ROR1-specific variant antigen-binding molecule

Abstract

The present invention relates to receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific variant antigen binding molecules and related fusion proteins, chimeric antigen receptors, nucleic acid sequences, vectors, host cells, pharmaceutical compositions, medical uses and conjugates and methods of preparing and using same.

Description

[Technical field]

[0001] The present invention relates to receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen binding molecules and related fusion proteins and conjugates. In a further aspect, the present invention relates to conjugated immunoglobulin-like shark variable novel antigen receptors (VNARs). [Background technology]

[0002] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a glycosylated type I single-pass transmembrane protein of 937 amino acids. The extracellular region consists of three distinct domains constituting an N-terminal immunoglobulin domain (Ig), followed by a cysteine-rich fizzled domain (fz), which in turn associates with a juxtamembrane kringle domain (kr). The intracellular region of the protein contains a pseudokinase domain followed by two Ser / Thr-rich domains interspersed with proline-rich regions; this same overall domain architecture is conserved in the closely related family member ROR2, with which it shares high sequence identity.

[0003] ROR1 is expressed during embryonic development, and is prominent in neural crest cells and in necrotic and interdigital zones during late development. However, this expression is rapidly silenced after birth and is largely absent in normal adult tissues. ROR1 expression has been observed at both mRNA and protein levels across a wide range of solid tumors and hematological malignancies, including lung, endometrial, pancreatic, ovarian, colon, head and neck, and prostate cancer, melanoma and renal cell carcinoma, breast cancer, and chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (AML). Furthermore, increased ROR1 expression has been reported to correlate with poor clinical outcomes for many cancer indications, including breast cancer, ovarian cancer, colorectal cancer, lung adenocarcinoma, and CLL.

[0004] Consistent with the expression pattern of ROR1 and its link to poor clinical prognosis, a functional role for ROR1 in tumor development and disease progression has been demonstrated for a number of different cancer indications. ROR1 promotes epithelial-mesenchymal transition and metastasis in breast cancer models and spheroid formation and tumor engraftment in ovarian cancer models. ROR1 is a transcriptional target of the NKX2-1 / TTF-1 lineage survival factor oncogene in lung adenocarcinoma, where it sustains EGFR signaling and suppresses proapoptotic signaling. ROR1 has also been shown to act as a scaffold to sustain caveolae structures and bypass signaling mechanisms that confer resistance to EGFR tyrosine kinase inhibitors. Signaling through the ROR1-HER3 complex modulates the Hippo-YAP pathway and promotes breast cancer bone metastasis, and this protein may promote Met-driven tumorigenesis. ROR1 expression is associated with chemotherapy resistance in breast cancer through activation of the Hippo-YAP / TAZ and BMI1 pathways. On the other hand, in CLL, it has been reported that ROR1 hetero-oligomerizes with ROR2 in response to Wnt5a to transmit signaling and enhance proliferation and migration.

[0005] Given the functional role of ROR1 in cancer pathology and the general lack of expression in normal adult tissues, this oncofetal protein is an attractive target for cancer therapy. Antibodies against ROR1 have been reported in the literature, including WO2021097313 (4A5 kipps), WO2014031174 (UC961), WO2016187220 (Five Prime), WO2010124188 (2A2), WO2012075158 (R11, R12), WO2011054007 (Oxford Bio), WO2011079902 (Bioinvent), WO2017127664, WO2017127664 (NBE Therapeutics, SCRIPPS), WO 2016094847 (Emergent), WO 2017127499), and the humanized murine anti-ROR1 antibody UC961 is in 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 using UC961 and CAR-T cells targeting ROR1 showed no overt toxicity, which is consistent with the general lack of expression of this protein in adult tissues (Choi M et al, Clinical Lymphoma, myeloma & leukemia, 2015, S167; Berger C et al, Cancer Immunol. Res., 2015, 3, 206).

[0006] Single domain binding molecules can be derived from a series of proteins from distinct species. Immunoglobulin isotope new antigen receptors (IgNARs) are homodimeric heavy chain complexes first found in the serum of the nurse shark (Ginglymostoma cirratum) and other shark and ray species. IgNARs do not contain light chains, which distinguishes them from typical immunoglobulin structures. Each molecule consists of one single variable domain (VNAR) and five constant domains (CNARs). Nomenclature in the literature refers to IgNARs as immunoglobulin isotope new antigen receptors or immunoglobulin isotope new antigen receptors, and the terms are synonymous.

[0007] There are three main defined types of shark IgNARs known as types I, II and III (Kovalena et al, Exp Opin Biol Ther 2014 14(10)1527-1539). These are classified based on the location of non-canonical cysteine ​​residues that are under strong selection pressure and therefore rarely replaced.

[0008] All three types have classical immunoglobulin canonical cysteines at positions 35 and 107, together with an invariant tryptophan at position 36, which stabilize the standard immunoglobulin fold. There is no defined CDR2 per se, but regions of sequence variation are defined in frameworks 2 and 3 that correspond more closely to TCR HV2 and HV4, respectively. Type I has germline-encoded cysteine ​​residues in frameworks 2 and 4, and an even number of additional cysteines in CDR3. Crystal structure studies of type I IgNARs isolated from and complexed with lysozyme have allowed the determination of the contribution of those cysteine ​​residues. Both cysteines in frameworks 2 and 4 form disulfide bridges with cysteines in CDR3, forming a tightly packed structure in which the CDR3 loops are held tightly down towards the HV2 region. To date, type I IgNAR has only been identified in the nurse shark; all other elasmobranchs, including members of the same order, only possess type II or variations of this type.

[0009] Type II IgNARs are defined as having cysteine ​​residues in CDR1 and CDR3 that form intramolecular disulfide bonds that hold the two regions in close proximity, resulting in a protruding CDR3 that promotes a binding pocket or groove. Type I sequences typically have longer CDR3s than type II, averaging 21 and 15 residues, respectively. This is believed to result from strong selection pressures that associate two or more cysteine ​​residues in type I CDR3 with their framework 2 and 4 counterparts. Studies of somatic mutation accumulation have shown that there are more mutations in CDR1 in type II than in type I, while the HV2 region of type I shows greater sequence variation than type II. This evidence correlates well with the determined positioning of these regions within the antigen-binding site. A third IgNAR type, known as type III, has been identified in newborns. This member of the IgNAR family lacks diversity within CDR3 due to germline fusion of the D1 and D2 regions (which form the CDR3) with the V gene. Nearly all known clones have a CDR3 length of 15 residues and little or no sequence diversity.

[0010] Another structural type of VNAR, designated type (IIb or IV), has only two canonical cysteine ​​residues (in the framework 1 and framework 3b regions). To date, this type has been found primarily in dogfish and was also isolated from a semi-synthetic V-NAR library derived from nurse shark.

[0011] ROR1-specific antigen binding molecules, including VNARs, are described in WO 2019 / 122447, which is incorporated by reference in its entirety. In particular, WO 2019 / 122447 describes the sequences of B1 and P3A1 G1, identified below.

[0012] B1 (chemical 1) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 113)

[0013] P3A1 G1 (chemical 2) TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 114)

[0014] ROR1-specific antigen binding molecules, e.g., conjugates of VNAR, are described in PCT / EP2020 / 067210, filed June 19, 2020, which is incorporated herein by reference in its entirety. PCT / EP2020 / 067210 describes anthracycline (PNU) derivatives suitable for use in drug conjugates. Specifically, derivatives of PNU159682 are provided that lack the C14 carbon and the attached hydroxyl functionality, and an ethylenediamino (EDA) group forms part of the linker region between the C13 carbonyl and the maleimide group of PNU159682. Alternatively, the same molecule may be described with EDA-PNU as a "warhead" such that the EDA group is not considered part of the linker region. When the linker includes val-cit-PAB, the maleimide group may be replaced with any reactive group suitable for conjugation reactions. Such payloads can react with free thiol groups on another molecule. If a free thiol is present on a protein, a protein-drug conjugate (PDC) can be formed.

[0015] The anthracycline derivative PNU-159682 has been described as a metabolite of nemorubicin (Quintieri et al. (2005) Clin. Cancer Res. 11, 1608-1617) and has been reported to show extremely high potency for in vitro cell killing in the picomolar to femtomolar range using one ovarian (A2780) and one breast cancer (MCF7) cell line (WO 2012 / 073217 A1). Derivatives of PNU-159682 have also been described in WO 2016 / 102679.

[0016] Conjugation of PNU-159682 derivatives to antibodies is described in WO 2009 / 099741, WO 2016 / 127081 and WO 2016 / 102679, Yu et al, Clin. Cancer Res 2015, 21, 3298 and Stefan et al, Mol. Cancer. Ther., 2017, 16, 879. Summary of the Invention [Means for solving the problem]

[0017] Described herein are ROR1-specific variant antigen binding molecules with improved properties and their conjugates to derivatives of PNU-159682.

