising blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, renal cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, Head and Neck cancer, bladder cancer, oesophageal cancer, stomach cancer or liver cancer.
  • blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL),
  • a method of imaging a site of disease in a subject comprising administration of a detectably labelled bi-specific antigen binding molecule of the ninth aspect to a subject.
  • a method of diagnosis of a disease or medical condition in a subject comprising administration of a bi-specific antigen binding molecule of the ninth aspect.
  • Stability of chemically-conjugated protein drug conjugates is an important consideration, since unintended release of a highly potent anthracycline toxin, like PNU-159682, in the circulation of a patient prior to targeting of the tumour cells would lead to off target effects and undesirable side effects.
  • Some example molecules released from PNU conjugates include release of PNU159682 derivative from different Val-Cit-PAB containing drug linkers. Potent toxins that can be linked to targeting proteins with high stability are therefore required in order to avoid, or at least reduce, unwanted side effects.
  • linker payloads are designed such that extracellular cleavage releases derivatives of the payload with attenuated potency.
  • Payloads of the present disclosure may use a maleimide group, which can react to any available thiol group on a conjugation partner using straightforward and standard conditions.
  • the use of maleimide/thiol chemistry for conjugation allows for site-specific conjugation to introduced thiol groups, for example on the side-chain of an engineered cysteine residue in a protein sequence.
  • a cysteine may be introduced via the introduction of his-myc tag containing an engineered cysteine (example sequences include, but are not limited to, QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) or QACGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 99)) at the C- or N-terminal of a protein.
  • cysteine is engineered into the Fc region of an Fc fusion protein.
  • PNU anthracycline
  • derivatives of PNU159682 are provided, which lack the C14 carbon and attached hydroxyl functionality, and are functionalised with an ethylenediamino (EDA) group at the C13 carbonyl of PNU159682.
  • EDA-PNU159682 can in turn be functionalised, through the amino group of the EDA moiety, with a maleimide containing linker.
  • a maleimide group is present in the anthracycline (PNU) derivatives of formula (V) and may also be present in the anthracycline (PNU) derivatives of formula (VI).
  • Such payloads are able to react with a free thiol group on another molecule.
  • a protein-drug conjugate may be formed.
  • PDC protein-drug conjugate
  • derivatives of PNU159682 functionalised with an ethylenediamino (EDA) group and linked to a thiol group via a maleimide group show higher stability compared to non-EDA payloads or liberated payload derivatives with slightly less potency. More stable payloads may be advantageous because of reduced off-target effects, which in turn may lead to reduced side effects and increased patient compliance.
  • EDA ethylenediamino
  • WO 2020/254640 describes anthracycline (PNU) derivatives of formula (V): wherein [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit- PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except g
  • the anthracycline (PNU) derivative of formula (V) may comprise [L1], [L2] or [L1] and [L2].
  • [L1] and/or [L2] are peptides
  • said peptides do not contain glycine.
  • [X] is selected from the group comprising polyethylene glycol, ’ , wherein represents the point of attachment to the rest of the molecule and wherein [R] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof.
  • [X] is polyethylene glycol.
  • the polyethylene glycol may be PEG4.
  • [L2] is p-aminobenzyloxycarbonyl (PAB) or Alanine.
  • the anthracycline (PNU) derivative comprises [L1] and/or [L2] and [X] is optional.
  • [L1] and/or [L2] may be linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH 2 ) n -, -(CH 2 CH 2 O) n -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof.
  • the anthracycline (PNU) derivative of formula (V) may comprise [L1], [L2] or [L1] and [L2].
  • the anthracycline (PNU) derivative of formula (V) may comprise [L1] and/or [L2].
  • WO 2020/254640 describes anthracycline (PNU) derivatives of formula (V): wherein [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and/or [L2] are linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit- PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except
  • [X] is polyethylene glycol.
  • the polyethylene glycol may be PEG4.
  • [L2] is p-aminobenzyloxycarbonyl (PAB) or Alanine.
  • PAB p-aminobenzyloxycarbonyl
  • the PNU derivative has a structure selected from:
  • [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; wherein [Z] is a reactive group.
  • the reactive group may be any reactive group suitable for use in a conjugation reaction, particularly a conjugation reaction to a target binding molecule. [Z] may therefore be a moiety comprising a functional group for use in bioconjugation reactions.
  • Functional groups for use in bioconjugation reactions include but are not limited to, ⁇ maleimides or alkyl halides for reaction with thiol groups or selenol groups on proteins through thioether and selonoether reactions; ⁇ sulphydryl groups for reaction with maleimide, alkyl halide or thiol functionalised molecules including the thiol groups of protein cysteine residues; ⁇ activated disulphides such as pyridyl dithiols (Npys thiols) or TNB thiols (5-thiol-2-nitrobenzoic acid) for reaction with thiol groups to form disulphide linkages through thiol disulphide exchange; ⁇ amino groups for attachment to carboxyl groups on proteins and biomolecules through amide bond forming reactions; ⁇ alkyne groups, particularly ring constrained alkynes such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (
  • Azido functionalities can be introduced into proteins through, for example, the incorporation of the unnatural amino acid para-azidomethy-L-phenyalanine or into protein glycans using enzyme mediated glycoengineering to attach azido-containing sugar analogues; ⁇ azido groups for reaction with alkyne functionalised target-binding molecule through strain promoted alkyne-azide cycloaddition copper free chemistry; ⁇ aminoxy groups for reactions with aldehyde and ketone groups on biomolecules through oxime forming ligations.
  • Ketones can be introduced into proteins through the use of amber stop codon technologies such as the incorporation of the non-natural amino acid, para-acetyl phenylalanine.
  • Aldehydes can be found on biomolecules through the presence of reducing sugars and can be introduced into proteins through periodate oxidation of N-terminal serine residues or periodate oxidation of cis-glycol groups of carbohydrates. Aldehyde groups can also be incorporated into proteins through the conversion of protein cysteines, within specific sequences, to formyl glycine by formylglycine generating enzyme.
  • formylglycine containing proteins have been conjugation to payloads via the Hydrazino-Pictet-Spengler (HIPS) ligation; ⁇ aldehyde or ketone groups for the reaction with aminoxy or hydrazide or hydrazinyl functionalized biomolecules through oxime or hydrazine bond forming ligation reactions.
  • HIPS Hydrazino-Pictet-Spengler
  • [Z] may therefore be selected from the group consisting of a maleimide, an alkyl halide, a sulphydryl group, an activated disulphide (such as pyridyl dithiols (Npys thiols) or TNB thiols (5-thiol-2-nitrobenzoic acid)), an amino group, an alkyne group (such as ring constrained alkynes such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (BCN)), an azido group, an aminoxy group, an aldehyde group and a ketone group.
  • a maleimide such as pyridyl dithiols (Npys thiols) or TNB thiols (5-thiol-2-nitrobenzoic acid)
  • an amino group such as pyridyl dithiols (Npys thiols)
  • [Z] may also be a moiety for enzyme mediated bioconjugation reactions.
  • Moieties for use in enzyme mediated conjugation reactions include but are not limited to polyGly [ (Gly)n] for use in sortase-enzyme mediated antibody conjugation or an appropriate primary amine for bacterial transglutaminase mediated conjugation to glutamine ⁇ -carboxyamide groups contained with sequences such as Lys-Lys-Gln-Gly and Lys-Pro-Glu-Thr-Gly. [Z] may therefore be selected from the group consisting of polyGly and a primary amine.
  • the PNU derivative according to formula (VI) may therefore correspond to a PNU derivative of formula (V) wherein L1 is Val-Cit-PAB, L2 is absent and wherein the maleimide group may be replaced with another Reactive Group as defined above.
  • [X] is selected from the group comprising polyethylene glycol, ’ , wherein represents the point of attachment to the rest of the molecule and wherein [R] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof.
  • [X] is polyethylene glycol.
  • the polyethylene glycol may be PEG4.
  • the PNU derivative according to formula (V) or formula (VI) may be conjugated to a ROR1 specific antigen binding molecule according to the present invention or to a recombinant fusion protein or recombinant fusion protein dimer of the invention.
  • the invention provides a target-binding molecule-drug conjugate, comprising (a) a PTK7-specific binding molecule of the first configuration of the invention, or a bi- specific antigen binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein of the third aspect or a recombinant fusion protein dimer of the fourth or fifth aspect or the second or third configuration, and (b) at least one toxin.
  • the at least one toxin may be any toxin suitable for use as a payload.
  • the toxin may be a topoisomerase inhibitor, a microtubule inhibitor or a DNA-targeting agent.
  • the topoisomerase inhibitor may be a topoisomerase I inhibitor or a topoisomerase II inhibitor.
  • the topoisomerase I or II inhibitor may be an anthracycline.
  • the anthracycline may be Doxorubicin, Daunorubicin, Epirubicin, or Idarubicin.
  • the anthracycline may be a PNU-derived anthracycline such as PNU-159682 or a derivative thereof.
  • the Topoisomerase I inhibitor may be a PNU-derived anthracycline such as PNU-159682 or a derivative thereof.
  • the Topoisomerase I inhibitor may be an exatecan for example deruxtecan
  • the microtubule inhibitor may be a taxane, a vinca alkaloid or an epothilone.
  • the microtubule inhibitor may be eribulin, paclitaxel, docetaxel or ixbepilone.
  • the microtubule inhibitor may be an auristatin, a maytansinoid or a tubulysin.
  • the DNA-targeting agent may for example be a calicheamicin, a duocarmycin, a pyrrolobenzodiazepine, an indolino-benzodiazepene, a cyclopropylpyrroloindole or a thienoindole
  • the at least one toxin may be one or more toxin selected from the group consisting of: • auristatins, • anthracyclines, preferably PNU-derived anthracyclines • maytansinoids, • amanitin derivatives, preferably ⁇ -amanitin derivatives • calicheamicins, • tubulysins • duocarmycins • radioisotopes - such as an alpha-emitting radionuclide, such as 227 Th and 225 Ac label • liposomes comprising a toxic payload, • protein toxins • taxanes • pyrrolbenzodiazepines and dimers thereof • indolinobenzodiazepine
  • the toxin may be an auristatin.
  • the auristatin may be Auristatin E (AE) or monomethylauristatin E (MMAE).
  • the auristatin may be an MMAE derivative.
  • Any of the spacer ([X]) and/or linker ([L1] and/or [L2]) groups described herein in connection with anthracycline toxins are also explicitly contemplated in connection with auristatins such as MMAE.
  • the target-binding molecule-drug conjugate may comprise Val-Cit-MMAE (vcMMAE).
  • the target-binding molecule-drug conjugate may comprise the structure of formula (VI): wherein, [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB), Val-C
  • the target-binding molecule-drug conjugate may comprise the structure of formula (VIII): wherein, Y comprises a bi-specific antigen binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein of the third aspect or a recombinant fusion protein dimer of the fourth or fifth aspect.
  • the toxin may be a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (NAMPTi), as described in Bohnke et al Bioconjugate Chem.2022, 33, 6, 1210–1221, hereby incorporated by reference in its entirety.
  • toxins and corresponding target-binding molecule-drug conjugates conjugated via a cysteine residue of a bi-specific antigen binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein or recombinant fusion protein of the third aspect or a recombinant fusion protein dimer of the fourth or fifth aspect may be selected from the following:
  • the target-binding molecule-drug conjugate may comprise (b) an anthracycline (PNU) derivative, wherein the target-binding molecule-drug conjugate has the structure of formula (III): wherein [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB)
  • the target-binding molecule-drug conjugate of formula (III) may comprise [L1], [L2] or [L1] and [L2].
  • target-binding molecule-drug conjugate where [L1] and/or [L2] are peptides, said peptides do not contain glycine. It will be clear to those of skill in the art that when optional spacers and/or optional linkers are absent a bond remains in their place.
  • the target-binding molecule-drug conjugate has a structure selected from:
  • the target-binding molecule-drug conjugate may comprise: (b) an anthracycline (PNU) derivative, wherein the target-binding molecule-drug conjugate has the structure of formula (IV): wherein [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; [Z] is a linker derived from a reactive group used to conjugate the anthracycline (PNU) derivative and the target-binding molecule; and Y comprises a bi-specific antigen binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein or recombinant fusion protein dimer according to the third
  • [Z] is a typically a moiety derived from a reactive group used to conjugate the anthracycline (PNU) derivative and the target-binding molecule.
  • [Z] may be a moiety derived from a reactive group selected from the group consisting of a maleimide, an alkyl halide, a sulphydryl group, an activated disulphide, an amino group, an alkyne group, an azido group, an aminoxy group, an aldehyde group and a ketone group.
  • [Z] may therefore be selected from the group consisting of a disulphide bond, an amide bond, an oxime bond, a hydrazone bond, a thioether bond, a 1, 2, 3 triazole and polyGly.
  • [X] is selected from the group comprising polyethylene glycol, ’ , wherein represents the point of attachment to the rest of the molecule and wherein [R] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof.
  • [X] is polyethylene glycol.
  • the polyethylene glycol may be PEG4.
  • the target-binding molecule is a protein or a nucleic acid.
  • target-binding proteins include but are not limited to an immunoglobulin or antibody, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobulin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), a scFv-Fc, (scFv) 2 , a diabody, a triabody, a tetrabody, a bispecific t-cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, a scaffold protein (affibodies, centyrin
  • target-binding nucleic acids include but are not limited to aptamers.
  • the target-binding molecule-drug conjugate is a protein and the anthracycline (PNU) derivative is conjugated to a thiol-containing amino acid residue in the amino acid sequence of a protein or to a thiol group introduced by chemical modification of the protein, for example incorporated at the N- terminus or C-terminus of the amino acid sequence of the specific antigen binding protein.
  • Thiol groups may also be introduced into other target-binding molecules, such as nucleic acids.
  • the target-binding molecule-drug conjugate, Y comprises a bi-specific antigen binding molecule according to the first or second aspects of the invention, conjugated to the PNU derivative via a human immunoglobulin Fc region or fragment thereof.
  • the fragment of the human immunoglobulin Fc region may be selected from the group consisting of an Fc heavy chain, a CH2 region and a CH3 region.
  • the target-binding molecule-drug conjugate according to the above aspects for use in therapy.
  • a target-binding molecule-drug conjugate according to the above aspects in the manufacture of a medicament for the treatment of a disease in a patient in need thereof.
  • a method of treatment of a disease in a patient in need of treatment comprising administration to said patient of a therapeutically effective dosage of a target-binding molecule-drug conjugate according to the above aspects.
  • the disease may be cancer.
  • the cancer is a ROR1-positive cancer type and/or an PTK7-positive cancer type.
  • the cancer is selected from the group comprising blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, renal cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, Head and Neck cancer, bladder cancer, oesophageal cancer, stomach cancer or liver cancer.
  • blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL),
  • the cancer may be mesothelioma or triple negative breast cancer (TNBC).
  • TNBC triple negative breast cancer
  • the mesothelioma may be pleural mesothelioma.
  • a pharmaceutical composition comprising a target-binding molecule-drug conjugate according to any of the above aspects, and at least one other pharmaceutically acceptable ingredient.
  • An antigen specific binding molecule of the invention comprises amino acid sequence derived from a synthetic library of VNAR molecules, or from libraries derived from the immunization of a cartilaginous fish.
  • VNAR, IgNAR and NAR may be used interchangeably also.
  • Amino acids are represented herein as either a single letter code or as the three letter code or both.
  • CDRs complementarity Determining Regions
  • CDR1 and CDR3 refers to the amino acid residues of a VNAR domain the presence of which are typically involved in antigen binding.
  • Each VNAR typically has two CDR regions identified as CDR1 and CDR3. Additionally, each VNAR domain comprises amino acids from a “hypervariable loop” (HV), which may also be involved in antigen binding.
  • HV hypervariable loop
  • a complementarity determining region can include amino acids from both a CDR region and a hypervariable loop.
  • antigen binding may only involve residues from a single CDR or HV.
  • a CDR2 region is not present.
  • “Framework regions” are those VNAR residues other than the CDR residues.
  • Each VNAR typically has five framework regions identified as FW1, FW2, FW3a, FW3b and FW4. The boundaries between FW, CDR and HV regions in VNARs are not intended to be fixed and accordingly some variation in the lengths and compositions of these regions is to be expected.
  • a “codon set” refers to a set of different nucleotide triplet sequences used to encode desired variant amino acids.
  • a set of oligonucleotides can be synthesized, for example, by solid phase synthesis, including sequences that represent all possible combinations of nucleotide triplets provided by the codon set and that will encode the desired group of amino acids.
  • a standard form of codon designation is that of the IUB code, which is known in the art and described herein.
  • a codon set is typically represented by 3 capital letters in italics, e.g. NNK, NNS, XYZ, DVK etc.
  • a “non- random codon set” therefore refers to a codon set that encodes select amino acids that fulfill partially, preferably completely, the criteria for amino acid selection as described herein.
  • Synthesis of oligonucleotides with selected nucleotide “degeneracy” at certain positions is well known in that art, for example the TRIM approach (Knappek et al.; J. Mol. Biol. (1999), 296, 57-86); Garrard & Henner, Gene (1993), 128, 103).
  • Such sets of oligonucleotides having certain codon sets can be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, CA), or can be obtained commercially (for example, from Life Technologies, Rockville, MD).
  • a set of oligonucleotides synthesized having a particular codon set will typically include a plurality of oligonucleotides with different sequences, the differences established by the codon set within the overall sequence.
  • Oligonucleotides used according to the present invention have sequences that allow for hybridization to a VNAR nucleic acid template and also may where convenient include restriction enzyme sites.
  • Cell Cell
  • cell line cell culture
  • progeny of a cell or cell line.
  • terms like “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
  • Control sequences when referring to expression means DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, etc.
  • Eukaryotic cells use control sequences such as promoters, polyadenylation signals, and enhancers.