[0018] The present invention relates generally to specific antigen-binding molecules.

[0019] According to a first aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, CDR3 is 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), Y a CDR sequence having an amino acid sequence selected from the group consisting of PWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22) and YPWGAGAPWLVQWY (SEQ ID NO: 10); CDR1 is 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); FW1 is the framework region; FW2 is the framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO:6) and SSNKERISIS (SEQ ID NO:7); FW3a is the framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO:8) and NKGTM (SEQ ID NO:9); FW3b is the framework region; FW4 is the framework region; When CDR3 is YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLSA (SEQ ID NO: 3) and GANYDLAA (SEQ ID NO: 4) The present invention provides a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprising an amino acid sequence represented by:

[0020] According to a second aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, 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) and GTKYGLYAS (SEQ ID NO: 30); FW1 is the framework region; FW2 is the framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERISIS (SEQ ID NO:31), STDEERISIG (SEQ ID NO:32), SPNKDRMIIG (SEQ ID NO:33), and STDKERIIIG (SEQ ID NO:34); FW3a is the framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKGTK (SEQ ID NO: 35), NKGSK (SEQ ID NO: 36), NNGTK (SEQ ID NO: 37) and NNRSK (SEQ ID NO: 38); FW3b is the framework region; CDR3 is a CDR sequence having an amino acid sequence according to REARHPWLRQWY (SEQ ID NO: 39); FW4 is the framework area) The present invention provides a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprising an amino acid sequence represented by:

[0021] According to a third aspect, the present invention provides a recombinant fusion protein comprising a specific antigen-binding molecule according to the first or second aspect of the invention.

[0022] According to a fourth aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is the CDR sequence, FW2 is the framework region, HV2 is a hypervariable sequence, FW3a is the framework region, HV4 is a hypervariable sequence, FW3b is a framework region, CDR3 is a CDR sequence, FW4 is the framework area) or a functional variant thereof, wherein the antigen-binding molecule is fused to a fragment of an immunoglobulin Fc region, and the fragment of the immunoglobulin Fc region is engineered to dimerize with a second fragment of the immunoglobulin Fc region.

[0023] According to a fifth aspect, the present invention provides a method for producing a composition comprising the steps of: (a) a first recombinant fusion protein, the first recombinant fusion protein being a recombinant fusion protein according to the third or fourth aspect, and (b) a second recombinant fusion protein comprising a second antigen-binding molecule fused to a second fragment of an immunoglobulin Fc region that is engineered to dimerize with the first fragment of the immunoglobulin Fc region. The present invention provides a recombinant fusion protein dimer comprising:

[0024] According to a sixth aspect, the present invention provides a ROR1-specific chimeric antigen receptor (CAR) comprising at least one ROR1-specific antigen binding molecule as defined by the first or second aspect of the invention, fused or conjugated to at least one transmembrane region and at least one intracellular domain.

[0025] The present invention also provides a cell comprising a chimeric antigen receptor according to the sixth aspect, which is preferably an engineered T cell.

[0026] In a seventh aspect of the invention there is provided a nucleic acid sequence comprising a polynucleotide sequence encoding a 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 aspect of the invention.

[0027] Also provided is a vector comprising a nucleic acid sequence according to the seventh aspect and a host cell comprising such nucleic acid.

[0028] Provided is a method for preparing the 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 culturing or maintaining a host cell comprising the polynucleotide or vector under conditions such that the host cell produces the specific antigen-binding molecule, recombinant fusion protein, or chimeric antigen receptor, and optionally further comprising isolating the specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor.

[0029] In an eighth aspect of the present invention, a pharmaceutical composition is provided comprising the specific antigen-binding molecule, fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspect. The pharmaceutical composition may contain various pharma- ceutical acceptable carriers. The pharmaceutical composition of the present 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. In a preferred embodiment, the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule or foam.

[0030] The 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 may be for use in a therapeutic method. More specifically, the 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 may be for use in the treatment of cancer. Preferably, the cancer is a ROR1-positive cancer type. More preferably, the cancer is selected from the group comprising hematological cancers such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, kidney 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, esophageal cancer, gastric cancer or liver cancer.

[0031] There is also provided herein the use of the 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 in the manufacture of a medicament for the treatment of a disease in a patient in need thereof.

[0032] The specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth, sixth aspect or pharmaceutical composition of the eighth aspect may be administered in a single dose. As used herein, "single dose" refers to a dosage regimen consisting of one dose. Alternatively, a multiple dose regimen may be used. Without being bound by theory, 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 aspect or pharmaceutical composition of the eighth aspect may be particularly evident when administered in a single dose.

[0033] Further according to the present invention there is provided a method of treating a disease in a patient in need of such treatment, comprising administering to said patient a therapeutically effective dose of the 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, or the pharmaceutical composition of the eighth aspect.

[0034] Preferably, the cancer is a ROR1 positive cancer type. More preferably, the cancer is selected from the group including blood cancers such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML) and solid tumors including neuroblastoma, kidney 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, esophageal cancer, gastric cancer or liver cancer.

[0035] Also provided herein is a method of assaying for the presence of a target analyte in a sample, comprising adding a detectably labeled specific antigen-binding molecule of the first or second aspect, or a recombinant fusion protein of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect to a sample, and detecting binding of the molecule to the target analyte.

[0036] Further provided herein is a method of imaging a site of disease in a subject, the method comprising administering to the subject a detectably labeled specific antigen-binding molecule of the first or second aspect, or a detectably labeled recombinant fusion protein of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect.

[0037] Also provided herein is a method of diagnosing a disease or medical condition in a subject, the method comprising administering a specific antigen-binding molecule of the first or second aspect, or a recombinant fusion protein of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect.

[0038] Also contemplated herein are antibodies, antibody fragments or antigen-binding molecules that compete for binding to ROR1 with the ROR1-specific antigen-binding molecule of the first or second aspect. The term "compete" when used in relation to an antigen-binding protein (e.g., a neutralizing antigen-binding protein or neutralizing antibody) refers to competition between antigen-binding proteins as determined by an assay in which the antigen-binding protein under test (e.g., an antibody or functional fragment thereof) prevents or inhibits specific binding of an antigen-binding molecule as defined herein (e.g., the 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).

[0039] Also described herein is a kit for diagnosing a subject suffering from or susceptibility to cancer or for providing a prognosis of a condition in a subject, comprising a detection means for detecting the concentration of an antigen present in a sample from a test subject, the detection means comprising a ROR1-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect, a chimeric antigen receptor of the sixth aspect, or a nucleic acid sequence of the seventh aspect, each optionally derivatized, and the presence of the antigen in the sample indicates that the subject suffers from cancer. Preferably, the antigen comprises the ROR1 protein, more preferably the extracellular domain thereof. More preferably, the kit is used to identify the presence or absence of ROR1-positive cells in a sample or to determine their concentration in a sample. The kit may also comprise a positive control and / or a negative control to which the assay is compared and / or a label that can be detected.

[0040] The present invention also provides a method of diagnosing a subject suffering from or susceptibility to cancer or providing a prognosis of a condition in a subject, comprising detecting the concentration of an antigen present in a sample obtained from the subject, wherein the detection is achieved using a ROR1-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect, a chimeric antigen receptor of the sixth aspect, or a nucleic acid sequence of the seventh aspect, each optionally derivatized, and wherein the presence of the antigen in the sample is indicative that the subject is suffering from cancer.

[0041] Also contemplated herein is a method of killing or inhibiting the growth of a cell expressing ROR1 in vitro or in a patient, comprising administering to the cell a pharma- ceutical effective amount or dose of (i) a ROR1-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect, a nucleic acid sequence of the seventh aspect, or a CAR or cell according to the sixth aspect, or (ii) a pharmaceutical composition of the eighth aspect. Preferably, the cell expressing ROR1 is a cancer cell. More preferably, the ROR1 is human ROR1.

[0042] According to a ninth aspect, the present invention provides a compound of formula (II): X-FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4-Y (II) (In the formula, 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 specific antigen-binding molecule comprises an amino acid sequence represented by:

[0043] According to a tenth aspect, the present invention provides a method for producing a composition comprising the steps of: (a) a ROR1-specific antigen-binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein according to the third or fourth aspect, or a recombinant fusion protein dimer according to the fifth aspect, and (b) Anthracycline (PNU) derivatives A target binding molecule-drug conjugate comprising the formula (III): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, 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), peptide, -(CH2)n-, -(CH2CHO)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; and Y comprises a ROR1-specific antigen-binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein according to the third or fourth aspect, or a recombinant fusion protein dimer according to the fifth aspect. The present invention provides a target binding molecule-drug conjugate having the structure:

[0044] According to an eleventh aspect, the present invention provides a method for producing a composition comprising the steps of: (a) a ROR1-specific antigen-binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein according to the third or fourth aspect, or a recombinant fusion protein-dimeric recombinant fusion protein according to the fifth aspect, and (b) Anthracycline (PNU) derivatives A target binding molecule-drug conjugate comprising the formula (IV): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [Z] is a linker derived from a reactive group used to conjugate an anthracycline (PNU) derivative and a target binding molecule; and Y comprises a ROR1-specific antigen-binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein according to the third or fourth aspect, or a recombinant fusion protein dimer according to the fifth aspect. The present invention provides a target binding molecule-drug conjugate having the structure: [Brief description of the drawings]

[0045] [Figure 1] Design of the B1 Loop Library: The sequence of B1 is shown with "X" indicating amino acids within CDR1 and CDR3 that were randomized. [Diagram 2] Cell surface binding of B1 VNAR loop variants (His6Myc tagged) to A549 (ROR1hi) lung cancer cells by flow cytometry. [Diagram 3] Cell surface binding of B1 VNAR loop variants (His6Myc tagged) to A427 (ROR1low) 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. [Diagram 5] Binding of P3A1G1 loop variants to human ROR1 by ELISA. Data are plotted as the OD signal obtained at 450 nm for a fixed concentration (5 ug / mL) of each of the loop variants. [Figure 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 engineered cysteine ​​substitutions in the hIgG1 Fc sequence, e.g., at positions S239C or S442C (EU numbering), to allow for site-specific labeling. [Figure 8] Cell surface binding of B1 loop variant-hFc fusion proteins to A549(ROR1hi) lung cancer cells by flow cytometry. [Figure 9] Analysis of ROR1 biparatopic VNAR-hFc fusions by SDS-PAGE (4-12% Bis Tris gel, MOPS buffer, ±50 mM DTT). Lane 1 G3CP-P3A1 hFc(S239C+KIH) and lane 2 G3CPG4-P3A1 hFc(S239C+KIH). [Figure 10] Cell surface binding of ROR1 biparatopic VNAR-hFc fusions to A549 (ROR1hi) and A427 (ROR1low) lung cancer cells by flow cytometry. [Figure 11] Structures of PNU-linker payload MA-PEG-vc-PAB-EDA-PNU159682 and MA-PEG-vc-EDA-PNU159682. [Figure 12] Dose response showing binding of G3CP-hFc and G3CPG4-hFc PNU conjugates and the corresponding parent proteins to human ROR1 by ELISA. [Figure 13] Efficacy of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in killing ROR1 positive and ROR1 knockout PA-1 cell lines. [Figure 14]In vivo efficacy of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in the ROR1+HBCx-28 patient-derived TNBC xenograft model. Data plotted to the time when the first animal in the vehicle group reached humane tumor burden. [Figure 15] Sequence alignment for B1, B1 G4, B1V15, G3CP and G3CPG4. Points of variation in the CDR and HV regions are highlighted with underlines. Note that 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 96 hours of incubation at 37° C. in PBS pH 7.4 buffer. [Figure 17] Size exclusion analysis (SEC) of B1-hFc, B1G4-hFc, G3CP-hFc and G3CPG4-hFc after 96 hours of incubation at 37° C. in PBS pH 7.4 buffer. [Figure 18] Efficacy of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in killing ROR1low 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 a ROR1+HBCx-10 patient-derived TNBC xenograft model. Data plotted to the time point when the first animal in the vehicle group reached humane tumor burden. [Figure 20] Cell surface binding of ROR1 biparatopic VNAR-hFc drug conjugates to A549 (ROR1hi) and A427 (ROR1low) lung cancer cells by flow cytometry. [Figure 21] Efficacy of biparatopic G3CP-P3A1-hFc PNU and G3CPG4-P3A1-hFc PNU conjugates in killing ROR1-positive and ROR1-knockout PA-1 cell lines. [Figure 22] In vivo efficacy of biparatopic G3CP-P3A1 hFc PNU and G3CPG4-P3A1-hFc PNU conjugates in a ROR1+HBCx-28 patient-derived TNBC xenograft model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present invention generally relates to specific antigen binding molecules. Specifically, the present invention provides immunoglobulin-like shark variable novel antigen receptors (VNARs) specific for receptor tyrosine kinase-like orphan receptor 1 (ROR1) and related fusion proteins, chimeric antigen receptors, conjugates and nucleic acids and methods associated therewith. ROR1-specific VNAR domains are herein referred to as ROR1-specific antigen binding molecules.

[0047] Novel or neoantigen receptors (IgNARs) are homodimeric proteins of approximately 160 kDa found in the serum of cartilaginous fish (Greenberg AS, et al., Nature, 1995. 374(6518): p. 168-173; Dooley, H., et al., Mol. Immunol, 2003. 40(1): p. 25-33; Mueller, MR, et al., mAbs, 2012. 4(6): p. 673-685). Each molecule consists of a single N-terminal variable domain (VNAR) and five constant domains (CNARs). IgNAR domains are members of the immunoglobulin-superfamily. VNARs are tightly folded domains with structural and some sequence similarity to immunoglobulin and T-cell receptor variable domains and cell adhesion molecules, and are so named due to their similarity to the N-variable terminal domains of classical immunoglobulins and T-cell receptors. VNARs share limited sequence homology with immunoglobulins, e.g., 25-30% similarity between VNARs and human light chain sequences.

[0048] 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 provide an overview of the structural characterization and generation of VNARs and are incorporated herein by reference.

[0049] VNARs are not thought to have evolved from a classical immunoglobulin antibody ancestor. A distinguishing structural feature of VNARs is the truncation of sequences equivalent to the CDR2 loop present in conventional immunoglobulin variable domains and the lack of hydrophobic VH / VL interface residues not present in the IgNAR structure that would normally allow association with the light chain domain. Furthermore, unlike classical immunoglobulins, some VNAR subtypes contain extra cysteine ​​residues in the CDR regions, which are observed to form disulfide bridges in addition to the canonical immunoglobulin superfamily bridges between cysteines in the framework 1 and 3 regions adjacent N-terminally to CDR1 and 3.

[0050] To date, there are three defined types of shark IgNAR, known as types I, II and III, which are classified based on the location of non-canonical cysteine ​​residues that are under strong selection pressure and therefore rarely replaced.

[0051] All three types have classical immunoglobulin canonical cysteines at positions 35 and 107 (numbering as in Kabat, EA et al. Sequences of proteins of immunological interest. 5th ed. 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH) that stabilize the standard immunoglobulin fold, together with an invariant tryptophan at position 36. There is no defined CDR2 per se, but regions of sequence variation that correspond more closely to TCR HV2 and HV4 are defined in frameworks 2 and 3, respectively. Type I has germline-encoded cysteine ​​residues in frameworks 2 and 4, and an even number of additional cysteines in CDR3. Crystal structure studies of type I IgNARs isolated from and complexed with lysozyme have allowed the determination of the contribution of these cysteine ​​residues. Both cysteines in frameworks 2 and 4 form disulfide bridges with cysteines in CDR3, forming a tightly packed structure in which the CDR3 loop is held tightly down towards the HV2 region. To date, type I IgNAR has only been identified in the nurse shark; all other elasmobranchs, including members of the same order, have only type II or variations of this type.

[0052] Type II IgNARs are defined as having cysteine ​​residues in CDR1 and CDR3 that form intramolecular disulfide bonds that hold the two regions in close proximity, resulting in a protruding CDR3 that promotes a binding pocket or groove. Type I sequences typically have longer CDR3s than type II, averaging 21 and 15 residues, respectively. This is believed to be due to strong selection pressures that associate two or more cysteine ​​residues in type I CDR3 with their framework 2 and 4 counterparts. Studies of somatic mutation accumulation have shown that there are more mutations in CDR1 in type II than in type I, while the HV2 region of type I shows greater sequence variation than type II. This evidence correlates well with the determined positioning of these regions within the antigen-binding site.

[0053] A third IgNAR type, known as type III, has been identified in newborns. This member of the IgNAR family lacks diversity within the CDR3 due to germline fusion of the D1 and D2 regions (which form the CDR3) with the V gene. Nearly all known clones have a CDR3 length of 15 residues and little or no sequence diversity.

[0054] Another structural type of VNAR, designated type (IIb or IV), has only two canonical cysteine ​​residues (in the framework 1 and framework 3b regions). To date, this type has been found primarily in dogfish and was also isolated from a semi-synthetic V-NAR library derived from nurse shark.

[0055] The VNAR binding surface differs from the variable domains in other natural immunoglobulins in that it is derived from four diversity regions: CDR1, HV2, HV4 and CDR3, which are linked by intervening framework sequences in the order FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4. The combination of the lack of a natural light chain partner and the lack of CDR2 makes VNARs the smallest naturally occurring binding domain in the vertebrate kingdom.