  • coat protein means a protein, at least a portion of which is present on the surface of the virus particle. From a functional perspective, a coat protein is any protein which associates with a virus particle during the viral assembly process in a host cell, and remains associated with the assembled virus until it infects another host cell.
  • the “detection limit” for a chemical entity in a particular assay is the minimum concentration of that entity which can be detected above the background level for that assay.
  • the “detection limit” for a particular phage displaying a particular antigen binding fragment is the phage concentration at which the particular phage produces an ELISA signal above that produced by a control phage not displaying the antigen binding fragment.
  • a “fusion protein” and a “fusion polypeptide” refer to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property.
  • the property may be a biological property, such as activity in vitro or in vivo.
  • the property may also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc.
  • the two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other.
  • the two portions of the polypeptide are obtained from heterologous or different polypeptides.
  • the term “fusion protein” in this text means, in general terms, one or more proteins joined together by chemical means, including hydrogen bonds or salt bridges, or by peptide bonds through protein synthesis or both. Typically fusion proteins will be prepared by DNA recombination techniques and may be referred to herein as recombinant fusion proteins.
  • “Heterologous DNA” is any DNA that is introduced into a host cell.
  • the DNA may be derived from a variety of sources including genomic DNA, cDNA, synthetic DNA and fusions or combinations of these.
  • the DNA may include DNA from the same cell or cell type as the host or recipient cell or DNA from a different cell type, for example, from an allogenic or xenogenic source.
  • the DNA may, optionally, include marker or selection genes, for example, antibiotic resistance genes, temperature resistance genes, etc.
  • a “highly diverse position” refers to a position of an amino acid located in the variable regions of the light and heavy chains that have a number of different amino acid represented at the position when the amino acid sequences of known and/or naturally occurring antibodies or antigen binding fragments are compared. The highly diverse positions are typically in the CDR or HV regions.
  • Identity describes the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness (homology) between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs.
  • Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. (1990) 215, 403).
  • the amino acid sequence of the protein has at least 45% identity, using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. (1990) 215, 403-410) provided by HGMP (Human Genome Mapping Project), at the amino acid level, to the amino acid sequences disclosed herein.
  • the protein sequence may have at least 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90% and still more preferably 95% (still more preferably at least 96%, 97%, 98% or 99%) identity, at the nucleic acid or amino acid level, to the amino acid sequences as shown herein.
  • the protein may also comprise a sequence which has at least 45%, 46%, 47%, 48%, 49%, 50%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with a sequence disclosed herein, using the default parameters of the BLAST computer program provided by HGMP, thereto
  • a “library” refers to a plurality of VNARs or VNAR fragment sequences (for example, polypeptides of the invention), or the nucleic acids that encode these sequences, the sequences being different in the combination of variant amino acids that are introduced into these sequences according to the methods of the invention.
  • “Ligation” is the process of forming phosphodiester bonds between two nucleic acid fragments.
  • the ends of the fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary first to convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.
  • the DNA is treated in a suitable buffer for at least 15 minutes at 15°C with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of the four deoxyribonucleotide triphosphates.
  • the DNA is then purified by phenol- chloroform extraction and ethanol precipitation or by silica purification.
  • the DNA fragments that are to be ligated together are put in solution in about equimolar amounts.
  • the solution will also contain ATP, ligase buffer, and a ligase such as T4 DNA ligase at about 10 units per 0.5 ⁇ g of DNA.
  • the vector is first linearized by digestion with the appropriate restriction endonuclease(s).
  • the linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.
  • a “mutation” is a deletion, insertion, or substitution of a nucleotide(s) relative to a reference nucleotide sequence, such as a wild type sequence.
  • “Natural” or “naturally occurring” VNARs refers to VNARs identified from a non-synthetic source, for example, from a tissue source obtained ex vivo, or from the serum of an animal of the Elasmobranchii subclass. These VNARs can include VNARs generated in any type of immune response, either natural or otherwise induced. Natural VNARs include the amino acid sequences, and the nucleotide sequences that constitute or encode these antibodies.
  • nucleic acid construct generally refers to any length of nucleic acid which may be DNA, cDNA or RNA such as mRNA obtained by cloning or produced by chemical synthesis.
  • the DNA may be single or double stranded. Single stranded DNA may be the coding sense strand, or it may be the non-coding or anti-sense strand.
  • the nucleic acid construct is preferably in a form capable of being expressed in the subject to be treated.
  • “Operably linked” when referring to nucleic acids means that the nucleic acids are placed in a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promotor or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contingent and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accord with conventional practice.
  • the term “protein” means, in general terms, a plurality of amino acid residues joined together by peptide bonds. It is used interchangeably and means the same as peptide, oligopeptide, oligomer or polypeptide, and includes glycoproteins and derivatives thereof.
  • protein is also intended to include fragments, analogues, variants and derivatives of a protein wherein the fragment, analogue, variant or derivative retains essentially the same biological activity or function as a reference protein.
  • protein analogues and derivatives include peptide nucleic acids, and DARPins (Designed Ankyrin Repeat Proteins).
  • a fragment, analogue, variant or derivative of the protein may be at least 25 preferably 30 or 40, or up to 50 or 100, or 60 to 120 amino acids long, depending on the length of the original protein sequence from which it is derived. A length of 90 to 120, 100 to 110 amino acids may be convenient in some instances.
  • the fragment, derivative, variant or analogue of the protein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably, a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or auxiliary sequence which is employed for purification of the polypeptide.
  • a conserved or non-conserved amino acid residue preferably, a conserved amino acid residue
  • substituted amino acid residue may or may not be one encoded by the genetic code
  • one or more of the amino acid residues includes a substituent group
  • the additional amino acids are fused to the mature polypeptide, such as a leader or auxiliary sequence which is employed for purification of the polypeptide.
  • Oligonucleotides are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (such as phosphotriester, phosphite, or phosphoramidite chemistry, using solid-phase techniques). Further methods include the polymerase chain reaction (PCR) used if the entire nucleic acid sequence of the gene is known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue. The oligonucleotides can be purified on polyacrylamide gels or molecular sizing columns or by precipitation.
  • PCR polymerase chain reaction
  • DNA is “purified” when the DNA is separated from non-nucleic acid impurities (which may be polar, non-polar, ionic, etc.).
  • a “source” or “template” VNAR refers to a VNAR or VNAR antigen binding fragment whose antigen binding sequence serves as the template sequence upon which diversification according to the criteria described herein is performed.
  • An antigen binding sequence generally includes within a VNAR preferably at least one CDR, preferably including framework regions.
  • a “transcription regulatory element” will contain one or more of the following components: an enhancer element, a promoter, an operator sequence, a repressor gene, and a transcription termination sequence.
  • Transformation means a process whereby a cell takes up DNA and becomes a “transformant”.
  • the DNA uptake may be permanent or transient.
  • a “transformant” is a cell which has taken up and maintained DNA as evidenced by the expression of a phenotype associated with the DNA (e.g., antibiotic resistance conferred by a protein encoded by the DNA).
  • a “variant” or “mutant” of a starting or reference polypeptide is a polypeptide that (1) has an amino acid sequence different from that of the starting or reference polypeptide and (2) was derived from the starting or reference polypeptide through either natural or artificial mutagenesis.
  • Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of the polypeptide of interest.
  • a fusion polypeptide of the invention generated using an oligonucleotide comprising a nonrandom codon set that encodes a sequence with a variant amino acid (with respect to the amino acid found at the corresponding position in a source VNAR or antigen binding fragment) would be a variant polypeptide with respect to a source VNAR or antigen binding fragment.
  • a variant CDR refers to a CDR comprising a variant sequence with respect to a starting or reference polypeptide sequence (such as that of a source VNAR or antigen binding fragment).
  • a variant amino acid in this context, refers to an amino acid different from the amino acid at the corresponding position in a starting or reference polypeptide sequence (such as that of a source VNAR or antigen binding fragment). Any combination of deletion, insertion, and substitution may be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics.
  • the amino acid changes also may alter post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites.
  • a “wild-type” or “reference” sequence or the sequence of a “wild-type” or “reference” protein/polypeptide, such as a coat protein, or a CDR of a source VNAR, may be the reference sequence from which variant polypeptides are derived through the introduction of mutations.
  • the “wild-type” sequence for a given protein is the sequence that is most common in nature.
  • a “wild-type” gene sequence is the sequence for that gene which is most commonly found in nature. Mutations may be introduced into a “wild-type” gene (and thus the protein it encodes) either through natural processes or through man induced means. The products of such processes are “variant” or “mutant” forms of the original “wild-type” protein or gene.
  • a “humanised” antigen specific antigen binding molecule may be modified at one or more amino acid sequence position to reduce the potential for immunogenicity in vivo, while retaining functional binding activity for the specific epitopes on the specific antigen.
  • Humanization of antibody variable domains is a technique well-known in the art to modify an antibody which has been raised, in a species other than humans, against a therapeutically useful target so that the humanized form may avoid unwanted immunological reaction when administered to a human subject.
  • the methods involved in humanization are summarized in Almagro J.C and William Strohl W.
  • Antibody Engineering Humanization, Affinity Maturation, and Selection Techniques in Therapeutic Monoclonal Antibodies: From Bench to Clinic.
  • IgNARs have distinct origins compared to immunoglobulins and have very little sequence homology compared to immunoglobulin variable domains there are some structural similarities between immunoglobulin and IgNAR variable domains, so that similar processes can be applied to the VNAR domain.
  • WO2013/167883 incorporated by reference, provides a description of the humanization of VNARs, see also Kovalenko O.V., et al. J Biol Chem.2013.288(24): p.17408-19.
  • a humanised antigen specific binding molecule may differ from a wild-type antigen specific binding molecule by substituting one or more framework amino acid residues with a corresponding framework amino acid residue of DPK-9.
  • DPK-9 is a human germline VL scaffold, a member of the variable kappa subgroup 1 (V ⁇ 1).
  • DPK-9 has a sequence according to: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGSG TDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIK (SEQ ID NO:132)
  • CARs chimeric antigen receptors
  • CARs may be employed to impart the specificity of an antigen-specific binding protein, such as a monoclonal antibody or VNAR, onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy.
  • CARs may direct the specificity of the cell to a tumour associated antigen, for example.
  • CARs may comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumour associated antigen binding region.
  • CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies fused to CD3-zeta transmembrane and endodomains.
  • CARs comprise fusions of the VNAR domains described herein with CD3-zeta transmembrane and endodomains.
  • the specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins.
  • one can target malignant B cells by redirecting the specificity of T cells by using a CAR specific for the B-lineage molecule, CD 19.
  • the spacing of the antigen-recognition domain can be modified to reduce activation-induced cell death.
  • CARs comprise domains for additional co-stimulatory signalling, such as CD3-zeta, FcR, CD27, CD28, CD 137, DAP 10, and/or OX40.
  • molecules can be co- expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.
  • conjugation as used herein may refer to any method of chemically linking two or more chemical moieties. Typically, conjugation will be via covalent bond.
  • At least one of the chemical moieties will be a polypeptide and in some cases the conjugation will involve two or more polypeptides, one or more of which may be generated by recombinant DNA technology.
  • a number of systems for conjugating polypeptides are known in the art. For example, conjugation can be achieved through a lysine residue present in the polypeptide molecule using N- hydroxy-succinimide or through a cysteine residue present in the polypeptide molecule using maleimidobenzoyl sulfosuccinimide ester.
  • conjugation occurs through a short- acting, degradable linkage including, but not limited to, physiologically cleavable linkages including ester, carbonate ester, carbamate, sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal, hydrazone, oxime and disulphide linkages.
  • linkers that are cleavable by intracellular or extracellular enzymes, such as cathepsin family members, cleavable under reducing conditions or acidic pH are incorporated to enable releases of conjugated moieties from the polypeptide or protein to which it is conjugated.
  • a particularly preferred method of conjugation is the use of intein-based technology (US2006247417) Briefly, the protein of interest is expressed as an N terminal fusion of an engineered intein domain (Muir 2006 Nature 442, 517–518). Subsequent N to S acyl shift at the protein-intein union results in a thioester linked intermediate that can be chemically cleaved with bis-aminoxy agents or amino-thiols to give the desired protein C-terminal aminoxy or thiol derivative, respectively.
  • C-terminal aminoxy and thiol derivatives can be reacted with aldehyde / ketone and maleimide functionalised moieties, respectively, in a chemoselective fashion to give the site-specific C-terminally modified protein.
  • the VNARs are directly expressed with an additional cysteine at or near the C-terminal region of the VNAR or incorporated within a short C-terminal tag sequence enabling conjugation with thiol reactive payloads such as maleimide functionalised moieties.
  • Conjugation as referred to herein is also intended to encompass the use of a linker moiety, which may impart a number of useful properties.
  • Linker moieties include, but are not limited to, peptide sequences such as poly-glycine, gly-ser, val-cit or val-ala.
  • the linker moiety may be selected such that it is cleavable under certain conditions, for example via the use of enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, or oxidizing reagents, or the linker may be specifically selected to resist cleavage under such conditions.
  • Polypeptides may be conjugated to a variety of functional moieties in order to achieve a number of goals.
  • Examples of functional moieties include, but are not limited to, polymers such as polyethylene glycol in order to reduce immunogenicity and antigenicity or to improve solubility. Further non-limiting examples include the conjugation of a polypeptide to a therapeutic agent or a cytotoxic agent.
  • the term “detectable label” is used herein to specify that an entity can be visualized or otherwise detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means. The detectable label may be selected such that it generates a signal which can be measured and whose intensity is proportional to the amount of bound entity.
  • a wide variety of systems for labelling and/or detecting proteins and peptides are known in the art.
  • a label may be directly detectable (i.e., it does not require any further reaction or manipulation to be detectable, e.g., a fluorophore is directly detectable) or it may be indirectly detectable (i.e., it is made detectable through reaction or binding with another entity that is detectable, e.g., a hapten is detectable by immunostaining after reaction with an appropriate antibody comprising a reporter such as a fluorophore).
  • Suitable detectable agents include, but are not limited to, radionuclides, fluorophores, chemiluminescent agents, microparticles, enzymes, colorimetric labels, magnetic labels, haptens, molecular beacons, and aptamer beacons.
  • an alkyl group is a straight chain or branched, substituted or unsubstituted group (preferably unsubstituted) containing from 1 to 40 carbon atoms.
  • alkyl group may optionally be substituted at any position.
  • alkenyl denotes a group derived from the removal of a single hydrogen atom from a straight- or branched-chain aliphatic moiety having at least one carbon- carbon double bond.
  • alkynyl refers to a group derived from the removal of a single hydrogen atom from a straight- or branched-chain aliphatic moiety having at least one carbon- carbon triple bond.
  • alkyl’, ‘aryl’, ‘heteroaryl’ etc also include multivalent species, for example alkylene, arylene, ‘heteroarylene’ etc.
  • alkylene groups include ethylene (-CH 2 -CH 2 -), and propylene (-CH 2 - CH2-CH2-).
  • An exemplary arylene group is phenylene (-C6H4-), and an exemplary heteroarylene group is pyridinylene (-C5H3N-).
  • Aromatic rings are cyclic aromatic groups that may have 0, 1, 2 or more, preferably 0, 1 or 2 ring heteroatoms. Aromatic rings may be optionally substituted and/or may be fused to one or more aromatic or non-aromatic rings (preferably aromatic), which may contain 0, 1, 2, or more ring heteroatoms, to form a polycyclic ring system. Aromatic rings include both aryl and heteroaryl groups.
  • Aryl and heteroaryl groups may be mononuclear, i.e. having only one aromatic ring (like for example phenyl or phenylene), or polynuclear, i.e. having two or more aromatic rings which may be fused (like for example napthyl or naphthylene), individually covalently linked (like for example biphenyl), and/or a combination of both fused and individually linked aromatic rings.
  • the aryl or heteroaryl group is an aromatic group which is substantially conjugated over substantially the whole group.
  • Aryl groups may contain from 5 to 40 ring carbon atoms, from 5 to 25 carbon atoms, from 5 to 20 carbon atoms, or from 5 to 12 carbon atoms.
  • Heteroaryl groups may be from 5 to 40 membered, from 5 to 25 membered, from 5 to 20 membered or from 5 to 12 membered rings, containing 1 or more ring heteroatoms selected from N, O, S and P.
  • An aryl or heteroaryl may be fused to one or more aromatic or non-aromatic rings (preferably an aromatic ring) to form a polycyclic ring system.
  • Aryl and heteroaryl preferably denote a mono-, bi- or tricyclic aromatic or heteroaromatic group with up to 25 ring atoms that may also comprise condensed rings and is optionally substituted.
  • Preferred aryl groups include, without limitation, benzene, biphenylene, triphenylene, [1,1':3',1'']terphenyl-2'-ylene, naphthalene, anthracene, binaphthylene, phenanthrene, pyrene, dihydropyrene, chrysene, perylene, tetracene, pentacene, benzopyrene, fluorene, indene, indenofluorene, spirobifluorene, etc.
  • Preferred heteroaryl groups include, without limitation, 5-membered rings like pyrrole, pyrazole, silole, imidazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, furan, thiophene, selenophene, oxazole, isoxazole, 1,2-thiazole, 1,3-thiazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3- thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 6-membered rings like pyridine, pyridazine, pyrimidine, pyrazine, 1,3,5-triazine, 1,2,4-triazine, 1,2,3-triazine, 1,2,4,5-tetrazine, 1,2,3,4- tetraz
  • heteroaryl groups may be substituted with alkyl, alkoxy, thioalkyl, fluoro, fluoroalkyl or further aryl or heteroaryl substituents.
  • a heteroaryl group is thiophene.
  • Particularly preferred heteroatoms are selected from O, S, N, P and Si.
  • hydrogen will complete the valency of a heteroatom included in the molecules of the invention, e.g. for N there may be -NH- or -NH2 where one or two other groups are involved.