[0056] IgNARs share some additional features with the heavy chain immunoglobulins (HCAbs) found in Camelidae (camels, dromedaries and llamas). Unlike IgNARs, HCAbs are clearly derived from the immunoglobulin family and share significant sequence homology with standard immunoglobulins. Importantly, one key distinction of VNARs is that the molecule, unlike classical immunoglobulins or HCAbs, has never had a partner light chain at any point in its evolution. Flajnik MF et al PLoS Biol 2011.9(8):e1001120 and Zielonka S. et al mAbs 2015.7(1):p.15-25 provide insight into the similarities and differences between VNARs and immunoglobulin-derived VHH single binding domains from Camelidae and their possible distinct evolutionary origins.

[0057] Although antibodies against ROR1 have been reported in the literature, the high sequence identity between the extracellular domains of human, mouse and rat ROR1 and between human ROR1 and ROR2 family members means that high affinity hROR1-specific binding agents are not readily generated. Furthermore, the large size of antibodies impairs their ability to penetrate solid tumors and renders regions of target proteins inaccessible due to steric factors, which may be particularly severe for cell surface proteins where oligomerization or receptor clustering is observed.

[0058] As a result, there is a need in the art for the development of improved anti-ROR1 binding protein agents that have functional or physical characteristics or properties distinct from antibodies, as well as therapeutic and diagnostic agents for malignancies associated with ROR1 expression. The present invention provides such agents in the form of the ROR1-specific antigen binding molecules described herein.

[0059] Without being bound by theory, it is believed that the presently described ROR1-specific antigen binding molecules bind to both human and mouse ROR1. A number of variants, such as G3CP, 1E5, 1B11, C3CP, 1G9, 1H8, G11CP, D9CP, 1B6, 1F10, F2CP, B6CP, 1E1, and 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 of the present invention can bind to the deglycosylated form of ROR1. Furthermore, they cannot bind to a number of linear peptides associated with anti-ROR1 antibodies described in the prior art. Thus, the presently described ROR1-specific antigen binding molecules are believed to bind to distinct epitopes in the ROR1 sequence compared to those prior art anti-ROR1 antibodies.

[0060] Binding and internalization of the ROR1-specific antigen binding molecules of the present invention into cancer cell lines has been demonstrated, supporting the potential use of such molecules in the treatment of cancer, particularly cancers that express ROR1.

[0061] Various forms of ROR1-specific antigen-binding molecules are described, including several types of fusion proteins. Both homo- and heterodimers and fusion proteins containing immunoglobulin Fc regions are also described. Fusing a protein to an Fc domain can improve protein solubility and stability, significantly increase plasma half-life, and improve overall therapeutic efficacy.

[0062] The inventors have also created VNAR molecules conjugated to various moieties and payloads. Thus, the present invention also provides chemically conjugated VNARs. More specifically, ROR1-specific antigen binding molecules in several conjugate formats are provided.

[0063] According to a first aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, CDR3 is 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), Y a CDR sequence having an amino acid sequence selected from the group consisting of PWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22) and YPWGAGAPWLVQWY (SEQ ID NO: 10); CDR1 is 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); FW1 is the framework region; FW2 is the framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO:6) and SSNKERISIS (SEQ ID NO:7); FW3a is the framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO:8) and NKGTM (SEQ ID NO:9); FW3b is the framework region; FW4 is the framework region; When CDR3 is YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLSA (SEQ ID NO: 3) and GANYDLAA (SEQ ID NO: 4) The present invention provides a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprising an amino acid sequence represented by:

[0064] In one embodiment of the ROR1 specific antigen binding molecule, the CDR3 is 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), The CDR sequence has an amino acid sequence selected from the group consisting of YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22) and YPWGAGAPWLVQWY (SEQ ID NO: 10).

[0065] When CDR3 is not YPWGAGAPWLVQWY (SEQ ID NO: 10), 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). Thus, a ROR1-specific antigen-binding molecule may have the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, 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), YPWGAGAPSQVQWY (SEQ ID NO:18), YPWGAGAPSSVQWY (SEQ ID NO:19), YPWGAGAPWQVQWY (SEQ ID NO:21) and YPWGAGAPWSVQWY (SEQ ID NO:22); CDR1 is 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); FW1 is the framework region; FW2 is the framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO:6) and SSNKERISIS (SEQ ID NO:7); FW3a is the framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO:8) and NKGTM (SEQ ID NO:9); FW3b is the framework region; FW4 is the framework area) It may be defined as including the amino acid sequence represented by:

[0066] In one embodiment of the ROR1-specific antigen binding molecule, 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).

[0067] In one embodiment of the ROR1-specific antigen binding molecule, the CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23).

[0068] 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 the 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).

[0069] 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 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).

[0070] In one embodiment of the ROR1-specific antigen binding molecule, 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).

[0071] In a preferred embodiment, the ROR1-specific antigen-binding molecule is Herein referred to as G3CP (C5) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO:50); Herein referred to as B1G4 (6) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO:51); Herein referred to as 1E2 (C7) ASVNQTPRTATKETGESLTINCVVTGANYDLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPSGAGAPRPVQWYDGAGTVLTVN (SEQ ID NO:52); Herein referred to as 1E5 (8) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPCLVQWYDGAGTVLTVN (SEQ ID NO:53); Herein referred to as 1B11 (9) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRLVQWYDGAGTVLTVN (SEQ ID NO:54); Herein referred to as C3CP (C10) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRQVQWYDGAGTVLTVN (SEQ ID NO:55); Herein referred to as 2G5 (Chem.11) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRSVQWYDGAGTVLTVN (SEQ ID NO:56); Herein referred to as 1G12 (C12) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRSVQWYDGAGTVLTVN (SEQ ID NO:57); Herein referred to as G5CP (C13) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSLVQWYDGAGTVLTVN (SEQ ID NO:58); Referred to herein as 2F4 (C14) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSNVQWYDGAGTVLTVN (SEQ ID NO:59); Herein referred to as 1G9 (C15) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSQVQWYDGAGTVLTVN (SEQ ID NO: 60); Herein referred to as 1H8 (C16) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN (SEQ ID NO: 61); Herein referred to as G11CP (C17) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 62); Herein referred to as D9CP (C18) ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 63); Herein referred to as 1B6 (C19) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWNVQWYDGAGTVLTVN (SEQ ID NO: 64); Herein referred to as 1F10 (20) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 65); Herein referred to as E6CP (21) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 66); Herein referred to as F2CP (22) ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 67); Herein referred to as B6CP (23) ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 68); Herein referred to as 1G1 (24) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 69); and Herein referred to as A10CP (25) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 70) or CDR1, HV2, HV4 and CDR2 sequences selected from the group consisting of:

[0072] In a particularly preferred embodiment, the ROR1-specific antigen-binding molecule is (C26) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50) It may comprise an amino acid sequence according to

[0073] ROR1-specific antigen-binding molecules (27) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (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 at least 45% combined sequence identity with the combined FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO:50.

[0074] Particular advantages associated with SEQ ID NO:50 ("G3CP") and its functional variants include increased expression yields and hydrophilicity in non-optimized aqueous buffer systems for these proteins, as well as ease of analysis, purification, and increased monomericity. Without being bound by theory, these advantages may be particularly evident in VNAR-hFc fusion proteins that include the G3CP sequence or a functional variant thereof. Thus, the G3CP sequence and its functional variants may provide improved manufacturing and / or handling properties. Furthermore, G3CP-hFc shows superior in vivo efficacy in patient-derived xenograft models of triple-negative breast cancer (TNBC) when conjugated to a cytotoxic anthracycline (PNU) derivative. Surprisingly, the efficacy of G3CP-hFc is also improved over B1-hFc, which itself shows superior in vivo efficacy.

[0075] In a particularly preferred embodiment, the ROR1-specific antigen-binding molecule is (28) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51) It may comprise an amino acid sequence according to

[0076] Particular advantages associated with SEQ ID NO:51 ("B1G4") and its functional variants include increased expression yields and monomericity in aqueous buffer systems for fusion proteins, such as VNAR-hFc fusion proteins, that include the B1G4 sequence or functional variants thereof. Thus, the B1G4 sequence and its functional variants may provide fusion proteins with improved manufacturing and / or handling properties.

[0077] ROR1-specific antigen-binding molecules (C29) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (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 at least 45% combined sequence identity with the combined FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO:51.

[0078] In a preferred embodiment, the ROR1-specific antigen-binding molecule is Herein referred to as G3CP G4 (30) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO:71); Herein referred to as G3CP V15 (31) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK (SEQ ID NO:72); Herein referred to as 1H8 G4 (32) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIK (SEQ ID NO:73); Herein referred to as 1H8 V15 (33) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVK (SEQ ID NO:74); Herein referred to as C3CP G4 (34) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPRQVQWYDGAGTKVEIK (SEQ ID NO: 75); and Herein referred to as C3CPV15 (35) ASVTQSPRSASKETGESLITITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPRQVQWYDGQGTKLEVK (SEQ ID NO: 76) or CDR1, HV2, HV4 and CDR2 sequences selected from the group consisting of:

[0079] In a particularly preferred embodiment, the ROR1-specific antigen-binding molecule is (36) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) It may comprise an amino acid sequence according to

[0080] ROR1-specific antigen-binding molecules (37) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (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 at least 45% combined sequence identity with the combined FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO: 71.

[0081] Particular advantages associated with SEQ ID NO: 71 ("G3CP G4") and its functional variants include increased expression yields and hydrophilicity in non-optimized aqueous buffer systems for those proteins, as well as ease of analysis, purification, and increased monomericity. Without being bound by theory, these advantages may be particularly evident in VNAR-hFc fusion proteins that include the G3CP G4 sequence or a functional variant thereof. Thus, the G3CP G4 sequence and its functional variants may provide improved manufacturing and / or handling properties. Furthermore, G3CPG4-hFc shows superior in vivo efficacy in patient-derived xenograft models of triple-negative breast cancer (TNBC) when conjugated to a cytotoxic anthracycline (PNU) derivative. Surprisingly, the efficacy of G3CPG4-hFc is also improved over B1-hFc, which itself shows superior in vivo efficacy.

[0082] The sequences of G3CP and G3CPG4 share two single amino acid changes relative to the sequence of B1, both of which are located within CDR3: 1. W residue in Y residue, and 2. L residue at N residue It is a substitution.

[0083] Compared to G3CP, G3CPG4 has an additional single amino acid change in each of CDR1, HV2 and HV4 relative to B1 (which also appears in B1 G4) as well as changes that humanize the framework regions (some of which also appear in B1V15, SEQ ID NO:115, as shown in Figure 15, where B1V15 has the same CDR1, HV2, HV4 and CDR3 sequences as B1, i.e., it is not a loop library variant; the changes in B1V15 relative to B1 are only in the framework regions).

[0084] Without being bound by theory, it is believed that any improvement over B1 exhibited by both G3CP and G3CPG4 that is not exhibited by either B1 G4 or B1 V15 arises from one or both of the two mutations that they share in CDR3. Thus, it is believed that the advantage of G3CP and G3CPG4 derives from the CDR3 containing the sequence YPWGAGAPYNVQWY (SEQ ID NO:23).

[0085] Without being bound by theory, the surprising advantage associated with YPWGAGAPYNVQWY (SEQ ID NO: 23) may represent a synergistic effect of both the W to Y and L to N substitutions. Alternatively, the surprising advantage may be derived primarily from the W to Y substitution and is therefore shared by YPWGAGAPYLVQWY (SEQ ID NO: 20). 1B6 has an L to N mutation and the CDR3 sequence of YPWGAGAPWNVQWY (SEQ ID NO: 24) has a lower elution volume than B1, therefore the L to N mutation in CDR3 results in improved manufacturing and / or handling properties.

[0086] According to a second aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, 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) and GTKYGLYAS (SEQ ID NO: 30); FW1 is the framework region; FW2 is the framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERISIS (SEQ ID NO:31), STDEERISIG (SEQ ID NO:32), SPNKDRMIIG (SEQ ID NO:33), and STDKERIIIG (SEQ ID NO:34); FW3a is the framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKGTK (SEQ ID NO: 35), NKGSK (SEQ ID NO: 36), NNGTK (SEQ ID NO: 37) and NNRSK (SEQ ID NO: 38); FW3b is the framework region; CDR3 is a CDR sequence having an amino acid sequence according to REARHPWLRQWY (SEQ ID NO: 39); FW4 is the framework area) The present invention provides a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprising an amino acid sequence represented by:

[0087] In a preferred embodiment, the ROR1-specific antigen-binding molecule is Herein referred to as P3A1 G1 AE3 (38) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO:77); Herein referred to as P3A1 G1 AE3.S (39) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO:78); Herein referred to as P3A1 G1 NAC6 (40) TRVDQSPSSLSASVGDRVTITCVLTDTRYALYSTYWYRKNPGSTDEERISIGGRYSESVNKGSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO:79); Referred to herein as P3A1 G1 NAC6.S (C41) TRVDQSPSSLSASVGDRVTITCVLTDTRYALYSSTYWYRKNPGSTDEERISIGGRYSESVNKGSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 80); Herein referred to as P3A1 G1 NAG8 (C42) TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYATYWYRKNPGSPNKDRMIIGGRYSESVNNGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 81); Referred to herein as P3A1 G1 NAG8.S (C43) TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYASTYWYRKNPGSPNKDRMIIGGRYSESVNNGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 82); Herein referred to as P3A1 G1 AF7.S (C44) TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYASTYWYRKNPGSTDKERIIIGGRYSESVNNRSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 83) or CDR1, HV2, HV4 and CDR2 sequences selected from the group consisting of:

[0088] Particular advantages associated with affinity matured variants of P3A1G1 and functional variants thereof include improved binding to hROR1-Fc compared to the parent P3A1G1, as illustrated by the Examples.

[0089] The ROR1-specific antigen-binding molecule may comprise the CDR and HV sequences of the clones shown in Table 1 below. In the first and / or second preferred embodiment of the present invention, the ROR1-specific antigen-binding molecule has any combination sequence of the clones shown in Table 1 below.

[0090] [Table 1] JPEG2024500405000005.jpg199170JPEG2024500405000006.jpg206170JPEG2024500405000007.jpg132170

[0091] The ROR1-specific antigen-binding molecule may comprise the CDR and HV sequences of the clones shown in Table 2 below. In the first and / or second preferred embodiments of the present invention, the ROR1-specific antigen-binding molecule has any combination sequence of the clones shown in Table 2 below.

[0092] [Table 2] JPEG2024500405000009.jpg87170

[0093] All possible combinations and permutations of the framework regions, complementarity determining regions and hypervariable regions listed herein are expressly contemplated herein.

[0094] The sequence identity referred to in relation to the molecules of the invention can be determined at the level of individual CDRs, HVs or FWs, combined CDRs, HVs or FWs, or it can be determined over the length of the entire molecule. The CDR, HV and FW sequences described can be longer or shorter, whether due to addition or deletion of amino acids at the N- or C-terminus of the sequence, or insertion or deletion of amino acids in the sequence.

[0095] The framework region FW1 is preferably 20 to 28 amino acids long, more preferably 22 to 26 amino acids long, and even more preferably 23 to 25 amino acids long. In one preferred embodiment, FW1 is 26 amino acids long. In another preferred embodiment, FW1 is 25 amino acids long. In another even more preferred embodiment, FW1 is 24 amino acids long.

[0096] In an alternative definition, the CDR region CDR1 is preferably 7 to 11 amino acids long, more preferably 8 to 10 amino acids long. In one preferred embodiment, CDR1 is 9 amino acids long. In another preferred embodiment, CDR1 is 8 amino acids long.

[0097] The framework region FW2 is preferably 6 to 14 amino acids long, more preferably 8 to 12 amino acids long. In one preferred embodiment, FW2 is 12 amino acids long. In another preferred embodiment, FW2 is 10 amino acids long. In another preferred embodiment, FW2 is 9 amino acids long. In another preferred embodiment, FW2 is 8 amino acids long.

[0098] In alternative definitions, the hypervariable sequence HV2 is preferably 4 to 11 amino acids long, more preferably 5 to 10 amino acids long. In one preferred embodiment, HV2 is 10 amino acids long. In one preferred embodiment, HV2 is 9 amino acids long. In another preferred embodiment, HV2 is 6 amino acids long.

[0099] The framework region FW3a is preferably 6 to 10 amino acids long, more preferably 7 to 9 amino acids long. In a preferred embodiment, FW3a is 8 amino acids long. In a preferred embodiment, FW3a is 7 amino acids long.

[0100] In an alternative definition, the hypervariable sequence HV4 is preferably 3 to 7 amino acids long, more preferably 4 to 6 amino acids long. In one preferred embodiment, HV4 is 5 amino acids long. In another preferred embodiment, HV4 is 4 amino acids long.

[0101] The framework region FW3b is preferably 17 to 24 amino acids long, more preferably 18 to 23 amino acids long, and even more preferably 19 to 22 amino acids long. In one preferred embodiment, FW3b is 21 amino acids long. In another preferred embodiment, FW3b is 20 amino acids long.

[0102] In an alternative definition, the CDR region CDR3 is preferably 8 to 21 amino acids long, more preferably 9 to 20 amino acids long, even more preferably 10 to 19 amino acids long. In one preferred embodiment, CDR3 is 17 amino acids long. In another preferred embodiment, CDR3 is 14 amino acids long. In yet another preferred embodiment, CDR3 is 12 amino acids long. In another even more preferred embodiment, CDR3 is 10 amino acids long.

[0103] The framework region FW4 is preferably 7 to 14 amino acids long, more preferably 8 to 13 amino acids long, and even more preferably 9 to 12 amino acids long. In one preferred embodiment, FW4 is 12 amino acids long. In another preferred embodiment, FW4 is 11 amino acids long. In another even more preferred embodiment, FW4 is 10 amino acids long. In yet another preferred embodiment, FW4 is 9 amino acids long.