  • the term “optionally substituted” means that one or more of the hydrogen atoms in the optionally substituted moiety is replaced by a suitable substituent.
  • an "optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable compounds.
  • stable refers to compounds that are chemically feasible and can exist for long enough at room temperature (i.e.16-25°C) to allow for their detection, isolation and/or use in chemical synthesis.
  • the optional substituents may comprise all chemically possible combinations in the same group and/or a plurality of the aforementioned groups (for example amino and sulfonyl if directly attached to each other represent a sulfamoyl radical).
  • the substituent is not acyl.
  • acyl refers to an acyl group which is a moiety derived by the removal of one or more hydroxyl groups from an oxoacid, such as a carboxylic acid. It contains a double-bonded oxygen atom and an alkyl group. In some embodiments the groups may be unsubstituted.
  • the anthracycline (PNU) derivative may be of formula (V): wherein [X] is an optional spacer selected from the group comprising unsubstituted alkyl groups, unsubstituted heteroalkyl groups, unsubstituted aryl groups, unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit- PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof.
  • [X] is an optional spacer
  • [X] is preferably selected from the group comprising polyethylene glycol and , wherein represents the point of attachment to the rest of the molecule and wherein [R] is an optional spacer selected from the group comprising unsubstituted alkyl groups, unsubstituted heteroalkyl groups, unsubstituted aryl groups, unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof.
  • PAB is intended to mean p-aminobenzyloxycarbonyl. Occasionally in the literature, the term PAB may be used to indicated p-aminobenzyl.
  • PAB is intended to indicate p-aminobenzyloxycarbonyl.
  • target-binding molecule refers to any molecule that binds to a given target.
  • target and antigen may be used interchangeably.
  • target-binding molecules include natural or recombinant proteins including immunoglobulins or antibodies, immunoglobulin Fc regions, immunoglobulin Fab regions, Fab, Fab’, Fv, Fv-Fc, single chain Fv (scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific t-cell engagers , inteins, intein fusions, VNAR domains, single domain antibodies (sdAb), VH domains, scaffold proteins (affibodies, centyrins, darpins etc.) and nucleic acids including aptamers or small molecules or natural products that have been developed to bind to the target or naturally bind to the target.
  • natural or recombinant proteins including immunoglobulins or antibodies, immunoglobulin Fc regions, immunoglobulin Fab regions, Fab, Fab’, Fv, Fv-Fc, single chain Fv (scFv),
  • Methods include reaction of amine groups with 2-iminothiolane (Traut’s reagent), modification of amine groups with NHS-ester containing heterobifunctional agents such as N-succinimidyl S-acetylthiolate (SATA) or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), followed by treatment with hydroxylamine and reducing agents respectively and cleavage of engineered intein-fusion proteins with cysteamine to generate C-terminal thiol proteins and peptides.
  • SATA N-succinimidyl S-acetylthiolate
  • SPDB N-succinimidyl-4-(2-pyridyldithio)butanoate
  • Preferred embodiments include target-binding molecule-drug conjugate selected from the group consisting of: ⁇ G3CP / P2A7 each fused to hFc (S239C+S442C) comprising a [G4S]3 linker and KIH technology, conjugated to vcMMAE via each S239C and each S442C ⁇ G3CP / 4D2 each fused to hFc (S239C+S442C) comprising a [G4S]3 linker and KIH technology, conjugated to vcMMAE via each S239C and each S442C ⁇ G3CP / E02 each fused to hFc (S239C+S442C) comprising a [G4S]3 linker and KIH technology, conjugated to vcMMAE via each S239C and each S442C ⁇ 1H8 / P2A7 each fused
  • ADCC antibody-dependent cellular toxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • ADCP is mediated by phagocytotic cells, for example macrophages, monocytes and neutrophils, when the Fc region of the antibody on antibody-labelled cells interacts with corresponding activatory Fc ⁇ Rs on the surface of these phagocytic cells. This causes activation of the phagocytic cell, promoting clearance of the Fc-labelled cells from the body by phagocytosis.
  • CDC occurs when sufficient Fc molecules engage the 6 globular heads of the C1q protein, initiating the proteolytic cascade of complement proteins and ultimately resulting in release of anaphylatoxins C3a and C5a, and formation of membrane attack complex (MAC).
  • the MAC forms pores in the plasma membrane of target cells, leading to osmolysis.
  • Fc mediated effector function(s) may be undesirable.
  • tissue expression of the antibody target which can lead to undesired on-target off-tumour immune activation and thereby potential toxicity issues.
  • a number of strategies can be used to reduce or silence Fc effector activity.
  • IgG isotypes have different affinities for the Fc ⁇ Rs (IgG1 > IgG3 > IgG2 > IgG4) and IgG2 and IgG4 backbones have been used to reduce Fc effector functions in therapeutics (Yu 2020 J Hematol & Oncol 13:45).
  • Ser is naturally found at positions 330 and 331 in IgG4, and results in reduced Fc ⁇ R binding when incorporated into IgG2 (Lund 1991 J Immunol 147:2657).
  • Methods for reducing Fc effector function in the IgG1 isotype include a glycosylation through amino acid substitution at N297 position (N297Q/A/G) (Jacobsen 2017 JBC 292(5) 1865), because glycans attached at this position of the Fc are critical for efficient binding to Fc ⁇ Rs and C1q.
  • cell free protein expression e.g. Sutro XpressCF TM
  • sugar remodelling / conjugation platforms e.g. Synaffix GlycoConnect TM
  • amino acid substitutions or modifications within the Fc:Fc ⁇ R/C1q binding interface can be used to reduce IgG1 Fc effector activity (Sonderman 2020 Nature 406(6793):267). At least 39 human IgG1 residues are relevant to binding Fc ⁇ Rs, with substitutions between positions 232-239 of particular interest and many antibody variants in the clinic have substitutions in this area.
  • L234A/L235A LALA
  • FES L234F/L235E/P331S
  • LALAPG Schott al.
  • an antibody of the invention does not display the effector function or functions associated with a normal Fc region.
  • the Fc region of an antibody of the invention does not bind to or has reduced binding to one or more Fc receptors.
  • an antibody of the invention may show reduced binding to all Fc receptors.
  • an antibody of the invention does not bind to any Fc receptors.
  • the antibody does bind to one or more types of Fc receptor.
  • an antibody of the invention does not bind or has reduced binding to one or more Fc ⁇ Rs.
  • an antibody of the invention may not bind or may have reduced binding to Fc ⁇ RIIIa.
  • an antibody of the invention does not bind or has reduced binding to C1q.
  • an antibody of the invention does not bind or has reduced binding to one or more Fc ⁇ Rs or C1q.
  • an antibody of the invention does not bind or has reduced binding to one or more Fc ⁇ Rs, but does bind C1q.
  • an antibody of the invention in general may comprise an Fc region modification(s) that alter the half-life of the antibody.
  • an antibody of the invention comprises a mutated Fc region, in particular, an Fc region comprising a mutation described herein.
  • the Fc mutation is selected from the group comprising a mutation to remove, reduce or enhance binding of the Fc region to an Fc receptor, a mutation to increase, reduce or remove an effector function, a mutation to increase or decrease half-life of the antibody and a combination of the same.
  • where reference is made to the impact of a modification it may be demonstrated by comparison to an equivalent antibody lacking the modification.
  • antibodies of the invention may comprise multiple modifications, for example modifications which reduce or silence effector function may be present in addition to modifications which alter the half-life of the antibody and/or modifications which promote heterodimerisation. Therefore, any of the Fc regions disclosed herein may be further modified to comprise any of the Fc “silencing” mutations or strategies described above.
  • the mutations may be symmetric or asymeteric.
  • the Fc region comprises a first fragment of an immunoglobulin Fc region and a second fragment of an immunoglobulin Fc region containing the same Fc “silencing” mutations.
  • the first fragment of an immunoglobulin Fc region and the second fragment of an immunoglobulin Fc region may contain different Fc “silencing” mutations.
  • any of the Fc regions disclosed herein may additionally comprise the following mutations or any combination thereof: ⁇ L234A ⁇ L234F ⁇ L234S ⁇ L234D ⁇ L234R ⁇ L235A ⁇ L235E ⁇ L235T ⁇ L235R ⁇ P331S ⁇ P329G ⁇ G236R
  • any of the Fc regions disclosed herein may additionally comprise the following groups of mutations: ⁇ L234A/L235A ⁇ L234F/L235E/P331S ⁇ L234A/L235A/P329G ⁇ L234S/L235T/G236R ⁇ L234D/L235E ⁇ E233K/L234R/L235R Preferred hFc regions that may be incorporated into any of the bi-specific
  • a bi-specific antigen binding molecule comprising: (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO:11), YPWGAGAPCLVQWY (SEQ ID NO: 12), YPWGAGAPRLVQWY (SEQ ID NO:13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGA
  • CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20) and YPWGAGAPWNVQWY (SEQ ID NO: 24), and/or CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1) and DANYGLAA (SEQ ID NO: 5).
  • CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23).
  • CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23);
  • CDR1 is a CDR sequence having an amino acid sequence according to GANYGLAA (SEQ ID NO: 1);
  • HV2 is a hypervariable sequence having an amino acid sequence according to SSNQERISIS (SEQ ID NO: 6);
  • HV4 is a hypervariable sequence having an amino acid sequence according to NKRTM (SEQ ID NO: 8).
  • CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23);
  • CDR1 is a CDR sequence having an amino acid sequence according to DANYGLAA (SEQ ID NO: 5);
  • HV2 is a hypervariable sequence having an amino acid sequence according to SSNKERISIS (SEQ ID NO: 7); and
  • HV4 is a hypervariable sequence having an amino acid sequence according to NKGTM (SEQ ID NO: 9).
  • CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPWLVQWY (SEQ ID NO: 10);
  • CDR1 is a CDR sequence having an amino acid sequence according to DANYGLAA (SEQ ID NO: 5);
  • HV2 is a hypervariable sequence having an amino acid sequence according to SSNKERISIS (SEQ ID NO: 7);
  • HV4 is a hypervariable sequence having an amino acid sequence according to NKGTM (SEQ ID NO: 9).
  • the bi-specific antigen binding molecule of any preceding clause further comprising an additional domain.
  • the bi-specific antigen binding molecule of clause 9 wherein the additional domain is a human Fc region.
  • the bi-specific antigen binding molecule of clause 10 wherein the additional domain is hIgG1 (SEQ ID NO: 216). 12. The bi-specific antigen binding molecule of clause 10 wherein the additional domain is hIgG1 (S239C) (SEQ ID NO: 217). 13. The bi-specific antigen binding molecule of clause 10 wherein the additional domain is hIgG1 (S442c) (SEQ ID NO: 218). 14. The bi-specific antigen binding molecule of clause 10 wherein the additional domain is hIgG1 (S239C+S442C) (SEQ ID NO: 219). 15.
  • the bi-specific antigen binding molecule of any preceding clause further comprising a linker region between the ROR1-specific antigen binding molecule and PTK7-specific antigen binding molecule.
  • the linker comprises [G 4 S] x , where x is 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, or 10.
  • a bi-specific antigen binding molecule comprising: (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30) and DTSYGLYS (SEQ ID NO: 207); FW1 is a framework region; FW2 is a framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERIS
  • FW1 is a framework region of from 20 to 28 amino acids
  • FW2 is a framework region of from 6 to 14 amino acids
  • FW3a is a framework region of from 6 to 10 amino acids
  • FW3b is a framework region of from 17 to 24 amino acids
  • FW4 is a framework region of from 7 to 14 amino acids.
  • FW1 has an amino acid sequence selected from the group consisting of: ASVNQTPRTATKETGESLTINCVVT (SEQ ID NO: 40), TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 41) and ASVTQSPRSASKETGESLTITCRVT (SEQ ID NO: 42), or a functional variant of any thereof with a sequence identity of at least 45%;
  • FW2 has an amino acid sequence according to TYWYRKNPG (SEQ ID NO: 43), or a functional variant of any thereof with a sequence identity of at least 45%;
  • FW3a has an amino acid sequence selected from the group consisting of: GRYVESV (SEQ ID NO: 44) and GRYSESV (SEQ ID NO: 45), or a functional variant of any thereof with a sequence identity of at least 45%;
  • FW3b has an amino acid sequence selected from the group consisting of: SFSLRIKDLTVADSATYYCKA (SEQ ID NO:
  • ROR1-specific antigen binding molecule comprises an amino acid sequence selected from the group consisting of: ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50); TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPWLVQWY DGAGTKVEIK (SEQ ID NO: 51); ASVNQTPRTATKETGESLTINCVVTGANYDLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPSGAGAPRPVQWYDGAGTVLTVN (SEQ ID NO:
  • the bi-specific antigen binding molecule of clause 38 wherein the additional domain is an immunoglobulin, an immunoglobulin Fc region, an immunoglobulin Fab region, a single chain Fv (scFv), a diabody, a triabody, a tetrabody, a bispecific t-cell engager (BiTE), an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, or a scaffold protein.
  • the additional domain is an Fc region.
  • the bi-specific antigen binding molecule of clause 41 wherein the additional domain is hIgG1 (SEQ ID NO: 216).
  • 45. The bi-specific antigen binding molecule of clause 41 wherein the additional domain is hIgG1 (S239C+S442C) (SEQ ID NO: 219). 46.
  • the bi-specific antigen binding molecule of any preceding clause further comprising a linker region between the ROR1-specific antigen binding molecule and PTK7-specific antigen binding molecule.
  • the linker comprises [G4S]x, where x is 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, or 10.
  • the linker comprises [G 4 S] 3 , [G4S]5, or G4S. 49.
  • the bi-specific antigen binding molecule of clause 47 wherein the linker comprises PGVQPSPGGGGS (SEQ ID NO: 89) (Wobbe-G 4 S) or PGVQPAPGGGGS (SEQ ID NO: 90) (Wobbe- G4S GM).
  • the linker comprises PGVQPSPGGGGS (SEQ ID NO: 89) (Wobbe-G 4 S) or PGVQPAPGGGGS (SEQ ID NO: 90) (Wobbe- G4S GM).
  • the bi-specific antigen binding molecule of clause 50 further comprising a C- terminal tag sequence selected from QASGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 98), QACGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 99), QACKAHHHHGAEFEQKLISEEDL (SEQ ID NO: 97), AAAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 100), ACAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 101), QASGAHHHHHH (SEQ ID NO: 102) QACGAHHHHHH (SEQ ID NO: 103), QACKAHHHHHH (SEQ ID NO: 104), AAAHHHHHH (SEQ ID NO: 105), ACAHHHHHH (SEQ ID NO: 106), QASGA (SEQ ID NO: 107), QACGA (SEQ ID NO: 108), QACKA (SEQ ID NO: 109), ACA (SEQ ID NO: 110), and SAPSA (SEQ ID NO:
  • a recombinant fusion protein comprising a bi-specific antigen binding molecule as defined in any one of clauses 1 to 51.
  • at least one biologically active protein is an immunoglobin, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobin Fab region
  • the at least one biologically active protein is a fragment of an immunoglobulin Fc region selected from the group consisting of an Fc heavy chain, a CH2 region and a CH3 region.
  • the fragment of an immunoglobulin Fc region is an Fc heavy chain, optionally wherein the Fc heavy chain is engineered to comprise one or more cysteine residues suitable for bioconjugation. 59.
  • a recombinant fusion protein dimer comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 12), YPWGAGAPRLVQWY (SEQ ID NO:
  • the one or more amino acid substitution is selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V. 71.
  • the recombinant fusion protein dimer according to any one of clauses 65 to 72 wherein the second specific antigen binding molecule is an immunoglobin, an immunoglobin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv)2, a diabody, a triabody, a tetrabody, a bispecific t-cell engager, an intein, a VNAR domain, a single domain antibody (sdAb) or a VH domain.
  • the second specific antigen binding molecule is an immunoglobin, an immunoglobin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv)2, a diabody, a triabody, a tetrabody, a bispecific t-cell engager,
  • the recombinant fusion protein dimer according to any one of clauses 65 or 73 wherein (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 50 (G3CP), SEQ ID NO: 61 (1H8) and SEQ ID NO: 71 (G3CP G4), and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 215. 75.
  • the recombinant fusion protein dimer according to any one of clauses 65 or 73 wherein (a) the first recombinant fusion protein comprises G3CP-hFc, 1H8-hFc or G3CPG4-hFc, and (b) the second recombinant fusion protein comprises P2A7-hFc, E02-hFc, or 4D2-hFc. 76.
  • the recombinant fusion protein dimer according to any one of clauses 65 or 73 wherein (a) the first recombinant fusion protein comprises G3CP-hFc (S239C & S442C), 1H8-hFc (S239C & S442C) or G3CPG4-hFc (S239C & S442C), and (b) the second recombinant fusion protein comprises P2A7-hFc (S239C & S442C), E02-hFc (S239C & S442C) or 4D2-hFc (S239C & S442C). 77.
  • the recombinant fusion protein dimer according to any one of clauses 65 or 73 wherein (a) the first recombinant fusion protein comprises G3CP-hFc (S239C & S442C), 1H8-hFc (S239C & S442C) or G3CPG4-hFc (S239C & S442C), and (b) the second recombinant fusion protein comprises P2A7-hFc (S239C & S442C), E02-hFc (S239C & S442C) or 4D2-hFc (S239C & S442C). 78.