[0104] In one embodiment of the ROR1-specific antigen binding molecule, FW1 is a framework region of 20-28 amino acids; FW2 is a framework region of 6 to 14 amino acids; FW3a is a framework region of 6-10 amino acids; FW3b is a framework region of 17 to 24 amino acids; and / or FW4 is a framework region of 7 to 14 amino acids.

[0105] In one embodiment of the ROR1-specific antigen binding molecule, 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 any functional variant thereof having at least 45% sequence identity; FW2 has an amino acid sequence according to TYWYRKNPG (SEQ ID NO: 43) or any functional variant thereof having at least 45% sequence identity; FW3a has an amino acid sequence selected from the group consisting of GRYVESV (SEQ ID NO: 44) and GRYSESV (SEQ ID NO: 45), or any functional variant thereof having at least 45% sequence identity; FW3b has an amino acid sequence selected from the group consisting of SFSLRIKDLTVADSATYYCKA (SEQ ID NO: 84), SFTLTISSLQPEDSATYYCRA (SEQ ID NO: 46) and SFSLRISSLTVEDSATYYCKA (SEQ ID NO: 47), or any functional variant thereof having at least 45% sequence identity; and / or FW4 has an amino acid sequence selected from the group consisting of DGAGTVLTVN (SEQ ID NO: 48), DGAGTKVEIK (SEQ ID NO: 49) or DGQGTKLEVK (SEQ ID NO: 85), or any functional variant thereof having at least 45% sequence identity.

[0106] The ROR1-specific antigen-binding molecules of the present invention may be humanized. The ROR1-specific antigen-binding molecules of the present invention may be deimmunized. The B1 loop variants on the humanized backbone G4 and V15 described herein are humanized. If P3A1G1 is already humanized, all loop variants of P3A1G1 are humanized. Examples of humanized sequences of the present 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 Examples include:

[0107] It will be appreciated by those skilled in the art that the humanized ROR1-specific antigen binding molecules described herein can be further humanized, for example, by replacing additional FW region amino acids with amino acids of DPK-9.

[0108] The ROR1-specific antigen binding molecules of the present invention may be conjugated to a detectable label, dye, toxin, drug, prodrug, radionuclide or biologically active molecule.

[0109] Preferably, 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 mouse ROR1 (mROR1). Even more preferably, the ROR1-specific antigen-binding molecule binds to deglycosylated ROR1.

[0110] A ROR1-specific antigen-binding molecule of the present invention is YMESLHMQGEIENQI (SEQ ID NO: 91) CQPWNSQYPHTHTFTALRFP (SEQ ID NO: 92) RSTIYGSRLRIRNLDTTDTGYFQ (SEQ ID NO: 93) QCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYE (SEQ ID NO: 94) The compound is unable to bind to a linear peptide sequence selected from the group consisting of:

[0111] Preferably, the ROR1-specific antigen-binding molecule selectively interacts with the ROR1 protein with an affinity constant of approximately 0.01-50 nM, preferably 0.1-30 nM, even more preferably 0.1-10 nM. The affinity constant can be measured by biolayer interferometry (BLI). For monomers, the interaction is 1:1. For the VNAR-hFc format, the inventors used two approaches. One is to immobilize ROR1, so that the bivalent VNAR-hFc binds with an apparent KD due to the avidity effect. The other approach is a 1:1 format, where VNAR-hFc is immobilized and ROR1 is flowed over the surface, thus giving a KD for a "true" 1:1 binding. Typically, as used herein, affinity constant refers to that measured by biolayer interferometry (BLI) using a 1:1 binding format. By this method, for example, G3CP and G3CP G4 are in the range of 0.1-10 nM. Among the P3A1 G1 loop variants, examples have KD values ​​of 5.0 nM (AE3), 13.8 nM (NAC6) and 12.2 nM (NAG8).

[0112] Furthermore, ROR1-specific antigen binding molecules may preferably mediate killing of ROR1-expressing tumor cells or inhibit cancer cell proliferation.

[0113] The ROR1-specific antigen-binding molecule may also be endocytosed upon binding to ROR1. In other embodiments, the ROR1-specific antigen-binding molecule may not be endocytosed upon binding to ROR1.

[0114] In a third aspect of the present invention, a recombinant fusion protein is provided comprising the specific antigen-binding molecule of the first or second aspect. Preferably, in the recombinant fusion protein of the third aspect, the 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]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). These linkers may be particularly useful when the recombinant fusion protein is expressed in expression systems with different glycosylation patterns, such as CHO and insect, as well as expression systems that do not glycosylate the expressed protein (e.g., E. coli). Any recombinant fusion protein sequence disclosed herein that includes a [G4S]3 linker may alternatively have any other linker sequence disclosed herein.

[0115] It is also recognized that the fusion proteins of the present invention can be constructed in any order, i.e., the ROR1-specific antigen-binding molecule is present at the N-terminus, C-terminus, or not at either terminus (e.g., internal to a longer amino acid sequence).

[0116] Preferred biologically active proteins include, but are not limited to, immunoglobulins, immunoglobulin Fc regions, fragments of immunoglobulin Fc regions, Fc heavy chains, CH2 regions, CH3 regions, immunoglobulin Fab regions, Fab', Fv, Fv-Fc, single chain Fv (scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific t cell engagers, inteins, VNAR domains, single domain antibodies (sdAbs), VH domains, or scaffold proteins (such as affibodies, centyrin, darpin, etc.). Another preferred biologically active protein is an immunoglobulin Fc region. Other preferred fusion proteins include VNAR-VNAR and VNAR-VNAR-VNAR.

[0117] In one embodiment, the at least one biologically active protein is an immunoglobulin Fc region.

[0118] Thus, 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 or SEQ ID NO:185. G3CP-hFc (C45) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 186) G3CPG4-hFc (C46) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 187) 1H8-hFc (Formula 47) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 183) 1H8 G4-hFc (Formula 48) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 184) 1H8 V15-hFc (C49) ASVTQSPRSASKETGESLTITCRVTGNYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 185)

[0119] In a further embodiment, the at least one biologically active protein is an immunoglobulin Fc region further modified to include an S to C mutation.

[0120] Thus, 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. G3CP-hFc(S239C) (50) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 178) G3CPG4-hFc(S239C) (51) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 179) 1H8-hFc(S239C) (52) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 180) 1H8 G4-hFc(S239C) (Formula 53) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 181) 1H8 V15-hFc(S239C) (Formula 54) ASVTQSPRSASKETGESLTITCRVTGNYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 182)

[0121] In one embodiment, 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.

[0122] In one embodiment, the fragment of an immunoglobulin Fc region is an Fc heavy chain.

[0123] In one embodiment, a fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region.

[0124] As used herein, an immunoglobulin Fc region that is "engineered to dimerize" may contain at least one amino acid substitution. Typically, the at least one amino acid substitution promotes and / or makes more energetically favorable the interaction and / or association of the immunoglobulin Fc region with a second fragment, which therefore promotes and / or makes more energetically favorable dimerization. Such recombinant fusion proteins may have particular utility in the preparation of bispecific and / or biparatopic binders.

[0125] Methods are known in the art for generating Fc-based bispecific and / or biparatopic binders through pairing of two distinct Fc heavy chains that are engineered to dimerize. These methods allow for the assembly of Fc regions 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 multispecific binders and / or to different regions or epitopes on the same target to generate biparatopic binding proteins. Multiple binding domains or sequences can be fused to the Fc region to generate multispecific or multiparatopic binders or both multispecific multiparatopic binders within the same protein. Methods for generating these asymmetric bispecific and / or biparatopic binders via heterodimerization of two different Fc heavy chains or fragments thereof include, but are not limited to, knobs-into-holes (YT), 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. See, e.g., 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 1868 140250; each of which is incorporated herein by reference in its entirety.

[0126] In one embodiment, a fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs-into-holes (YT), 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. Features of each of these methods are detailed in relation to the fourth aspect of the invention and are contemplated to be relevant to the third aspect of the invention as well.

[0127] In one embodiment, one or more residues of the fragment of an immunoglobulin Fc region contain one or more amino acid substitutions suitable for heterodimerization with a second fragment of an immunoglobulin Fc region that contains one or more corresponding amino acid mutations.

[0128] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.

[0129] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0130] Any part of the fusion protein of the present invention may be engineered to allow conjugation. In a preferred example where an immunoglobulin Fc region is used, 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. In some embodiments, any of the fusion proteins disclosed herein may include an S239C point mutation. In some embodiments, any of the fusion proteins disclosed herein may include an S442C point mutation. In some embodiments, any of the fusion proteins disclosed herein may include both an S239C and an S442C point mutation. It is expressly contemplated herein that any of the sequences of the fusion proteins disclosed herein may be modified to include an S239C and / or an S442C point mutation.