  • the recombinant fusion protein dimer according to any one of clauses 65 or 73 wherein the recombinant fusion protein dimer is selected from the group consisting of: G3CP-hFc (S239C & S442C) and P2A7-hFc (S239C & S442C), G3CP-hFc (S239C & S442C) and E02-hFc (S239C & S442C), and G3CP-hFc (S239C & S442C) and 4D2-hFc (S239C & S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G 4 S] 3 linker or a G 4 S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises
  • recombinant fusion protein dimer according to any one of clauses 65 or 73 wherein the recombinant fusion protein dimer is selected from the group consisting of: G3CPG4-hFc (S239C & S442C) and P2A7-hFc (S239C & S442C), G3CPG4-hFc (S239C & S442C) and E02-hFc (S239C & S442C), and G3CPG4-hFc (S239C & S442C) and 4D2-hFc (S239C & S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G 4 S] 3 linker or a G 4 S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK
  • recombinant fusion protein dimer according to any one of clauses 65 or 73 wherein the recombinant fusion protein dimer is selected from the group consisting of: 1H8-hFc (S239C & S442C) and P2A7-hFc (S239C & S442C), 1H8-hFc (S239C & S442C) and E02-hFc (S239C & S442C), and 1H8-hFc (S239C & S442C) and 4D2-hFc (S239C & S442C); wherein the ROR1-specific binding molecule is fused to the hFc region via a [G 4 S] 3 linker or a G 4 S linker and wherein (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution and the hFc region fused to the PTK7-specific binding molecule comprises
  • a recombinant fusion protein dimer comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecule comprising an amino acid sequence represented by the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) wherein CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30) and DTSYGLYS (SEQ ID NO: 207); FW1 is a framework region; FW2 is a framework region
  • the recombinant fusion protein dimer of clause 82 wherein: (a) the first recombinant fusion protein comprises P3A1, and (b) the second recombinant fusion protein comprises P2A7, E02, or 4D2. 84. The recombinant fusion protein dimer of clause 82 wherein: (a) the first recombinant fusion protein comprises P3A1-hFc, and (b) the second recombinant fusion protein comprises P2A7-hFc, E02-hFc or 4D2-hFc. 85.
  • the recombinant fusion protein dimer of clause 82 wherein: (a) the first recombinant fusion protein comprises P3A1-hFc (S239C & S442C), and (b) the second recombinant fusion protein comprises P2A7-hFc (S239C & S442C), E02-hFc (S239C & S442C) or 4D2-hFc (S239C & S442C).
  • the first recombinant fusion protein comprises P3A1-hFc (S239C & S442C)
  • the second recombinant fusion protein comprises P2A7-hFc (S239C & S442C), E02-hFc (S239C & S442C) or 4D2-hFc (S239C & S442C).
  • the recombinant fusion protein dimer of clause 82 wherein: (a) the first recombinant fusion protein comprises P3A1-hFc (S239C & S442C), and (b) the second recombinant fusion protein comprises P2A7-hFc (S239C & S442C), E02-hFc (S239C & S442C) or 4D2-hFc (S239C & S442C). 87.
  • a bi-specific chimeric antigen receptor comprising at least one bi-specific antigen binding molecule as defined in any one of clauses 1 to 32, fused or conjugated to at least one transmembrane region and at least one intracellular domain.
  • a cell comprising a chimeric antigen receptor according to clause 88, which cell is preferably an engineered T-cell.
  • a nucleic acid sequence comprising a polynucleotide sequence that encodes a bi-specific antigen binding molecule, recombinant fusion protein or chimeric antigen receptor according to any one of clauses 1 to 88.
  • a vector comprising a nucleic acid sequence as defined in clause 90, optionally further comprising one or more regulatory sequences.
  • a host cell comprising a vector as defined in clause 91.
  • a method for preparing a bi-specific antigen binding molecule, recombinant fusion protein or chimeric antigen receptor comprising cultivating or maintaining a host cell comprising the polynucleotide of clause 90 under conditions such that said host cell produces the binding molecule, optionally further comprising isolating the binding molecule.
  • a pharmaceutical composition comprising the bi-specific antigen binding molecule, recombinant fusion protein or chimeric antigen receptor of any one of clauses 1 to 88.
  • blood cancers such as lymphomas and leukemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic
  • the cancer is selected from the group consisting of blood cancers such as lymphomas and leukaemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, renal cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, Head and Neck cancer, bladder cancer, oesophageal cancer, stomach cancer or liver cancer.
  • blood cancers such as lymphomas and leukaemias, chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukaemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphomas (
  • a method of assaying for the presence of a target analyte in a sample comprising the addition of a detectably labelled bi-specific antigen binding molecule of any one of clauses 1 to 51 or a recombinant fusion protein of clause 52 to 64, or a recombinant fusion protein dimer of any one of clauses 65 to 87 to the sample and detecting the binding of the molecule to the target analyte. 104.
  • a method of imaging a site of disease in a subject comprising administration of a detectably labelled bi-specific antigen binding molecule as defined in any one of clauses 1 to 51 or a detectably labelled recombinant fusion protein of any one of clause 52 to 64, or a detectably labelled recombinant fusion protein dimer of any one of clauses 65 to 87 to a subject.
  • a method of diagnosis of a disease or medical condition in a subject comprising administration of a bi-specific antigen binding molecule as defined in any one of clauses 1 to 51 or a recombinant fusion protein of clause 52 to 64, or a recombinant fusion protein dimer of any one of clauses 65 to 87.
  • kits for diagnosing a subject suffering from cancer, or a pre-disposition thereto, or for providing a prognosis of the subject's condition comprising detection means for detecting the concentration of antigen present in a sample from a test subject, wherein the detection means comprises a bi-specific antigen binding molecule as defined in any one of clauses 1 to 51, a recombinant fusion protein as defined in any one of clauses 52 to 64, a recombinant fusion protein dimer as defined in any one of clauses 65 to 87, a CAR as defined in clause 88, or a nucleic acid as defined in clause 90, each being optionally derivatized, wherein presence of antigen in the sample suggests that the subject suffers from cancer.
  • the antigen comprises ROR1 protein, more preferably an extracellular domain thereof.
  • the kit is used to identify the presence or absence of ROR1-positive cells and/or PTK7-positive cells in the sample, or determine the concentration thereof in the sample.
  • the kit comprises a positive control and/or a negative control against which the assay is compared.
  • the kit further comprises a label which may be detected. 112.
  • a method for diagnosing a subject suffering from cancer, or a pre-disposition thereto, or for providing a prognosis of the subject's condition comprising detecting the concentration of antigen present in a sample obtained from a subject, wherein the detection is achieved using a bi- specific antigen binding molecule as defined in any one of clauses 1 to 51, a recombinant fusion protein as defined in any one of clauses 52 to 64, a recombinant fusion protein dimer as defined in any one of clauses 65 to 87, a CAR as defined in clause 88, or a nucleic acid sequence as defined in clause 90, each being optionally derivatized, and wherein presence of antigen in the sample suggests that the subject suffers from cancer.
  • a method of killing or inhibiting the growth of a cell expressing ROR1 and/or PTK7 in vitro or in a patient comprises administering to the cell a pharmaceutically effective amount or dose of (i) bi-specific antigen binding molecule as defined in any one of clauses 1 to 51, a recombinant fusion protein as defined in any one of clauses 52 to 64, a recombinant fusion protein dimer as defined in any one of clauses 65 to 87, a nucleic acid as defined in clause 90, or the CAR or cell according to clause 88 or 89, or (ii) of a pharmaceutical composition according to clause 64.
  • 114 A method of killing or inhibiting the growth of a cell expressing ROR1 and/or PTK7 in vitro or in a patient, which method comprises administering to the cell a pharmaceutically effective amount or dose of (i) bi-specific antigen binding molecule as defined in any one of clauses 1 to 51, a recombinant fusion protein as defined in any one of clauses
  • a bi-specific antigen binding molecule comprising an amino acid sequence represented by the formula (II): X-FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4-Y (II) wherein FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 is a ROR1-specific antigen binding molecule according to any one of clauses 1 to 25 X and Y are optional amino acid sequences wherein the ROR1-specific antigen binding molecule is conjugated to a second moiety and wherein the bi-specific antigen binding molecule further comprises a PTK7-specific antigen binding molecule.
  • X or Y are individually either absent or selected from the group comprising an immunoglobulin, an immunoglobulin Fc region, an immunoglobulin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv) 2 , a diabody, a triabody, a tetrabody, a bispecific t-cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, or a scaffold protein.
  • X or Y are individually either absent or selected from the group comprising an immunoglobulin, an immunoglobulin Fc region, an immunoglobulin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv) 2 , a diabody, a tri
  • X or Y are individually either absent or selected from the group comprising an immunoglobin, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv)2, a diabody, a triabody, a tetrabody, a bispecific t-cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, or a scaffold protein.
  • X or Y are individually either absent or selected from the group comprising an immunoglobin, an immunoglobulin Fc region, a fragment of an immunoglobulin Fc region, an Fc heavy chain, a CH2 region, a CH3 region, an immunoglobin Fab region, a
  • bi-specific antigen binding molecule of clause 120 to 122 wherein X or Y are individually either absent or a fragment of an immunoglobulin Fc region engineered to dimerize with the second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs- into-holes (Y-T), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab-arm exchange, SEED technology, BEATtechnology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT and Triomab. 124.
  • X or Y are individually either absent or a fragment of the immunoglobulin Fc region that comprises one or more amino acid substitution suitable for heterodimerization with a second fragment of an immunoglobulin Fc region comprising one or more corresponding amino acid mutation.
  • 125 The bi-specific antigen binding molecule of clause 124 wherein the one or more amino acid substitution is selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.
  • the bi-specific antigen binding molecule of any one of clause 117 to clause 127, wherein the specific antigen binding molecule comprises SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, or SEQ ID NO: 182 and/or SEQ ID NO: 224. 129.
  • the bi-specific antigen binding molecule of any one of clause 117 to clause 127, wherein the specific antigen binding molecule comprises SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, and/or SEQ ID NO: 236. 131.
  • the bi-specific antigen binding molecule of any one of clause 117 to clause 127, wherein the specific antigen binding molecule comprises SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 494, SEQ ID NO: 495 and/or SEQ ID NO: 496. 135.
  • the second moiety is selected from the group comprising an immunoglobulin or antibody, an immunoglobulin Fc region, an immunoglobulin Fab region, a Fab’, a Fv, a Fv-Fc, a single chain Fv (scFv), scFv-Fc, (scFv
  • bi-specific antigen binding molecule of any one of clauses 116 to 134, wherein the second moiety is selected from the group comprising detectable label, dye, toxin, drug, pro-drug, radionuclide or biologically active molecule. 137.
  • the bi-specific antigen binding molecule according to any one of clauses 116 to 134 or 136, wherein the second moiety is at least one toxin selected from the group comprising: • auristatins, • anthracyclines, preferably PNU-derived anthracyclines • maytansinoids, • amanitin derivatives, preferably ⁇ -amanitin derivatives • calicheamicins, • tubulysins • duocarmycins • radioisotopes - such as an alpha-emitting radionuclide, such as 227 Th and 225 Ac label • liposomes comprising a toxic payload, • protein toxins • taxanes • pyrrolbenzodiazepines and dimers thereof • indolinobenzodiazepine pseudodimers • spliceosome inhibitors • CDK11 inhibitors • Nicotinamide phosphoribosyltransferase inhibitors (NAMPTi) • Pyridinobenzodiazepines and
  • a target-binding molecule-drug conjugate comprising (a) a bi-specific antigen binding molecule according to any one of clauses 1 to 51, or a recombinant fusion protein of any one of clauses 52 to 64, or a recombinant fusion protein dimer of any one of clauses 65 to 87, and (b) at least one toxin. 139.
  • the target-binding molecule-drug conjugate of clause 130 or clause 139 comprising (b) an anthracycline (PNU) derivative, wherein the target-binding molecule-drug conjugate has the structure of formula (III): wherein [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-C
  • the target-binding molecule-drug conjugate of clause 140 wherein the target-binding molecule-drug conjugate of formula (III) comprises [L1], [L2] or [L1] and [L2].
  • PAB p-aminobenzyloxycarbonyl
  • 143. The target-binding molecule-drug conjugate of clause 140, wherein the target-binding molecule-drug conjugate has a structure selected from: 144.
  • the target-binding molecule-drug conjugate of clause 140 comprising (b) an anthracycline (PNU) derivative, wherein the target-binding molecule-drug conjugate has the structure of formula (IV): wherein [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof; [Z] is a linker derived from a reactive group used to conjugate the anthracycline (PNU) derivative and the target-binding molecule; and Y comprises a bi-specific antigen binding molecule according to any one of clauses 1 to 51, or a recombinant fusion protein of any one of clauses 52 to 64, or a recombinant fusion protein dimer of any one of clauses 65 to
  • PNU anthracycline
  • [X] is an optional spacer selected from the group comprising substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, one or more heteroatoms, polyethylene glycol, or a combination thereof;
  • [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), a peptide, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB), Val-
  • EXAMPLE 1 Generation of the anti-ROR1 VNAR B1 loop library sequences B1 protein library design. To gain a better understanding of the interaction between B1 and ROR1, we solved the crystal structure of B1 in complex with the ROR1 Ig domain (data not shown). This crystal structure informed which positions to change in the protein library that was expressed and screened.
  • B1 Tryptophan residues at positions 88 and 94 to Alanine caused loss of function or expression of the protein. From the crystal structure it was observed that these residues in the CDR3 loop appear to be important for ROR1 binding.
  • a B1 loop library was therefore designed to modify the biophysical properties of the protein through changing selected positions within the CDR1 and CDR3 regions. The set of mutations made at each particular loop position was informed from the structural analysis of the B1:ROR1 complex, with a view to changing the biophysical properties whilst maintaining structural integrity and high affinity binding.
  • Library construction Sequence and loop library design of B1 are shown in Figure 1. Library was synthesised by controlled mutagenesis of CDR1 and CDR3.
  • Residues 30, 32, 88, 94 and 95 located within CDR loops were randomised.
  • Libraries construction B1 loop library DNA was amplified by PCR using specific primers to introduce SfiI restriction sites for cloning into pEDV1 phagemid vector.
  • Library DNA ligated into pEDV1 was transformed into electrocompetent TG1 E.coli (Lucigen). The library size was calculated to be 8 x 104. 84 single clones were picked and sequenced as a quality control of the library. One sequence has been found to be WT B1 clone. In total 70 unique clones based on CDR1 and CDR3 diversity were identified.
  • VNARs were tested at various concentrations and the Ka (M -1 s -1 ), Kd (s -1 ) and KD (nM) values were determined using Octet Data Analysis High Throughput software (ForteBio) for Biolayer Interferometry. Table 5. summarise the BLI data for the affinity of these molecules for human and mouse ROR1.
  • VNARs For expression as intein fusions, DNA encoding VNARs was optimised for E. coli expression (GeneArt, Thermo) and cloned in frame into an intein expression vector. This results in a gene encoding the VNAR protein of interest fused to an engineered intein domain which in turn is fused to a chitin binding domain (CBD) to enable purification on a chitin column.
  • CBD chitin binding domain
  • VNAR intein fusion protein was purified from clarified cell lysate by immobilising on chitin beads (NEB, S6651).
  • VNARs were washed extensively with lysis buffer followed by cleavage buffer (50mM sodium phosphate pH6.9, 200mM NaCl) and VNARs released from the beads by overnight chemical cleavage in 400mM dioxyamine, or O,O’- 1,3-propanediylbishydroxylamine, or 100mM cysteine or cysteamine to generate the corresponding C- terminal aminoxy, C-terminal cysteine or C-terminal thiol derivative of the VNARs. Cleaved VNAR supernatant was then further purified by SEC (Superdex7526/60 GE healthcare) and / or IMAC (HisTrap HP, GE Healthcare).
  • SEC Superdex7526/60 GE healthcare
  • IMAC HisTrap HP, GE Healthcare
  • Cells were re-suspended in ice-cold PBS/2%FCS in 15ml tubes and centrifuged at 1500rpm for 5 mins at 4 °C. Supernatant was removed and the cell pellet re-suspended in PBS/2%FCS. A cell count was performed using a Z1 Coulter Particle Counter (Beckman Coulter) or Chemometec Nucleocounter NC-202 and 5 x 10 ⁇ 5 cells were aliquoted per test sample into a 96 well plate. Cells were incubated with 100 ⁇ l of test agents at a range of concentrations, plus controls for 1hr on ice. The sample plate was centrifuged at 2000 rpm for 5mins.
  • the supernatant was removed and a wash performed by re-suspending the cell pellets in 0.25mL of ice-cold PBS/2%FCS using a multichannel pipette. Samples were again centrifuged at 2000rpm for 5min at 4°C. Supernatant was removed and two further washes performed as described. After the final wash and centrifugation step, excess liquid was removed by blotting the plate on tissue paper. Binding of VNARs was determined by adding 100 ⁇ l of anti-x6His tag Ab (Abcam) per cell pellet sample as appropriate and incubated on ice for 30mins. Wash steps were performed as described previously.
  • PE-anti-mouse antibody JIR was used to detect binding of the VNAR (His6 tagged) agents and corresponding drug-conjugates by incubating with the appropriate samples for 30min on ice in the dark. Wash steps were performed as described previously. All cell pellets were finally re-suspended in 0.3ml of ice-cold PBS/2%FCS and left on ice in the dark prior to analysis on a Cytek Biosciences Guava EasyCyte HT or Thermo Fisher Attune NxT flow cytometer.
  • the loop library variants bind to the ROR1 hi human cancer cell-line A549 but not to the ROR1 low human cancer cell-line A427.2V is a control VNAR sequence, derived from a na ⁇ ve VNAR library, so is representative of this protein class but has no known target.
  • EXAMPLE 2 Humanisation and further engineering of B1 loop library variants Humanised sequence derivatives of three lead ROR1 binding B1 loop library VNARs were generated using two different strategies. Humanised sequences were designed based on the human germ line V ⁇ 1 sequence, DPK-9. For example, in P3A1 V1 the framework regions 1, 3 and 4 of the VNAR were mutated to align with the framework regions of DPK-9.