[0131] According to a third aspect, a recombinant fusion comprising multiple VNAR domains is provided. Thus, the recombinant fusion of the present invention may be a dimer, trimer or higher multimer of VNAR. In such a recombinant fusion, the specificity of each VNAR may be the same or different. The recombinant fusion of the present invention includes, but is not limited to, bispecific or trispecific molecules in which each VNAR domain binds to a different antigen or binds to a different epitope on a single antigen (biparatopic binders). The term "biparatopic" as used herein is intended to encompass molecules that bind to multiple epitopes on a given antigen. Molecules that bind to three or more epitopes on a given antigen are also contemplated herein, and it should be understood that when the term "biparatopic" is used, the possibility of triparatopic or multiparatopic molecules is also contemplated.

[0132] According to a third aspect, there is also provided a recombinant fusion comprising a ROR1-specific antigen binding molecule of the first aspect and a humanized VNAR domain, which may be referred to as a soloMER, including but not limited to VNAR BA11, a humanized VNAR that binds human serum albumin with high affinity.

[0133] Examples of biparatopic and multivalent fusion proteins include, but are not limited to, B1-G3CP G3CP-BA11 BA11-G3CP 1H8-BA11 ·BA11-1H8 G3CP V15-BA11 G3CP G4-BA11 B1-G3CP Cys G3CP-BA11 Cys BA11-G3CP Cys 1H8-BA11 Cys BA11-1H8 Cys G3CP V15-BA11 Cys G3CP G4-BA11 Cys P3A1G1AE3-(L2)-G3CPG4 G3CPG4(L2)--P3A1G1AE3 P3A1G1AE3-(L2)-G3CPG4 Cys G3CPG4-(L2)-P3A1G1AE3 Cys P3A1-(L2)-BA11-(L2)-G3CP P3A1-(L2)-G3CP-(L2)-BA11 BA11-(L2)-G3CP-(L2)-P3A1 BA11-(L2)-P3A1-(L2)-G3CP P3A1-(L2)-BA11-(L2)-1H8 BA11-(L2)-P3A1-(L2)-1H8 ·P3A1-(L2)-BA11-(L2)-G3CP Cys ·P3A1-(L2)-G3CP-(L2)-BA11 Cys ·BA11-(L2)-G3CP-(L2)-P3A1 Cys ·BA11-(L2)-P3A1-(L2)-G3CP Cys P3A1-(L2)-BA11-(L2)-1H8 Cys ·BA11-(L2)-1P3A1-(L2)-1H8 Cys The following are some of the reasons: G3CP is (55) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50) and 1H8 is, (56) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN (SEQ ID NO: 61) and G3CP G4 is (57) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) and The G3CP V15 is (58) ASVTQSPRSASKETGESLITITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK (SEQ ID NO: 72) and BA11 is, (59) TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (SEQ ID NO: 95) and P3A1 G1 AE3 is (60) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO:77) and If the linker is not defined, (-) corresponds to the linker Wobbe-G5S, which in turn is PGVQPSPGGGGGS (SEQ ID NO:96); -(L2)- corresponds to the linker Wobbe-G4S-GM, which in turn is PGVQPAPGGGGS (SEQ ID NO: 90); Cys- corresponds to a Cys-containing C-terminal tag, for example, QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO:97).

[0134] Recombinant biparatopic fusion protein dimers can also be made by fusing any of the recombinant fusion proteins disclosed herein, particularly the loop library variants disclosed herein, onto one arm of an Fc fusion, and a binder to a different ROR1 epitope onto the other.

[0135] In some embodiments, the specific binding molecules or recombinant fusions of the present invention can be expressed with N- or C-terminal tags to aid in purification. Examples include, but are not limited to, His6 and / or Myc. Furthermore, the N- or C-terminal tags can be further engineered to contain additional cysteine ​​residues to serve as conjugation points. Thus, it is recognized that references to specific binding molecules or recombinant fusions in all aspects of the present invention are intended to encompass such molecules with various N- or C-terminal tags, which may also contain additional cysteines for conjugation.

[0136] Additional recombinant fusions are listed below. It is recognized that not all combinations of linkers and VNARs or fusion partners are listed below. However, all such combinations are expressly encompassed by the present invention.

[0137] [Table 3]

[0138] The linkers between the VNAR domains are preferentially, but not limited to, (G4S)5 (SEQ ID NO:87), (G4S)3 (SEQ ID NO:86), (G4S)7 (SEQ ID NO:116), PGVQPSPGGGGS (SEQ ID NO:89) (Wobbe-G4S), PGVQPAPGGGGS (SEQ ID NO:90) (Wobbe-G4S GM), PGVQPCPGGGGGS (SEQ ID NO:177) (WobbeCys-G4S), and different combinations of different linkers can be combined in the same construct. The WobbeCys-G4S sequence also contains a single cysteine ​​residue to facilitate site-selective bioconjugation of payloads to proteins in this linker using a thiol-mediated chemical coupling strategy. The use of this linker sequence for bioconjugation is advantageous because reoxidation and capping of reduced cysteines is minimal, resulting in high-yield conversion of proteins to the corresponding conjugates in the bioconjugation reaction.

[0139] Thus, an additional C-terminal (or N-terminal) tag sequence may or may not be present.

[0140] C-terminal tags include, but are not limited to, tags containing a poly-histidine sequence (e.g., His6) to facilitate purification, tags containing a c-Myc sequence (e.g., EQKLISEEDL (SEQ ID NO: 112)) to allow detection, and / or tags containing a cysteine ​​residue to allow labeling 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) The following are some of the reasons: G3CP is (61) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50) and G3CP G4 is (62) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) and BA11 is, (63) TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (SEQ ID NO: 95) and P3A1 G1 AE3 is (64) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO:77) and B1G4 is, (65) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51) It is.

[0141] As stated above, all combinations of VNARs and linkers are expressly encompassed herein. Humanized derivatives of VNARs are also encompassed herein.

[0142] According to a third aspect, there is also provided a recombinant fusion comprising the ROR1-specific antigen binding molecule of the first aspect and a recombinant toxin, examples of which include, but are not limited to, Pseudomonas exotoxin PE38 and diphtheria toxin.

[0143] According to a third aspect, there is also provided a recombinant fusion comprising the ROR1-specific antigen binding molecule of the first aspect and a recombinant CD3 binding protein. Examples of recombinant ROR1 and CD3 binding agents include, but are not limited to: B1G4-[WGM]-CD3 (66) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (SEQ ID NO: 117) G3CP-[WGM]-CD3 (67) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (SEQ ID NO: 118) G3CPG4-[WGM]-CD3 (68) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (SEQ ID NO: 119) P3A1G1AE3-[WGM]-CD3 (69) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (SEQ ID NO: 120) B1G4-[WGM]-BA11-[G4S]-CD3 (70) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIKPGVQPAPGGGGS TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELAR PGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (SEQ ID NO: 121) G3CP-[WGM]-BA11-[G4S]-CD3 (71) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 122) G3CPG4-[WGM]-BA11-[G4S]-CD3 (Chemical formula 72) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKPGVQPAPGGGGS TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELAR PGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (SEQ ID NO: 123) P3A1G1AE3-[WGM]-BA11-[G4S]-CD3 (73) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGST RVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARP GASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (SEQ ID NO: 124) P3A1-[WGM]-G3CP-[G4S]-CD3 (74) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNPGVQPAPGGGGSASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 125) P3A1-[WGM]-G3CPG4-[G4S]-CD3 (Chemical Formula 75) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNPGVQPAPGGGGSTRV DQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSDIKLQQSGAEL ARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (sequence number 126) P3A1G1AE3-[WGM]-G3CP-[G4S]-CD3 (76) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 127) P3A1G1AE3-[WGM]-G3CPG4-[G4S]-CD3 (Chemical Formula 77) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 128) P3A1G1AE3-[WGM]-D3-[G4S]-CD3 (Chemical formula 78) TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSASVN QTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAISTGSGHGYNWYDGAGTVLTVNGGGGSDIKLQQSGAE LARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH (sequence number 129) Examples include:

[0144] Any CD3 binding sequence known in the art and variants thereof can be substituted for the above. For example, UCL OKT3 sequence (International Publication No. 2019008379) (79) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYCLDYWGQGTMVTVSSVEGGSGGSGGSGGSGGVDDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPFTFGQGTKVEIK (SEQ ID NO: 130) Harpoon ID20 (International Publication No. 2016187594 Brochure) (80) DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 131) It is.

[0145] According to a fourth aspect, the present invention provides a compound of formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, FW1 is the framework region, CDR1 is the CDR sequence, FW2 is the framework region, HV2 is a hypervariable sequence, FW3a is the framework region, HV4 is a hypervariable sequence, FW3b is a framework region, CDR3 is a CDR sequence, FW4 is the framework area) or a functional variant thereof, wherein the antigen-binding molecule is fused to a fragment of an immunoglobulin Fc region, and the fragment of the immunoglobulin Fc region is engineered to dimerize with a second fragment of the immunoglobulin Fc region.