  • the second strategy involved grafting the binding loops of the ROR1 binding VNARs onto a previously humanised VNAR framework (Kovalenko et al JBC 2013288(24) 17408-17419; WO2013/167883). But with further positions engineered based on the structure of the VNAR B1 in complex with the ROR1 Ig domain. Additional sites of engineering include amino acid changes in the CDR1, HV2 and HV4 regions of the protein. Similarly, a humanised variant of B1 was developed using this approach, which accordingly contains amino acid changes in its CDR1, HV2 and HV4 regions as well as the framework regions.
  • So B1G4 is, by de facto, a loop library derivative of B1 or a loop library variant of humanised variants of B1 whereby the CDR1, HV2, HV4 and CDR3 sequences are the same as in the parental protein.
  • humanised / grafted loop library VNAR sequences are below: G3CP G4 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTL TISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71)
  • G3CP V15 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSL RISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK SEQ ID NO: 72
  • DNA encoding the humanised constructs was codon optimised for expression in E. coli and synthesised by GeneArt (Thermo). All humanised sequences were generated with the following C terminal His 6 tag: QASGAHHHHHH (SEQ ID NO: 102) G4 sequences were made without an additional C-terminal tag. DNA encoding these proteins was sub cloned into the intein expression vectors, expressed in E. coli and purified as described previously in “Typical method for expression of VNAR intein fusion proteins” section. Humanised ROR1 binding VNAR variants demonstrated high affinity binding to human ROR1 by BLI, good thermal stability and little evidence of aggregation by SEC.
  • BLI was performed as described previously using human ROR1 ECD – Fc immobilised to the chip surface. SEC was performed as previously described.
  • Thermal stability assays used Applied Biosystems StepOne Real Time PCR system with the Protein Thermal ShiftTM dye kit (Thermo). The assay mix was set up so that the protein was at a final concentration of 20 ⁇ M in 20 ⁇ L.5 ⁇ L of Thermal ShiftTM buffer was added alongside 2.5 uL 8x Thermal ShiftTM Dye. Assays were run using the StepOne software and data analysed using Protein Thermal ShiftTM software. All data are from first derivative analysis. BLI data for hROR1 binding and thermal stability by protein thermal shift is shown in Table 6.
  • Table 6 Thermal stability and hROR1 binding data for humanised VNAR loop variants Either grafting the HV and CDR loops of G3CP, 1H8 and C3CP onto a humanised VNAR framework coupled with additional mutations in the CDR1, HV2 and HV4 regions or substituting VNAR framework sequences with regions from the human DPK-9 sequence, yielded substantially engineered proteins that are stable, monomeric and maintain high affinity binding to hROR1.
  • EXAMPLE 3 Generation of the anti-ROR1 VNAR P3A1 G1 loop library sequences
  • Library design P3A1 G1 is a humanised version of the ROR1 binding VNAR P3A1.
  • the P3A1 G1 loop library was designed to improve ROR1 binding affinity of this humanised variant via randomisation of CDR1, HV2 and HV4 regions without any changes within frameworks. Choice of mutations was made based on the data analysis of VNAR sequences from Squalus acanthus. Sequence of P3A1 G1 and library design are shown in Figure 4. Library was synthesised by controlled mutagenesis of CDR1, HV2 and HV4. Residues 26-33, 44-52 and 61-65 located within CDR1, HV2 and HV4 loops respectively were changed to selected amino acids as specified in Fig.4 resulting in total library diversity of 8.2x10 6 combinations. Libraries construction.
  • P3A1 G1 library DNA was amplified by PCR using specific primers to introduce SfiI restriction sites for cloning into pEDV1 phagemid vector. This introduces an additional Ser residue into CD1.
  • Library DNA ligated into pEDV1 was transformed into electrocompetent TG1 E.coli (Lucigen). The library size was calculated to be 2 x 10 8 .192 single clones were picked and sequenced as a quality control of the library. Screening of P3A1 G1 library for antigen specific VNAR sequences. Recombinant human ROR1 protein was used for selections and screening of the P3A1 G1 library.
  • Selection on immunotubes consists of 2 rounds of panning with constant antigen concentration of 2 ng/ml. Following the selection process, outputs were screened for antigen-specific binding by monoclonal phage and periplasmic extract ELISAs against human or mouse ROR1.95% of monoclonal phage displaying the VNARs were specific to human and mouse ROR1 from selections with antigen directly immobilised to the immunotube and 4% for selections on biotinylated antigen immobilised on pre-decorated streptavidin-coated beads. In total 9 unique sequences from each selection campaign were expressed and analysed for binding and selectivity (Table 7 and 8).
  • Binding of P3A1 G1 loop variants to hROR1 by ELISA The binding of P3A1 G1 loop variants to human ROR1 was initially assessed by ELISA.
  • ELISA method as follows. Wells coated with 100ng of ROR1-hFc antigen and incubated, covered, at room temperature for 2hr. Plates washed 3x 400ul per well with PBST (PBS + 0.05% Tween 20 (v/v)), then blocked with 4% skimmed milk powder (w/v) in PBST for 1 hour at 37°C. Plates washed as before plus additional wash in PBS alone. HisMyc-tagged binding proteins were diluted in 4% milk PBST and incubated overnight at 4 °C.
  • Figure 5 shows the relative binding of different variants to human ROR1 with sequences NAG8.S, AF7.S, NAC6.S and AE3.S showing the strongest signal for binding.
  • the same ELISA method was also used to compare binding of variants NAG8.S, AF7.S, NAC6.S and AE3.S to human ROR1 with that of the parental P3A1 G1 sequence.
  • the dose response data shown in Figure 6 shows that these loop library sequences bind stronger to human ROR1 than the parental P3A1 G1 protein.
  • NAC6.S and AE3.S bound to mouse ROR1 Expression of P3A1 G1 loop library variants as intein fusion proteins
  • P3A1 G1 loop variant VNARs NAG8.S, NAC6.S and AE3.S were re-expressed using intein technology but with a Ser deletion from the CDR1 loop.
  • Expression as intein fusions was performed as described above with either a His tag QACKAHHHHHHG (SEQ ID NO: 163) or HisMyc tag QACKAHHHHHHGAEFEQKLISEEDLG (SEQ ID NO: 164) incorporated at the C-terminus of the VNAR domain.
  • VNARs were released from the beads by overnight chemical cleavage in 400mM dioxyamine, or O,O’-1,3-propanediylbishydroxylamine, or 100mM cysteine or cysteamine to generate the corresponding C-terminal aminoxy, C-terminal cysteine or C-terminal thiol derivative of the VNARs.
  • Cleaved VNAR supernatant was then further purified by SEC (Superdex7526/60 GE healthcare) and / or IMAC (HisTrap HP, GE Healthcare) to give the proteins NAG8, NAC6 and AE3.
  • the assay mix was set up so that the protein was at a final concentration of 20 ⁇ M in 20 ⁇ L in PBS pH 7.4.2.5 ⁇ L 8x Thermal ShiftTM Dye was added.
  • Assays were run using the StepOne software and data analysed using Protein Thermal ShiftTM software. All data are from first derivative analysis with the Tm values detailed in Table 9.
  • Size exclusion chromatography The monomericity and biophysical properties of P3A1 loop variants were assessed by size-exclusion chromatography (SEC) using an analytical SEC column (Superdex 75 increase 10/300 GL). Chromatography was carried out in PBS pH 7.4. The % monomericity and SEC elution volumes run under identical conditions are shown in Table 9.
  • Table 9 Summary of ROR1 binding and physical properties of P3A1 G1 variants NAG8, NAC6 and AE3 EXAMPLE 4 – VNAR Reformatting as multimers
  • ROR1 binding loop variant VNARs were successfully reformatted into hetero dimers and trimers by genetic fusion using different GlySer based linkers to generate bi-specific binders, ROR1 bi-paratopic binders and ROR1 bi-paratopic bi-specific binders.
  • bi-specific binders were developed by combining ROR1 loop-variant VNAR binders with the humanised VNAR BA11, which binds with high affinity to serum albumins, using a PGVQPSPGGGGGS (SEQ ID NO: 96) linker Proteins were expressed with a C-terminal tag QACKAHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) or QACKAHHHHHH (SEQ ID NO: 104) to aid purification and characterisation.
  • This tag also contains a single cysteine residue to facilitate site-selective bioconjugation of payloads to the proteins using thiol mediated chemical coupling strategies
  • Binding kinetics were determined using Biolayer interferometry (K2 Octet instrument / Pall ForteBio) as previously described.
  • ROR1-hFc, (extracellular domain) and HSA were immobilised in sodium acetate pH5 buffer to AR2G sensors using amine coupling.
  • VNAR-based molecules were tested at various concentrations and the Ka (M -1 s -1 ), Kd (s -1 ) and KD (nM) values were determined using the Octet data analysis HT software (Pall ForteBio).
  • Binding kinetics for hROR1 binding were also performed with saturating levels of HSA (200 nM) in the baseline, association and dissociation conditions. Binding to the ROR1hi A549 cancer-cell lines was determined by flow cytometry. A dose response was performed and the KDapp for cell-surface ROR1 binder determined using the change in median fluorescence intensity (background corrected) as a function of VNAR concentration. The characterisation of these bi-specific VNARs is shown in Table 10. Table 10: Characterisation of bi-specific proteins containing ROR1 VNAR loop library variants Bi-specific VNAR binders were further modified through conjugation to the single cysteine residue in the C-terminal tag.
  • VNAR domains were joined together using a PGVQPAPGGGGS (SEQ ID NO: 90) linker and proteins were expressed with a C-terminal tag QACKAHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) or QACKAHHHHHH (SEQ ID NO: 104) to aid purification and characterisation.
  • This tag also contains a single cysteine residue to facilitate site-selective bioconjugation of payloads to the proteins using thiol mediated chemical coupling strategies
  • Binding kinetics were determined using Biolayer interferometry (K2 Octet instrument / Pall ForteBio) as previously described.
  • ROR1-hFc extracelluar domain
  • VNAR-based molecules were tested at various concentrations and the Ka (M -1 s -1 ), Kd (s -1 ) and KD (nM) values were determined using the Octet data analysis HT software (Pall ForteBio).
  • bi-specific VNARs The characterisation of these bi-specific VNARs is shown in Table 11.
  • Table 11 Characterisation of bi-paratopic proteins containing ROR1 VNAR loop library variants Bi-paratopic binders show increased affinity for binding ROR1 as compared to the individual ROR1 binding monomers.
  • the constructs containing BA11 are examples of bi-paratopic bi-specific protein binders.
  • several bi-specific and bi-paratopic VNAR-based binders were developed by combining ROR1 loop-variant VNAR binders with the humanised VNAR BA11 or by combining different ROR1 loop-variant VNAR binders using a PGVQPCPGGGGGS (SEQ ID NO: 177) linker.
  • This linker sequence also contains a single cysteine residue to facilitate site-selective bioconjugation of payloads to the proteins, in this linker, using thiol mediated chemical coupling strategies.
  • Proteins were expressed with a C-terminal tag QASGAHHHHHH (SEQ ID NO: 102) or QACKAHHHHHH (SEQ ID NO: 104) to aid purification and characterisation.
  • Table 12 Characterisation of bi-specific and bi-paratopic proteins containing ROR1 VNAR loop library variants with cysteine containing linker sequences Bi-specific VNAR binders were further modified through conjugation to the single cysteine residue in the linker sequence.
  • VNAR loop variants were genetically fused via standard [G4S]3 linkers to engineered hIgG1 Fc domains that contained a cysteine substitution in the hIgG1 Fc sequence, S239C (EU numbering).
  • VNAR Fc fusion proteins were transiently expressed as secreted protein in CHO K1 cells and purified from the media using MabSelectTM SuReTM (Evitria, Switzerland). Purified proteins were exchanged into PBS pH 7.4 or PBS + 100 mM Arg pH 7.4 and analysed by SEC (AdvanceBio, Agilent, running buffer DPBS pH 7.4), SDS PAGE and mass spectrometry to confirm sequence and protein integrity. Binding kinetics were determined using a Pioneer Surface Plasmon Resonance (SPR) instrument (SensiQ/Pall ForteBio), or the Biolayer Interferometry (BLI) Octet K2 system (ForteBio).
  • SPR Surface Plasmon Resonance
  • BLI Biolayer Interferometry
  • ROR1-hFc fusion proteins were immobilised in sodium acetate pH5 buffer to COOH2 chips or AR2G sensors using amine coupling.
  • VNAR-Fc molecules were tested at various concentrations and the Ka (M -1 s -1 ), Kd (s -1 ) and KDapp (nM) values were determined using Octet Data Analysis High Throughput software (ForteBio) for Biolayer Interferometry.
  • the kinetic parameters for binding were determined by immobilising the VNAR-hFc fusion onto AHC sensors.
  • VNAR-Fc fusions Binding of the VNAR-Fc fusions to ROR1 on the surface of cancer cell lines was measured by flow cytometry using the methods described previously, with binding of VNAR-hFc fusion molecules determined by adding 100 ⁇ L of PE-anti-human antibody (JIR) and incubating on ice for 30mins. KDapp values were calculated from the increase in fluorescence intensity as a function of VNAR-hFc concentration.
  • Figure 8 shows the binding of different VNAR-Fc fusions to the ROR1 hi A549 lung adenocarcinoma cells.
  • G3CP-hFc, G3CPG4- hFc and B1G4-hFc and parental B1-hFc were incubated at 2 mg / mL in sterile PBS buffer pH 7.4 containing 0.05% sodium azide at 37°C for 96h.
  • VNAR loop variants were genetically fused via standard [G4S]3 linkers to hIgG1 Fcs engineered for heterodimerisation (Ridgway 1996 Protein Engineering 9(7):617-21).
  • the Knob variant has a tryptophan substitution at position 336 (T366Y) and the Hole variant has a Threonine substitution at position 407 (Y407T) (EU numbering).
  • This approach was used to generate bi-paratopic ROR1 binders where one arm comprises a VNAR loop variant and the other arm comprises a second ROR1 binding VNAR.
  • a cysteine substitution was incorporated in the hIgG1 Fc sequence [S239C (EU numbering)] of both Knob and Hole variants to facilitate bioconjugation with different payloads.
  • the VNAR Fc fusion proteins were transiently co-expressed as secreted protein in CHO K1 cells and purified from the media using MabSelectTM SuReTM (Evitria, Switzerland).
  • G3CP hFc(S239C+Y407T) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSL RIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKT HTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVD
  • Binding to ROR1 was determined using Biolayer interferometry (K2 Octet instrument / Pall ForteBio) as previously described. For BLI experiments ROR1-hFc, (extracelluar domain) was immobilised on the sensors. Data is shown in Table 15. Table 15: MS characterisation of bi-paratopic VNAR-Fc fusions and binding to human ROR1 by BLI Binding of the bi-paratopic VNAR-Fc fusions to ROR1 on the surface of cancer cell lines was measured by flow cytometry using the methods described previously. Binding of VNAR-hFc fusion molecules determined by adding 100 ⁇ L of PE-anti-human antibody (JIR) and incubating on ice for 30mins.
  • JIR PE-anti-human antibody
  • KDapp values were calculated from the increase in fluorescence intensity as a function of VNAR-hFc concentration.
  • Figure 10 shows the binding of the bi-paratopic VNAR-Fc fusions to the ROR1 hi A549 lung adenocarcinoma cells and the ROR1 low A427 cells.
  • G3CP-P3A1 hFc (S239C+KIH) and G3CPG4- P3A1 hFc (S239C+KIH) bind strongly to A549 cells with KD app of 0.06 nM and 0.20 nM respectively but show little binding to A427 cells.
  • VNAR-hFc drug conjugates Another approach for generating ADCs is to engineer cysteine substitutions or additions at positions on the light and heavy chains of antibodies and these cysteines provide reactive thiol groups for site specific labelling (Junutula 2008 Nature Biotechnology 26, 925 – 932, Jeffrey 2013, Sutherland 2016).
  • the anti ROR1 loop library VNAR -hFc fusions were generated with an additional cysteine engineered into the Fc region as described previously, which enabled site specific labelling with maleimide derivatives of fluorescent labels (AF488) and cytotoxic drugs (MA PEG4 vc PAB EDA PNU 159682 and MA PEG4 va PAB EDA PNU 159682) (Figure 11).
  • Generation of VNAR-hFc – drug conjugates Using a partial reduction, refolding and labelling method adapted from the literature [Junutula et al, 2008 Nat Biotech, Jeffrey et al, 2013 Bioconj Chem], these proteins were site specific labelled with the maleimide PNU derivatives.
  • VNAR hFc solutions were prepared in PBS +100mM L- Arginine pH7.4 with 1mM EDTA. 20 molar equivalents TCEP added and incubated at 4°C for a minimum of 48 hours. 30 molar equivalents DHAA added, pH adjusted to 6.5 and incubated at room temperature for 1 hour. Refolded VNAR Fc S239C was extensively dialysed or buffer exchanged into PBS +50mM L-Arginine and quantified by UV before reacting with 4 or 5 molar equivalents maleimide PNU solution, room temperature overnight. Conjugates were purified by SEC and analysed by analytical HIC, analytical SEC, and LC-MS.
  • Table 16 summaries the conjugates prepared. Table 16: Summary of characteristics of VNAR-PNU conjugates SDS-PAGE and mass spectrometry analysis of the final conjugates determined that the labelling had proceeded in a quantitative fashion to give highly pure homogenous protein drug conjugates with drug to antibody ratio (DAR) of 2. Binding of VNAR-hFc – drug conjugates to hROR1 by ELISA The binding of G3CP hFc and G3CPG4 hFc and their respective drug conjugates to human ROR1 was assessed by ELISA. In brief, ELISA method as follows. Wells were coated with 100ng of ROR1-his antigen and incubated, covered, at room temperature for 2hr.
  • PA-1 – human ovarian cancer cell line EMEM, 10% hiFCS
  • PA-1 ROR1 ko - human ovarian cancer cell line with ROR1 knock-out EMEM, 10% hiFCS HEK293 – human embryonic kidney cell line: EMEM, 10% FCS HEK293 stably transfected with human ROR1 (HEK293.hROR1) – human embryonic kidney cell line stably expressing hROR1: EMEM, 10% FCS Figure 13 shows dose response curves, with corresponding IC50 values (Table 17), for cell-killing of the ROR1 positive PA-1 ovarian cancer cells and PA-1 ROR1 ko cells by G3CP-hFc-PNU conjugates (PEG4-vc PAB EDA PNU159682 and PEG4-va-EDA-PNU159682) and G3CPG4-hFc-PNU conjugate (PEG4-vc PAB EDA PNU159682).
  • PA-1 ROR1 ko is PA-1 cancer cell-line where ROR1 expression has been knocked out.
  • Table 17 Calculated IC50 values (nM) for the cell-killing of PA-1 and PA1 ROR1 ko cancer cells by G3CP-hFc conjugates.
  • the ROR1 targeting VNAR-hFc conjugates show potent killing of PA-1 cell-lines, which is abrogated upon knockdown of the ROR1 receptor. There is > 100 fold window in the IC50 values for both of the G3CP-hFc PNU conjugates.
  • Figure 18 shows dose response curves, with corresponding IC50 values (Table 18), for cell-killing of the ROR1 low HEK293 cells and HEK293 cells stably transfected with human ROR1 (HEK293.hROR1) by G3CP-hFc-PNU, G3CPG4-hFc-PNU and 2V-hFc-PNU conjugates (PEG4-vc PAB EDA PNU159682).
  • 2V is a control VNAR sequence, derived from a na ⁇ ve VNAR library, so is representative of this protein class but has no known target.
  • Table 18 Calculated IC 50 values (nM) for the cell-killing of HEK293 WT and HEK293.hROR1 cells by G3CP-hFc, G3CPG4-hFc and 2V-hFc conjugates.
  • the ROR1 targeting VNAR-hFc conjugates show potent killing of the HEK293.hROR1 cell-line, which is stably transfected with the ROR1 receptor, but not the ROR1 low wild-type HEK293 cells.
  • Example 7 In vivo efficacy of protein-drug conjugates in patient-derived xenograft model of Triple Negative Breast Cancer (TNBC) An efficacy study in the ROR1+ HBCx-28 patient-derived TNBC xenograft model was performed by XenTech (Paris).
  • mice Outbred athymic (nu/nu) female mice (HSD: Athymic Nude-Foxn1 nu ) were implanted subcutaneously with tumours of the same in vivo passage. Mice were monitored until the tumour implants reached the study volume recruitment criteria of 60–200 mm 3 , preferably 75–196 mm 3 in a sufficient number of animals. Mice were randomised to treatment groups such that there was no statistical difference between tumour volumes in each group. Randomisation was designated as Day 0 of the experiment.
  • mice were treated with vehicle or with the protein-drug conjugates B1-hFc-vc-PAB-EDA-PNU, B1G4- hFc-vc-PAB-EDA-PNU, G3CP-hFc-vc-PAB-EDA-PNU or G3CPG4-hFc-vc-PAB-EDA-PNU by single dose 0.3 mg / kg i.v. injection on day 2. All mice pre-primed with mouse IgG 20h before first PDC dose. Tumour volume was evaluated by measuring perpendicular tumour diameters, with a calliper, three times a week during the experimental period.
  • Figure 14 shows the effect of the protein-drug conjugates on tumour growth versus vehicle control. All protein drug conjugates were well tolerated and show highly statistically significant in vivo efficacy in this ROR1+ TNBC PDX model.
  • B1G4-hFc-vc-PAB-EDA-PNU retains comparable levels of in vivo efficacy to B1-hFc-vc-PAB-EDA-PNU (data not shown).
  • Loop library variants G3CP-hFc-vc-PAB-EDA- PNU and G3CPG4-hFc-vc-PAB-EDA-PNU show improved efficacy over the parental B1 fusion with complete and durable regressions observed for both loop library variants for the 0.3 mg / kg single dose regimen.
  • An efficacy study was also performed in the ROR1+ HBCx-10 patient-derived TNBC xenograft model by XenTech (Paris).
  • mice Outbred athymic (nu/nu) female mice (HSD: Athymic Nude-Foxn1 nu ) were implanted subcutaneously with tumours of the same in vivo passage. Mice were monitored until the tumour implants reached the study volume recruitment criteria of 75–196 mm 3 in a sufficient number of animals. Mice were randomised to treatment groups such that there was no statistical difference between tumour volumes in each group. Randomisation was designated as Day 0 of the experiment.
  • mice were treated with vehicle or with the protein-drug conjugates B1-hFc-vc-PAB-EDA-PNU, B1G4-hFc-vc-PAB-EDA-PNU, G3CP-hFc-vc-PAB-EDA-PNU or G3CPG4-hFc-vc-PAB-EDA-PNU or G3CP-hFc-va-EDA-PNU at a dose of 0.3 mg / kg i.v. injection, three times, four days apart (3 x Q4D on day 2, 6 and 10). All mice were pre-primed with mouse IgG 20h before first PDC dose.
  • Tumour volume was evaluated by measuring perpendicular tumour diameters, with a calliper, three times a week during the experimental period.
  • Figure 19 shows the effect of the protein-drug conjugates on tumour growth versus vehicle control.
  • Bi-paratopic VNAR-hFc drug conjugates Bi-paratopic ROR1 binding proteins
  • G3CP-P3A1 hFc S239C+KIH
  • G3CPG4-P3A1 hFc S239C+KIH
  • MC-vc-PAB-MMAE or MA-PEG4-vc-PAB-EDA-PNU159682 using a partial reduction, refolding and labelling method as described in Example 6.
  • Conjugates were purified by SEC and analysed by analytical HIC, analytical SEC, and LC-MS. Table 19 summarizes the conjugates prepared.
  • Table 19 Summary of characteristics of Bi-paratopic VNAR-PNU conjugates Binding of the bi-paratopic VNAR-Fc-PNU conjugates to ROR1 on the surface of cancer cell lines was measured by flow cytometry using the methods described previously. Binding of VNAR-hFc-PNU molecules was determined by adding 100 ⁇ L of PE-anti-human antibody (JIR) and incubating on ice for 30mins. KDapp values were calculated from the increase in fluorescence intensity as a function of VNAR- hFc concentration.
  • JIR PE-anti-human antibody
  • Figure 20 a and b shows the binding of the bi-paratopic VNAR-Fc-PNU conjugates (PEG4-vc PAB EDA PNU159682) to the ROR1 hi A549 lung adenocarcinoma cells and the ROR1 low A427 cells along with the corresponding mono-paratopic PNU conjugates.
  • G3CP-P3A1 hFc (S239C+KIH)-PNU and G3CPG4-P3A1 hFc (S239C+KIH)-PNU bind strongly to A549 cells with KD app of 0.92 nM and 1.83 nM respectively but show little binding to A427 cells.
  • G3CP-P3A1 hFc (S239C+KIH)-PNU demonstrates a greater level of saturation binding to A549 cells as compared to the corresponding G3CP-hFc-PNU and P3A1-hFc-PNU conjugates ( Figure 20a).
  • G3CPG4-P3A1 hFc (S239C+KIH)-PNU demonstrates a greater level of saturation binding to A549 cells as compared to the corresponding G3CPG4-hFc-PNU and P3A1-hFc-PNU conjugates ( Figure 20b)
  • Figure 20b In vitro cell viability assays for cancer cells treated with anti ROR1 Bi-paratopic VNAR drug conjugates Cell Titre Glo assays were performed as described in Example 6. Cells were incubated with VNAR-hFc conjugates at 37°C, 5% CO2 for 96 hours and the % of cell viability determined as a function of dose response.
  • the % of control data was plotted against Log [Treatment] concentration and the IC50 value derived using non-linear regression fitting in GraphPad Prism software.
  • the following cell lines were used PA-1 (ECACC 90113101) – human ovarian cancer cell line: EMEM, 10% hiFCS PA-1 ROR1 ko - human ovarian cancer cell line with ROR1 knock-out: EMEM, 10% hiFCS Kasumi-2 (ACC 526; DSMZ-German Collection of Microorganisms and Cell Cultures GmbH)– human B cell precursor leukaemia cell line: RPMI 1640, 10% hiFCS MHH-ES1 (ACC 167; DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) – human Ewings sarcoma cell line: RPMI 1640, 10% hiFCS Figure 21 shows dose response curves for cell-killing of the ROR1 positive PA-1 ovarian cancer cells and PA-1 ROR1 ko cells by G3CP-P3A1
  • PA-1 ROR1 ko is PA-1 cancer cell- line where ROR1 expression has been knocked out.
  • Table 20 shows IC50 values, for cell-killing of Kasumi-2, MHH-ES1, PA-1 and PA-1 ROR1 ko cells by G3CP-P3A1 hFc (S239C+KIH)-PNU and G3CPG4-P3A1 hFc (S239C+KIH)-PNU conjugates (PEG4-vc PAB EDA PNU159682).
  • the cell-surface ROR1 receptor number was determined for each cell-line by flow cytometry using BD Biosciences Quantibrite beads.
  • Table 20 Calculated IC50 values (nM) for the cell-killing of PA-1 and PA1 ROR1 ko cancer cells by G3CP-P3A1 hFc (S239C+KIH)-PNU and G3CPG4-P3A1 hFc (S239C+KIH)-PNU conjugates.
  • the ROR1 targeting bi-paratopic VNAR-hFc conjugates show potent killing of the ROR1+ cancer cell- lines, but not the ROR1 negative PA1.ROR1ko cell-line.
  • EXAMPLE 9 In vivo efficacy of bi-paratopic protein-drug conjugates in patient-derived xenograft model of Triple Negative Breast Cancer (TNBC)
  • TNBC Triple Negative Breast Cancer
  • mice were treated with vehicle or with the Bi-paratopic protein-drug conjugates G3CP-P3A1 hFc (S239C+KIH)-vc-PAB-EDA- PNU and G3CPG4-P3A1 hFc (S239C+KIH) vc-PAB-EDA-PNU either by single dose 0.3 mg / kg i.v. injection on day 2 or by 3 x 0.1 mg / kg i.v. injections four days apart (3 x Q4D on day 2, 6 and 10). All mice were pre-primed with mouse IgG 20h before first PDC dose.
  • Tumour volume was evaluated by measuring perpendicular tumour diameters, with a caliper, three times a week during the experimental period.
  • Figure 22 shows the effect of the protein-drug conjugates on tumour growth versus vehicle control.
  • VNAR BA11 is an example of a HSA-binding VNAR.
  • Bi-specific molecules comprising a HSA-binding VNAR (such as BA11) and another specific binding molecule are discussed.
  • ROR1 x CD3 bispecific sequences combining N-terminal ROR1 VNARs with a C-terminal anti-CD3 scFv (clone OKT3) via 2 different length G4S linkers were expressed in CHO cells (Evitria) and purified by IMAC (HisTrap Excel, GE Healthcare) followed by SEC (Superdex 200 26/60, GE Healthcare).
  • biparatopic ROR1 x CD3 bispecific sequences combining N-terminal biparatopic ROR1 VNARs with the C-terminal anti-CD3 scFv were also expressed in CHO (Evitria).
  • CD3 BiTE-like approach examples of CD3 binding sequences for use as an ROR1 VNAR bispecific Anti CD3 scFv clone OKT3 (WO 2014028776 Zyngenia) and orientation and humanised derivatives thereof VH-[G4S]3-VL DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGG GGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSG SGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS (SEQ ID NO: 149) Humanised anti CD3 scFv UCHT1 (Arnett et al PNAS 2004101(46
  • engineered T cells expressing such a CAR may also be generated, which may then be used in, for example, adoptive cell therapy.
  • a nucleic acid construct encoding a ROR1-specific CAR may be produced.
  • the ROR1-specific CAR may include an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising the ROR1-specific antigen binding molecule described herein.
  • the nucleic acid construct may then be incorporated into a viral vector, such as a retroviral vector (e.g., a lentiviral vector).
  • T cells may be isolated from a patient in need of treatment, which may then be modified to express the nucleic acid construct encoding the CAR, for example by retroviral transfection or gene-editing using approaches such as CRISPR-CAS-9.
  • the engineered T cells may then be re-infused into the patient in order to treat the condition, such as treatment of cancer.
  • the phage was PEG-precipitated (20% PEG/2.5 M NaCl) twice from the bacterial culture and the resulting phage pellets were resuspended in 1 ml PBS. 4.
  • Library phage was de-selected by incubation with Dynabeads for 1 h rotating at room temperature and then added to the antigen-coated beads. 6.
  • Phage binders were detected using HRP-conjugated anti-M13 antibody (GE Healthcare, 27942101) and periplasmic protein was detected using HRP-conjugated anti-HIS antibody (Sigma A7058).
  • Recombinant protein expression and IMAC purification 1.
  • E.coli set up a culture of bacterial cells transformed with plasmid encoding VNAR sequence in 10 ml of 2xTY media. Grow overnight at 37 ⁇ C at 250 rpm. 2. Dilute overnight culture 1:50 in TB media supplemented with phosphate salts, 1% glucose and 100 ⁇ g/ml ampicillin. Incubate at 37 ⁇ C, 250 rpm all day or as long as possible. 3.
  • Pellet the cells by centrifugation at 4,000 x g for 15 min at 20 ⁇ C 4. Resuspend the cells in the same volume of TB media (same supplements as above) and incubate at 30 ⁇ C, 250 rpm overnight. 5. Pellet the cells by centrifugation at 4,000 x g for 20 min at 20 ⁇ C 6. Resuspend the cells in the same volume of TB supplemented with phosphate salts, 100 ⁇ g/ml ampicillin (NO GLUCOSE) and IPTG of a final concentration 1 mM IPTG. Incubate at 30 ⁇ C, 250rpm for 4-5 h. 7. Collect the cells by centrifugation at 4500 x g for 25 min and freeze the pellet. 8.
  • VNAR sequences specific to huPTK7 were isolated from ELLS1 and ELSS4 library respectively. All VNAR clones were expressed, purified and re-assessed for PTK7 specific binding. Fifteen clones obtained in selections on huPTK7 (ECD) recognise recombinant huPTK7 ECD in ELISA, but not recombinant huPTK7 (domains 5-7). One clone (4A12) didn’t show any specificity to huPTK7.
  • P2B1-QASGA-His-Myc (SEQ ID NO: 638) ASVDQTPRTATKETGESLTINCVLTDTWEEDMTTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSF SLRIKDLTVADSATYYCKAWWSPSYYSFMWYDGAGTVLTVNQASGAHHHHHHGAEFEQKLISEEDL P2B12-QASGA-His-Myc (SEQ ID NO: 639) ASVNQTPRTATKETGESLTINCVLTDTDDQVPATSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFS LRIKDLTVADSATYYCKAWWWDGNWVWVWYDGAGTVLTVNQASGAHHHHHHGAEFEQKLISEEDL P2C6-QASGA-His-Myc (SEQ ID NO: 640) TRVDQTPRTATKETGESLTINCVLTDTDDGWPTTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSF
  • Species cross-reactivity of lead clones was analysed with cynomolgus monkey and mouse PTK7 along with human antigen (both full ECD and PTK7 domains 5-7). Briefly 1. Coat 96 well plates with 1 ⁇ g/ml of huPTK7 ECD-huFc (or PTK7(ECD)-his for CCK4 and hu24 controls), huPTK7 ECD (5-7)-Fc (or PTK7(5-7)-his for CCK4 and hu24 controls), mouse PTK7 or cyno PTK7 in PBS. Incubated overnight at 4 o C. 2. Wash 2 x PBS and block with 200 ⁇ l/well of 4% MPBS for 1h at RT.
  • P2A7 bound to human PTK7 (ECD), but not human PTK7 (domains 5-7), and cross-reacted with mouse and cynomolgus PTK7.
  • 4D2 bound to human PTK7 (ECD) but not human PTK7 (domains 5-7) and cross-reacted with cynomolgus PTK7 only (did not bind mouse PTK7).
  • E02 bound to both human PTK7 ECD and PTK7 (5-7) but did not show any species cross-reactivity with either mouse or cyno antigen (Figure 24).
  • PTK7 binding VNARs were re-expressed using intein technology. For expression as intein fusions, DNA encoding VNARs was optimised for E.
  • coli expression (GeneArt, Thermo) and cloned in frame into an intein expression vector. This results in a gene encoding the VNAR protein of interest fused to an engineered intein domain which in turn is fused to a chitin binding domain (CBD) to enable purification on a chitin column.
  • CBD chitin binding domain
  • VNAR intein fusion protein was purified from clarified cell lysate by immobilising on chitin beads (NEB, S6651).
  • VNARs were washed extensively with lysis buffer followed by cleavage buffer (50mM sodium phosphate pH6.9, 200mM NaCl) and VNARs released from the beads by overnight chemical cleavage in 400mM dioxyamine, or O,O’- 1,3-propanediylbishydroxylamine, or 100mM cysteine or cysteamine to generate the corresponding C- terminal aminoxy, C-terminal cysteine or C-terminal thiol derivative of the VNARs. Cleaved VNAR supernatant was then further purified by SEC (Superdex7526/60 GE healthcare) and / or IMAC (HisTrap HP, GE Healthcare).
  • SEC Superdex7526/60 GE healthcare
  • IMAC HisTrap HP, GE Healthcare
  • a human IgG1 Fc sequence is shown below and further examples are shown in Figure 7.
  • Human IgG1 Fc (hFc) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 145) PTK7-specific VNARs were genetically fused via standard G4S linkers (typically [G4S]3) linkers to engineered hIgG1 Fc domains that contained a cysteine substitution in the hIgG1 Fc sequence, S239C (EU number
  • VNAR Fc fusion proteins were transiently expressed as secreted protein in CHO K1 cells and purified from the media using MabSelectTM SuReTM (Evitria, Switzerland). Purified proteins were exchanged into PBS pH 7.4 or PBS + 100 mM Arg pH 7.4 and analysed by SEC (Superdex 75 increase 10/300 GL, running buffer PBS pH 7.4), SDS PAGE and mass spectrometry to confirm sequence and protein integrity (Table 22).
  • Table 22 Characterisation of PTK7 VNAR-hFc fusions Binding to PTK7 was determined using Biolayer interferometry (K2 Octet instrument, Sartorius) as previously described.
  • PTK7-hFc extracellular domain
  • PTK7 Binding kinetics were determined using Biolayer Interferometry (BLI) on an Octet K2 system (Sartorius).
  • PTK7(ECD)-hFc fusion proteins extracellular domains
  • PTK7(ECD)-hFc fusion proteins were immobilised in sodium acetate pH5 buffer to COOH2 chips or AR2G sensors using amine coupling.
  • VNAR-Fc molecules were tested at various concentrations and the Ka (M-1s-1), Kd (s-1) and KDapp (nM) values were determined using Octet Data Analysis High Throughput software (Sartorius) for Biolayer Interferometry.
  • the kinetic parameters for binding were determined by immobilising the VNAR-hFc fusion onto AHC sensors.
  • Human PTK7 ECD
  • Ka M-1s-1
  • Kd s-1
  • KD nM
  • Binding parameters are shown in Table 23.
  • Table 23 Parameters for binding of VNAR-Fc fusions to PTK7 by BLI BLI data confirms that P2A7 and 4D2 Fc fusions bind with high affinity to both human and cynomolgus PTK7.
  • P2A7 hFc shows cross-reactivity with the mouse orthologue, whereas 4D2 hFc does not bind to mouse PTK7.
  • BLI experiments to assess competitive binding of the VNAR-hFc fusions showed that E02, P2A7 and 4D2 do not compete with each other for PTK7 binding. Combinations of these binders can therefore be used to generate bi-paratopic PTK7 binders.
  • Bi-paratopic VNAR Fc Fusion Proteins PTK7-specific VNARs were genetically fused via standard [G 4 S] 3 linkers to hIgG1 Fcs engineered for heterodimerisation (Ridgway 1996 Protein Engineering 9(7):617-21).
  • the Knob variant has a tryptophan substitution at position 366 (T366Y) and the Hole variant has a Threonine substitution at position 407 (Y407T) (EU numbering).
  • T366Y tryptophan substitution
  • Y407T Threonine substitution
  • This approach was used to generate bi-paratopic PTK7 binders where one arm comprises a PTK7 targeting VNAR and the other arm comprises a second PTK7 binding VNAR.
  • a cysteine substitution was incorporated in the hIgG1 Fc sequence [S239C (EU numbering) or S239C + S442C (EU numbering)] of both Knob and Hole variants to facilitate bioconjugation with different payloads.
  • VNAR Fc fusion proteins were transiently co-expressed as secreted protein in CHO K1 cells and purified from the media using MabSelectTM SuReTM (Evitria, Switzerland). Purified proteins were exchanged into PBS pH 7.4 and analysed by SEC (Superdex 75 increase 10/300 GL, running buffer PBS pH 7.4), SDS PAGE and mass spectrometry to confirm sequence and protein integrity (Table 24).
  • P2A7 hFc (S239C+T366Y) (SEQ ID NO: 509) TRVDQTPRTATKETGESLTINCVLTDTNYGLYSTSWFRKNPGTTDWERMSIGGRYVESVN KGAKSFSLRIKDLTVADSATYYCKAAYIERNGFLTWYDGAGTVLTVNGGGGSGGGGSGGG GSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D2 hFc (S239C+Y407T)
  • PTK7 VNAR-hFc proteins were generated with additional cysteines engineered into the Fc region as described previously, which enabled site-specific labelling with maleimide derivatives of labels and probes including cytotoxic payloads.
  • Generation of PTK7 VNAR-hFc MMAE conjugates Using a partial reduction, refolding and labelling method adapted from the literature (Junutula et al, 2008 Nat Biotech, Jeffrey et al, 2013 Bioconj Chem), the PTK7 hFc (S239C ⁇ S442C) series of proteins were site specifically labelled with MC-vc-PAB-MMAE (Levena # SET0201).
  • PTK7 hFc (S239C + S442C) 1.5 mg/ml PTK7 hFc protein solutions were prepared in PBS +100mM L-Arginine pH7.4 with 1mM EDTA. 40 molar equivalents TCEP added and incubated at 4°C for a minimum of 48 hours. 50 molar equivalents DHAA added, pH adjusted to 6.5 and incubated at room temperature for 1 hour. Refolded VNAR hFc was extensively dialysed into PBS +50mM L-Arginine before reacting with 8 equivalents MC-vc-PAB-MMAE, room temperature 2 hours.
  • PTK7 hFc S239C series of proteins were site specifically labelled using a similar partial reduction, refolding approach. Briefly, 2-5 mg/ml PTK7 hFc protein solutions were prepared in PBS +100mM L- Arginine pH7.4 with 1mM EDTA. 20 molar equivalents TCEP added and incubated at 4°C for a minimum of 48 hours. 30 molar equivalents DHAA added, pH adjusted to 6.5 with 400 mM sodium phosphate pH 6.0 at 1/30th of the reaction volume and incubated at room temperature for 1 hour.
  • Adherent cancer cells were detached from tissue culture flasks by incubating with 0.1% EDTA/PBS solution at 37 °C for ⁇ 10 minutes or until cells detached easily. Cells were re- suspended in ice-cold PBS/2%FCS in 15ml tubes and centrifuged at 1500rpm for 5 mins at 4 °C. Supernatant was removed and the cell pellet re-suspended in PBS/2%FCS. A cell count was performed using a Z1 Coulter Particle Counter (Beckman Coulter) or Chemometec Nucleocounter NC-202 and 5 x 10 ⁇ 5 cells were aliquoted per test sample into a 96 well plate.
  • Binding of VNAR-hFc molecules was determined by adding 100 ⁇ l of of PE-anti-human antibody (JIR) and incubating on ice for 30mins. Wash steps were performed as described previously and analysis on a Cytek Biosciences Guava EasyCyte HT or Thermo Fisher Attune NxT flow cytometer. KDapp values were calculated from the increase in fluorescence intensity as a function of VNAR-hFc concentration. Bmax values are the MFI values at saturation binding. Table 26 below summarizes the binding of the PTK7-hFc proteins and drug conjugates to PTK7 hi human cancer cell-line NCI-H661 (KDapp and Bmax).
  • Table 26 Summary of K D app and Bmax values for binding of PTK7-hFc proteins and conjugates to the human cancer cell-line NCI-H661 Note Bmax values can only be compared within a series As shown in Figure 25 and Figure 26, the unconjugated monoparatopic and biparatopic VNAR-hFc proteins bind with high affinity to the PTK7 hi human cancer cell-line NCI-H661 but do not bind to the PTK7 low human cancer cell-line AsPC1.
  • Binding to cell-surface PTK7 was saturable, with higher Bmax values observed for biparatopic binder P2A7-4D2 hFc compared to the corresponding P2A7-hFc and 4D2-hFc proteins, indicating increased cell surface binding on the surface of NCI-H661 (PTK7Hi) cells for the bi-paratopic protein.
  • the corresponding vc-PAB-MMAE and vc-PAB-EDA-PNU drug conjugates maintain the high affinity binding to the NCI-H661 cells with no-binding observed to AsPC1 cells (PTK7 Lo ).
  • Binding of the drug conjugates to cell-surface PTK7 was saturable and, as is the case for the non-conjugates, higher Bmax values were observed for the biparatopic P2A7-4D2 hFc drug conjugates as compared to the respective monoparatopic P2A7-hFc and 4D2-hFc conjugates. This indicates increased cell surface binding on the surface of NCI-H661 (PTK7Hi) cells for the bi-paratopic drug conjugates versus the monoparatopic counterparts ( Figures 27 to 29).
  • EXAMPLE 17 In vitro cell viability assays for cancer cells treated with anti PTK7 VNAR drug conjugates Cells were seeded into white, clear bottom 96 well plates (Costar) and incubated at 37°C, 5% CO 2 for 24 hours. On the following day, dilution series were set up for each test agent at x10 working stocks. The dose response X10 stock was: 10000, 5000, 1000, 500, 100, 50, 10, 5, 1, 0.5nM etc. 10 ⁇ L of the X10 stock solutions were added to the cell plates (90 ⁇ l per well) using a multichannel pipette.
  • PA-1 - human ovarian cancer cell line EMEM, 10% hiFCS
  • PA-1 PTK7 ko - human ovarian cancer cell line with PTK7 knock-out EMEM, 10% hiFCS
  • Figure 30 shows dose response curves, with corresponding IC 50 values (Table 27), for cell-killing of the PTK7 positive PA-1 ovarian cancer cells and PA-1 PTK7 ko cells by P2A7-hFc-PNU conjugates (PEG4- vc PAB EDA PNU159682), 4D2-hFc-PNU conjugates (PEG4-vc PAB EDA PNU159682) and P2A7- 4D2-hFc-PNU conjugates (PEG4-vc PAB EDA PNU159682) and P2A7-4D2-hFc-MMAE conjugate (vc- PAB-MMAE).
  • PA-1 PTK7 ko is PA-1 cancer cell-line where PTK7 expression has been knocked out.
  • Table 27 Calculated IC50 values (nM) for the cell-killing of PA-1 and PA1 PTK7 ko cancer cells by P2A7- hFc, 4D2-hFc and P2A7 conjugates.
  • the PTK7 targeting VNAR-hFc conjugates show potent killing of PA-1 cells, which is abrogated upon knockdown of the PTK7 receptor.
  • Reformatting as a biparatopic binder [P2A7-4D2-hFc (S239C) vcPAB- EDA-PNU] increases the potency of the drug conjugates with respect to the equivalent mono-paratopic binders [cf P2A7-hFc (S239C) vcPAB-EDA-PNU and 4D2-hFc (S239C) vcPAB-EDA-PNU].
  • Example 18 In vivo efficacy of PTK7-hFc protein-drug conjugates in patient-derived xenograft model of Ovarian Cancer (OvCa)
  • OvCa Ovarian Cancer
  • Outbred athymic (nu/nu) female mice (Crl:NMRI-Foxn1nu) were implanted subcutaneously with tumours of the same in vivo passage. Mice were monitored until the tumour implants reached the study volume recruitment criteria of 60–200 mm 3 , preferably 75–196 mm 3 in a sufficient number of animals.
  • mice were randomised to treatment groups such that there was no statistical difference between tumour volumes in each group. Randomisation was designated as Day 0 of the experiment. Mice were treated with vehicle or with the protein-drug conjugates P2A7-hFc-vc-PAB-EDA-PNU, 4D2-hFc-vc-PAB-EDA-PNU, P2A7-4D2-hFc-vc-PAB-EDA-PNU at a dose of 0.3 mg / kg i.v. injection, three times, four days apart (3 x Q4D on day 1, 5 and 9). Separately, P2A7-4D2-hFc-vc-PAB-EDA-PNU was also dosed at 0.1 mg / kg i.v.
  • mice were also treated with protein drug conjugate P2A7-4D2-hFc-vc-PAB-MMAE at a 2.5 mg / kg i.v. injection, four times, four days apart (4 x Q4D on day 1, 5, 9 and 13). All mice were pre-primed with mouse IgG 20h before first PDC dose. Tumour volume was evaluated by measuring perpendicular tumour diameters, with a calliper, three times a week during the experimental period.
  • Figure 31 shows the effect of the protein-drug conjugates on tumour growth versus vehicle control. All protein drug conjugates were well tolerated.
  • EXAMPLE 19 Generation of the anti-ROR1 x anti-PTK7 bi-specific proteins
  • ROR1 binding VNAR sequences as described previously (WO 2019/122447 and PCT/EP2021/086667, filed on 17 December 2021 were genetically fused to human IgG1 hFc sequence via standard [G4S]3 or short [G4S]1 linkers.
  • the human IgG1 sequence comprised 1 or 2 site specific Cys substitutions (S239C ⁇ S442C) for site specific conjugation and the T366Y knobs-into-holes (KIH) mutation for bi-specific chain pairing (EU numbering).
  • the PTK7 binding VNARs P2A7 and 4D2 were fused to human IgG1 hFc sequence via standard [G4S]3 or short [G4S]1 linkers.
  • the Fc region contains site-specific Cys substitutions (S239C ⁇ S442C) for site specific conjugation and the corresponding Y407T KIH mutation for bi-specific pairing with above ROR1 VNAR chains.
  • Bi-specific protein combinations were transiently expressed as secreted protein in CHO K1 cells and purified from the media using MabSelectTM SuReTM (Evitria, Switzerland).
  • HMW high molecular weight
  • ROR1 x PTK7 hFc bi-specific proteins containing the ROR1 binder B1 had increased elution volume as compared to the bi-specific proteins containing other ROR1 binders.
  • the non B1 containing bi-specifics therefore show advantageously reduced hydrophobicity relative to B1 containing bi-specifics, thus providing improved developability.
  • EXAMPLE 20 – ROR1 Target binding by ELISA The ROR1 x PTK7 hFc bi-specific proteins were assessed for ROR1 target binding by ELISA. Briefly, plates were coated with 2 ⁇ g/ml human ROR1 ECD His (Evitria) in PBS for 2hr at room temperature.
  • ROR1 x PTK7 hFc bi-specifics were titrated in PBSTM (PBS + 0.05% Tween 20 (v/v) + 4% skimmed milk powder (w/v)) and plates incubated at 4°C overnight. Binding was detected using anti human IgG HRP in PBSTM (Abcam #Ab6759, 1:130,000 dilution, 1hr at room temperature] and TMB substrate (Thermo Scientific, #12617087) and absorbance measured at 450nm. As can be seen in Figure 32 all ROR1 x PTK7 bi-specific proteins bind human ROR1.
  • EXAMPLE 21 – PTK7 Target binding by ELISA The ROR1 x PTK7 hFc bi-specific proteins were assessed for PTK7 target binding by ELISA. Briefly, plates were coated with 2 ⁇ g/ml human PTK7 ECD His (Evitria) in PBS for 2hr at room temperature. ROR1 x PTK7 hFc bi-specifics were titrated in PBSTM (PBS + 0.05% Tween 20 (v/v) + 4% skimmed milk powder (w/v)) and plates incubated at 4°C overnight.
  • PBSTM PBS + 0.05% Tween 20 (v/v) + 4% skimmed milk powder (w/v)
  • Binding was detected using anti human IgG HRP in PBSTM (Abcam #Ab6759, 1:130,000 dilution, 1hr at room temperature] and TMB substrate (Thermo Scientific, #12617087) and absorbance measured at 450nm. As can be seen in Figure 33 all ROR1 x PTK7 bi-specific proteins bind human PTK7.
  • Example 22 Binding of ROR1 x PTK7 bi-specific hFc proteins to ROR1 and PTK7 by BLI Binding to ROR1 or PTK7 was determined using Biolayer interferometry (R4 Octet instrument, Sartorius) as previously described.
  • Each ROR1 x PTK7 hFc protein (1 mg) was prepared at 2 mg/ml (except for 683 and 684 at 2.9 mg/ml and 821 at 1.5 mg/ml) in PBS pH 7.4 + 0.05% sodium azide. Half the protein was used for T0 analysis and the other half was incubated at 37°C for 96h (T96). Analysis of samples by SDS PAGE (intact and reduced), MS (glycosylated, reduced) and ELISA (PTK7 and ROR1 target binding as described above) showed no significant differences between T0 and T96 for all samples. Samples at T0 and T96 were also analysed by size exclusion chromatography (S200 Increase 10/300GL with PBS pH 7.4 running buffer).
  • An approach for generating ADCs is to engineer cysteine substitutions or additions at positions on the light and heavy chains of antibodies and these cysteines provide reactive thiol groups for site specific labelling (Junutula 2008 Nature Biotechnology 26, 925 – 932, Jeffrey 2013, Sutherland 2016).
  • ROR1 x PTK7 hFc bi-specific proteins were generated with additional cysteines engineered into the Fc region as described previously, which enabled site-specific labelling with maleimide derivatives of labels and probes including cytotoxic payloads.
  • EXAMPLE 25 Generation of Bi-specific ROR1 x PTK7 hFc MMAE conjugates Using a partial reduction, refolding and labelling method adapted from the literature (Junutula et al, 2008 Nat Biotech, Jeffrey et al, 2013 Bioconj Chem), the ROR1 x PTK7 hFc (S239C+S442C) series of proteins were site specifically labelled with MC-vc-PAB-MMAE (Levena # SET0201). Briefly, 2-5 mg/ml ROR1 X PTK7 hFc protein solutions were prepared in PBS +100mM L-Arginine pH7.4 with 1mM EDTA.
  • ROR1 x PTK7 hFc (S239C) series of proteins were site specifically labelled with MC-vc-PAB-MMAE (Levena # SET0201) using a similar partial reduction, refolding approach.
  • 2-5 mg/ml ROR1 X PTK7 hFc protein solutions were prepared in PBS +100mM L-Arginine pH7.4 with 1mM EDTA.
  • 20 molar equivalents TCEP added and incubated at 4°C for a minimum of 48 hours.
  • Example 26 Binding of ROR1 x PTK7 bi-specific hFc MMAE conjugates to ROR1 and PTK7 by BLI Binding to ROR1 or PTK7 was determined using Biolayer interferometry (R4 Octet instrument, Sartorius) as previously described.
  • PTK7 ECD His,or ROR1 ECD His was immobilised on AR2G sensors in MES-NaOH pH 5.2 using amine coupling.
  • the % of control data was plotted against Log [Treatment] concentration and the IC50 value derived using non-linear regression fitting in GraphPad Prism software (Table 40).
  • the following cell lines were used ⁇ PA-1 (ECACC 90113101) – human ovarian cancer cell line: EMEM, 10% hiFCS ⁇ PA-1 ROR1 ko - human ovarian cancer cell line with ROR1 knock-out: EMEM, 10% hiFCS ⁇ SW403 [SW-403] (ATCC CCL-230) - human colorectal cancer cells: L-15, 10% hiFCS ⁇ NCI-H1703 [H1703] (ATCC CRL-5889) – human squamous lung cancer cell line: RPMI1640, 10% hiFCS ⁇ NCI-H1975 [H-1975, H1975] (ATCC CRL-5908) – human lung cancer cell line: RPMI1640, 10% hiFCS ⁇ NHEK-Ad (Lonza 00
  • Quantibrite beads (BD Biosciences) were used as per the manufacturer’s instructions. Analysis was performed on Attune NxT flow cytometer (ThermoFisher). Cells were incubated with PE-conjugated anti-PTK7 OT12E7 mAb at 22 ⁇ g/ml for 1 hour on ice in the dark. Quantum Simply Cellular Beads (Bangs Laboratories) were used as per the manufacturer’s instructions. Analysis was performed on Attune NxT flow cytometer (ThermoFisher). Table 39: ROR1 and PTK7 receptor numbers for a panel of cancer and normal cell lines used in the cell 5 viability assays.
  • ROR1 x PTK7 targeting bi-specific hFc conjugates show potent killing of cancer cells expressing ROR1 or PTK7, with the most potent killing observed for PA-1, which expresses both ROR1 and PTK7 receptors (single digit nanomolar range for a number of conjugates).
  • PA-1 which expresses both ROR1 and PTK7 receptors (single digit nanomolar range for a number of conjugates).
  • the potencies were decreased for all the conjugates with respect to the PA-1 cell-line.
  • a 2-5- fold drop-off in IC 50 values was observed for these ROR1-negative, PTK7-positive cells.
  • all conjugates showed much weaker cell-killing of SW403 colorectal cancer cells which lack both ROR1 and PTK7 expression.
  • the bi-specific conjugates showed much poorer killing of primary adult keratinocytes (NHEK-Ad cells) and normal prostate epithelium cells (PNT2) despite high levels of PTK7 expression. Generally, the percentage of cell-killing did not reach an IC50 value over the large dose-response range used (in these instances the projected IC50 values reported are for comparative purposes only). Therefore, the ROR1 x PTK7 targeting bi-specific conjugates provide a large window for the killing of dual receptor positive cancer cells versus normal cells. The estimated IC50 values for NHEK-Ad showed that these normal cells are between 12 to 111 times less sensitive to ROR1 x PTK7-hFc MMAE conjugates than the PA- 1 cancer cells.
  • a C- terminal sequence including a 6xHis tag (QASGAHHHHHH (SEQ ID NO: 102) was applied to facilitate purification.
  • the nucleotide sequences of the constructs were synthesized by Geneart (Thermo Scientific) and inserted to the pET100/D-TOPO expression vector under the control of a T7 promoter.
  • Bi-specific protein combinations were expressed in Shuffle Xpress E. coli cells (NEB) and purified from inclusion bodies in the presence of 8 M Urea and 1 mM DTT using HisTrap Excel columns (Cytiva).
  • the purified proteins were oxidatively refolded by stepwise dialysis of the urea down to 0.5 M in the presence of 20 mM NaPi, pH 7.8, 250 mM NaCl, 3 mM Cysteine, 1 mM Cystine.
  • the refolded proteins were exchanged into PBS pH 7.4 and analysed by SEC (Superdex75 Increase 10/300, Cytiva, running buffer PBS pH 7.4).
  • the % high molecular weight (HMW) species was determined as the amount of protein eluting before 12.5 mL based on integration of Abs 280nm signal as a % of total protein eluting. (total Abs 280nm).
  • Example 29 Binding of ROR1 x PTK7 (BOS) proteins to ROR1 and PTK7 by BLI Binding to ROR1 or PTK7 was determined using Biolayer interferometry (R4 Octet instrument, Sartorius) as previously described. For BLI experiments, PTK7 ECD His or ROR1 ECD His was immobilised on AR2G sensors in MES-NaOH pH 5.2 using amine coupling.
  • ROR1 x PTK7 BOS proteins were tested at various concentrations and the Ka (M -1 s -1 ), Kdis (s -1 ) and KDapp (nM) values were determined using Octet Data Analysis High Throughput software (Sartorius) for Biolayer Interferometry (Table 42).
  • Table 42 Kinetic parameters for ROR1 x PTK7 BOS binding to human ROR1 and PTK7 by BLI ROR1 x PTK7 beads-on-a-string format bind with high affinity to both ROR1 and PTK7.
  • EXAMPLE 30 Generation of bi-specific ROR1 x PTK7 BOS MMAE conjugates
  • the ROR1 x P2A7 bi-specific BOS proteins were prepared at 0.4-0.8 mg/ml in PBS, pH 7.4.
  • TCEP was added at 2 mM and incubated at 22°C for 1 hour.
  • the buffer was exchanged to PBS, 100 mM L-Arginine pH 7.4, 1 mM EDTA using a HiPrep 26/10 desalting column (Cytiva).
  • 3 molar equivalents MC-vc-PAB-MMAE were added to the protein and incubated at 22 o C for 1 hour.
  • Tumours from a wide range of different cancer indications express both ROR1 and PTK7 proteins
  • PTK7 and ROR1 protein expression levels were investigated by Western blotting in a series of PDX models from a number of different cancer indications.
  • Tumour fragments were weighed and 3-10X volume of RIPA buffer, containing phosphatase and protease inhibitors, was added.
  • Tumours were homogenised at 4°C for 5min at 50Hz. Samples were sonicated for up to 30 minutes (30sec on - 30 sec off cycles) at 4°C. Lysates were centrifuged at 14000g for 15 minutes at 4 degrees and the supernatant was collected.
  • Protein concentrations were quantified using BCA Protein Assay and samples were prepared adding loading buffer and reducing agents. Samples ( ⁇ 60 ⁇ g/ml determined by BCA assay) were loaded on a pre-cast gel and run for approximately 1hr then transferred on PVDF or nitrocellulose membrane. Membranes were blocked in TBST with 5% milk for 1h at RT. Incubation with primary antibodies was performed overnight at 4degC in TBST + 5% milk. Commercial primary antibodies (anti ROR1 CST #16540 and anti PTK7 clone 4F9 (Merck Sigma) or anti-PTK7 clone OTI2E7 (Novus Biologicals) were used according to manufacturer’s recommendations. Beta Actin or Vinculin were used as a loading control.
  • Membranes were washed in TBST and incubated for 1h at room temperature with secondary antibodies in TBST+ 5% milk. Both chemiluminescence and fluorescence detection methods were used to detect protein bands. PA-1 and SW403 cells were used as ROR1/PTK7 positive and negative controls respectively.
  • PDX models from several different cancer indications were selected for analysis based on reported ROR1 mRNA expression levels. PDX models predicted to have high levels of ROR1 expression were screened for PTK7 and ROR1 protein expression by Western blot. A large number of PDX tumour samples, across different cancer indications, were shown to express both ROR1 and PTK7 proteins ( Figures 37 and 38).
  • Table 44a ROR1 and PTK7 relative protein expression in selected PDX models from TNBC, Breast adenocarcinoma, Ovarian cancer, Sarcoma and Endometrial cancer. Protein levels were quantified by fluorescence detections methods.
  • Table 44b ROR1 and PTK7 relative protein expression in selected PDX models from a variety of lung cancer indications. Protein levels were quantified by chemiluminescence detections methods. Relative protein expression levels were annotated in consideration of loading controls within each experiment Table 44a and Table 44b show a selection of PDX models expressing both ROR1 and PTK7 proteins (corresponding western blots are shown in Figure 37 and 38).
  • tumours with dual ROR1 / PTK7 expression can be found in a variety of cancer indications, including SCLC (model LXFS_650), LCLC (model LXFL_2228 and LXFL_625), lung adenocarcinoma (models LXFA_1647, LXFA_1125, LXFA_2184), Lung Squamous Cell Carcinoma (model LXFE_409), Pleuromesothelioma (model PXF- 1118), TNBC (models HBCx-28, HBCx-30, HBCx-33, HBCx-24 and HBCx-10), Breast adenocarcinoma (models MAXFHER_BR64 and MAXFTN_BR5), Ovarian cancer (models OVXF_OV55, OXVF_OV-003, OXVF_OV-006 and OVXF_899), Sarcoma (models SXFS_117, SXFS_1407 and SXFS_174) and
  • Dual expression of ROR1 x PTK7 is advantageous for treating indications, such as lung cancer, where mono-specific ROR1 targeted therapies are not especially effective.
  • EXAMPLE 32 Binding of bi-specific ROR1 x PTK7-hFc MMAE conjugates to ROR1+PTK7+ cancer cells (PA1) Cell surface binding of ROR1xPTK7 VNAR-hFc MMAE conjugates (G3CP-P2A7 & G3CP-4D2), bispecific and their respective ROR1 and PTK7 monospecific MMAE drug conjugates (G3CP, P2A7 & 4D2) was assessed in different cancer cell lines PA-1 (ROR1 hi & PTK7 hi ovarian cancer cell line) and SW403 (ROR1 neg & PTK7 low colorectal cancer cell line) and the resulting KDapp values determined.
  • PA-1 ROR1 hi & PTK7 hi ovarian cancer cell line
  • SW403 ROR1 neg & PTK7 low colore
  • Adherent cancer cells were detached from tissue culture flasks by incubating with 0.1% EDTA/PBS solution at 37 °C for ⁇ 10 minutes or until cells detached easily. Cells were re-suspended in ice-cold PBS/2%FCS in 15ml tubes and centrifuged at 1500rpm for 5 mins at 4 °C. Supernatant was removed and the cell pellet re-suspended in PBS/2%FCS. A cell count was performed using a Chemometec Nucleocounter NC-202 and 5 x 10 ⁇ 5 cells were aliquoted per test sample into a 96 well plate. Cells were incubated with 100 ⁇ l of test agents at a range of concentrations, plus controls for 1hr on ice.
  • the sample plate was centrifuged at 2000 rpm for 5mins. The supernatant was removed, and a wash performed by re-suspending the cell pellets in 0.25mL of ice-cold PBS/2%FCS using a multichannel pipette. Samples were again centrifuged at 2000rpm for 5min at 4°C. Supernatant was removed and two further washes performed as described. After the final wash and centrifugation step, excess liquid was removed by blotting the plate on tissue paper. Binding of VNAR-hFc MMAE conjugates was determined by adding 100 ⁇ l of of PE-anti-human antibody (JIR) and incubating on ice for 30mins.
  • JIR PE-anti-human antibody
  • Binding to cell-surface ROR1 & PTK7 was saturable, with higher Bmax values observed for bispecific binder G3CP-P2A7 hFc MMAE conjugate compared to the corresponding G3CP-hFc and P2A7-hFc MMAE monospecific bivalent conjugates, indicating increased cell surface binding on the surface of PA-1 (ROR1 hi & PTK7hi) cells for the bispecific conjugate.
  • EXAMPLE 33 Internalisation of bi-specific ROR1 x PTK7-hFc MMAE conjugates to ROR1+PTK7+ HEK293-ROR1 cells Internalisation of the following MMAE conjugates: ROR1 x PTK7-targeting, ROR1-hFc binders, PTK7- hFc binders and 2V-hFc non-targeting control was assessed in HEK293-ROR1 cells using an IncuCyte S3 live cell analysis instrument (Sartorius).
  • HEK293-ROR1 stable transfectants were generated using 293 [HEK-293] (ATCC CRL-1573) provided by the American Type Culture Collection (ATCC) grown in EMEM, 10% hiFCS. Cells were seeded at a density of 3000 cells/well into a black clear-bottom 96-well plate (Corning, #3340) and left to adhere at 37 °C and 5% CO2 for 24 hrs.
  • test agents were mixed with FabFluor- pH Red Antibody Labeling Reagent (Sartorius, #4722) at a molar ratio of 1:2 in media, x2 final assay concentration, and incubated for 15 minutes at 37 °C to allow conjugation.50 ⁇ l of the resulting mixtures was added to appropriate wells containing cells (50 ⁇ l) to result in a final concentration of 25nM of each test agent. Images were captured periodically for 30 hours and four regions of interest were imaged from each well. Cell-by-cell analysis was performed using Incucyte integrated module. Data is presented as average red mean intensity (Red Calibrated Unit; RCU) over time. Assay was performed in triplicates.
  • the ROR1xPTK7 VNAR-hFc MMAE conjugates (G3CP-P2A7 & G3CP-4D2), bispecifics have enhanced internalisation compared to ROR1 & PTK7 monospecific bivalent MMAE conjugates. Indicating that bispecific ROR1xPTK7 VNAR-hFc MMAE conjugates have enhanced internalisation compared to monospecific controls into a cell line which expresses ROR1 & PTK7.
  • the ROR1xPTK7 targeting VNAR-hFc conjugates show potent killing of NCI-H1975 cells.
  • EXAMPLE 35 – Generation of Bi-specific ROR1 x PTK7 hFc PNU conjugates A similar partial reduction, refolding and labelling method was used for PNU conjugation as above with some modifications.
  • VNAR hFc solutions were prepared in PBS, 100 mM L-Arg, 1 mM EDTA pH 7.4. 20 molar equivalents TCEP added and incubated at 4°C for 18 hours. 30 molar equivalents DHAA is then added, pH adjusted to 6.5 and incubated at room temperature for 3 hours. Refolded VNAR hFc was buffer exchanged into PBS, 50 mM L-Arg, pH 7.4 using NAP25 columns and concentrated to 3-4 mg/ml. Propylene glycol was then added to a 20% final concentration before addition of 4 molar equivalents maleimide PNU solution. This was incubated at room temperature for 2 hours.
  • EXAMPLE 36 Binding of bi-specific ROR1 x PTK7-hFc PNU conjugates to ROR1+PTK7+ cancer cells (PA1) Cell surface binding of ROR1xPTK7 VNAR-hFc PNU conjugates (G3CP-P2A7 & G3CP-4D2), bispecific and their respective ROR1 and PTK7 monospecific PNU drug conjugates (G3CP, P2A7 & 4D2) was assessed in different cancer cell lines PA-1 (ROR1 hi & PTK7 hi ovarian cancer cell line) and SW403 (ROR1 Neg & PTK7 Lo colorectal cancer cell line) and the resulting KDapp values determined as described in Example 32.
  • PA-1 ROR1 hi & PTK7 hi ovarian cancer cell line
  • SW403 ROR1 Neg & PTK7 Lo colorectal cancer cell line
  • the ROR1xPTK7 VNAR-hFc PNU conjugates (G3CP-P2A7 & G3CP-4D2), bispecifics bind with high affinity to the ROR1 hi & PTK7 hi human ovarian cancer cell-line PA-1 but do not bind to the ROR1 neg & PTK7 low human colorectal cancer cell-line SW403.
  • IC50 (nM) 96hr Examples of the dose response curves for cell-killing are shown in Figure 45.
  • ROR1 x PTK7 targeting bi-specific hFc PNU conjugates show potent killing of cancer cells expressing ROR1 and PTK7, with the most potent killing observed for PA-1, which expresses both ROR1 and PTK7 receptors (sub nanomolar range for a number of conjugates).
  • PA-1 which expresses both ROR1 and PTK7 receptors (sub nanomolar range for a number of conjugates).
  • the potencies were decreased for all the conjugates with respect to the PA-1 cell-line. In general, a 10-12 fold drop-off in IC50 values was observed for these ROR1-negative, PTK7-positive cells.

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Abstract

La présente invention concerne des molécules de liaison à l'antigène bispécifiques ayant une spécificité pour le récepteur orphelin 1 de type récepteur à tyrosine kinase (ROR1) et la protéine tyrosine kinase 7 inactive (PTK7, CCK4) et des protéines de fusion et des conjugués associés. Dans un autre aspect, la présente invention concerne de nouveaux récepteurs antigéniques à domaine variable dérivés du requin du type immunoglobulines conjuguées (VNAR).
PCT/EP2023/067045 2022-06-22 2023-06-22 Molécules de liaison à l'antigène bispécifiques ror1/ptk7 WO2023247731A1 (fr)

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WO2007067730A2 (fr) * 2005-12-08 2007-06-14 Medarex, Inc. Anticorps monoclonaux humains contre la proteine tyrosine kinase 7 (ptk7) et procedes pour utiliser des anticorps anti-ptk7
WO2019122445A1 (fr) * 2017-12-22 2019-06-27 Almac Discovery Limited Molécules bispécifiques de liaison à l'antigène

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007067730A2 (fr) * 2005-12-08 2007-06-14 Medarex, Inc. Anticorps monoclonaux humains contre la proteine tyrosine kinase 7 (ptk7) et procedes pour utiliser des anticorps anti-ptk7
WO2019122445A1 (fr) * 2017-12-22 2019-06-27 Almac Discovery Limited Molécules bispécifiques de liaison à l'antigène
WO2019122447A1 (fr) * 2017-12-22 2019-06-27 Almac Discovery Limited Molécules de fixation à l'antigène spécifique de ror1

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Title
RAIVOLA JUULI ET AL: "New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome", CMLS CELLULAR AND MOLECULAR LIFE SCIENCES, BIRKHAUSER VERLAG, HEIDELBERG, DE, vol. 79, no. 5, 1 May 2022 (2022-05-01), XP037815858, ISSN: 1420-682X, [retrieved on 20220504], DOI: 10.1007/S00018-022-04301-6 *

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