[0146] In one embodiment, the fragment of an immunoglobulin Fc region is selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[0147] In one embodiment, the fragment of an immunoglobulin Fc region is an Fc heavy chain.

[0148] The Fc region may be engineered to reduce FcγR binding. Thus, the Fc regions disclosed herein may be engineered to reduce FcγR binding.

[0149] As used herein, an immunoglobulin Fc region that is "engineered to dimerize" may contain at least one amino acid substitution. Typically, the at least one amino acid substitution promotes and / or makes more energetically favorable the interaction and / or association of the immunoglobulin Fc region with a second fragment, which therefore promotes and / or makes more energetically favorable dimerization. Such recombinant fusion proteins may have particular utility in the preparation of bispecific and / or biparatopic binders.

[0150] Methods are known in the art for generating Fc-based bispecific and / or biparatopic binders through pairing of two distinct Fc heavy chains that are engineered to dimerize. These methods allow for the assembly of Fc regions 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 multispecific binders and / or to different regions or epitopes on the same target to generate biparatopic binding proteins. Multiple binding domains or sequences can be fused to the Fc region to generate multispecific or multiparatopic binders or both multispecific multiparatopic binders within the same protein. Methods for generating these asymmetric bispecific and / or biparatopic binders via heterodimerization of two different Fc heavy chains or fragments thereof include, but are not limited to, knobs-into-holes (YT), 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. See, e.g., 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 1868 140250; each of which is incorporated herein by reference in its entirety.

[0151] In one embodiment, a fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs-into-holes (YT), knobs-into-holes (CW-CSAV), CH3 charge pairing, Fab arm exchange, SEED technology, BEAT technology, HA-TF, the ZW1 approach, the Biclonic approach, EW-RVT, and Triomab.

[0152] Knobs-into-holes (YT) can comprise a T366Y substitution in the first CH3 domain and a Y407T substitution in the second CH3 domain.

[0153] Knobs-into-holes (CW-CSAV) may include one or more (preferably all) of the following substitutions in the first CH3 domain: S354C, T366W. Knobs-into-holes (CW-CSAV) may include one or more (preferably all) of the following substitutions in the second CH3 domain: Y349C, T366S, L368A, Y407V. Knobs-into-holes (CW-CSAV) may include a disulfide bond in CH3.

[0154] The CH3 charge pairing may include one or more (preferably all) of the following substitutions in the first CH3 domain: K392D, K409D. The CH3 charge pairing may include one or more (preferably all) of the following substitutions in the second CH3 domain: E356K, D399K.

[0155] Fab arm exchange can include a K409R substitution in the first CH3 domain and a F405L substitution in the second CH3 domain. Fab arm exchange and DuoBody capture the same Fc changes. Thus, DuoBody technology can include a K409R substitution in the first CH3 domain and a F405L substitution in the second CH3 domain.

[0156] SEED technology can incorporate known substitutions and / or result in IgG / A chimeras. Complementarity in the CH3 interface that allows heterodimeric assembly of Fc chains was developed by designing strand-exchange engineered domain (SEED) heterodimers. These SEED CH3 domains are composed of alternating segments (AG SEED CH3 and GA SEED CH3) derived from human IgA and IgG CH3 sequences and were used to generate so-called SEED bodies, Davis et al (2010) PEDS 23,4,195-202, incorporated herein by reference in its entirety. Molecular models suggested that interaction with FcRn was compromised in AG SEED CH3, so residues at the CH2-CH3 junction were reverted to the IgG sequence. Pharmacokinetic studies confirmed that the half-life of SEED bodies was comparable to other Fc fusion proteins and IgG1.

[0157] The BEAT technology engineered constant α and β domains of the human T cell receptor into the IgG1 CH3 dimer interface to drive heterodimerization (Skegro et al (2017) JBC 292 (23) 9745-9759). An additional D410Q mutation could further increase heterodimer formation in this system (Stutz & Blein 2020 JBC 295 (28) 9392-9408).

[0158] HA-TF may comprise one or more (preferably all) of the following substitutions in the first CH3 domain: S364H, F405A. HA-TF may comprise one or more (preferably all) of the following substitutions in the second CH3 domain: Y349T, T394F.

[0159] The ZW1 approach may include one or more (preferably all) of the following substitutions in the first CH3 domain: T350V, L351Y, F405A, Y407V. The ZW1 approach may include one or more (preferably all) of the following substitutions in the second CH3 domain: T350V, T366L, K392L, T394W.

[0160] The biclonic approach may include one or more (preferably all) of the following substitutions in the first CH3 domain: 366K (+351K). The biclonic approach may include one or more (preferably all) of the following substitutions in the second CH3 domain: 351D or E or D at 349, 368, 349 or 349+355.

[0161] EW-RVT may comprise one or more (preferably all) of the following substitutions in the first CH3 domain: K360E, K409W. EW-RVT may comprise one or more (preferably all) of the following substitutions in the second CH3 domain: Q347R, D399V, F405T. EW-RVT may comprise a disulfide bond in CH3. The disulfide bridge may be supported by the further incorporation of Y349C in the first CH3 domain and S354C in the second CH3 domain.

[0162] Triomabs can be formed by fusing a mouse hybridoma with a rat hybridoma, resulting in the production of a bispecific asymmetric hybrid IgG molecule. Preferential pairing of a light chain with its corresponding heavy chain can then occur.

[0163] In one embodiment, one or more residues of the fragment of an immunoglobulin Fc region contain one or more amino acid substitutions suitable for knobs-in-holes (KIH) dimerization with a second fragment of an immunoglobulin Fc region that contains one or more corresponding amino acid mutations.

[0164] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.

[0165] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0166] In one embodiment, the antigen binding molecule is a ROR1-specific antigen binding molecule.

[0167] 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 (81) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK G3CPG4 hFc (S239C + Y407T) SEQ ID NO: 147 (82) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0168] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:194, SEQ ID NO:195 or SEQ ID NO:196. 1H8 hFc (S239C + Y407T) SEQ ID NO: 194 (83) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1H8 G4 hFc (S239C + Y407T) SEQ ID NO: 195 (84) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1H8 v15 hFc (S239C + Y407T) SEQ ID NO: 196 (85) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0169] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:148. P3A1 hFc (S239C + T366Y) SEQ ID NO: 148 (86) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0170] The recombinant fusion protein comprises a sequence according to SEQ ID NO:191 or SEQ ID NO:192. G3CP hFc (S239C + T366Y) SEQ ID NO: 191 (87) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK G3CPG4 hFc (S239C + T366Y) SEQ ID NO: 192 (88) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0171] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:197, SEQ ID NO:198 or SEQ ID NO:199. 1H8 hFc (S239C + T366Y) SEQ ID NO: 197 (89) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1H8 G4 hFc(S239C+T366Y) Sequence number 198 (Chemical formula 90) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1H8 v15 hFc(S239C+T366Y) Sequence number 199 (Chemical formula 91) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0172] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:193. P3A1 hFc (S239C+Y407T) SEQ ID NO: 193: (92) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0173] The recombinant fusion protein may be a biparatopic dimer comprising any one or any two of SEQ ID NOs: 146, 147, 194, 195, 196, 148, 191, 191, 192, 193, 197, 198, and 199. The biparatopic dimer may comprise one of SEQ ID NOs: 146, 147, 194, 195, 196, and 193 comprising a Y407T point mutation. The biparatopic dimer may comprise one of SEQ ID NOs: 148, 191, 192, 197, 198, and 199 comprising a T366Y point mutation. The biparatopic 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, and any of the biparatopic dimers disclosed herein may be associated with any of the linkers and payloads disclosed herein, and conjugation may be via any one or more S239C residues in the biparatopic dimer. Preferably, the biparatopic dimer may be associated with the linker and payload vc-PAB-EDA-PNU. Preferably, the biparatopic dimer comprises 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 more highly effective in vivo.

[0174] SEQ ID NOs: 146, 147, 194, 195, 196, 148, 191, 191, 192, 193, 197, 198 and 199 contain an S239C mutation for use in conjugation reactions. If the recombinant fusion protein is not conjugated (e.g., to an anthracycline (PNU) derivative), the S239C mutation is not required and position 239 can be S instead of C. Thus, in alternative embodiments, a recombinant fusion protein or biparatopic dimer can contain 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 contain an S239C mutation.

[0175] Thus, the recombinant fusion protein may comprise a sequence according to SEQ ID NO:165 or SEQ ID NO:166. G3CP-hFc(Y407T) SEQ ID NO:165: (93) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK G3CP G4-hFc(Y407T) SEQ ID NO:166: (94) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0176] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:200, SEQ ID NO:201 or SEQ ID NO:202. 1H8 hFc(Y407T) SEQ ID NO:200 (95) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1H8 G4 hFc (Y407T) SEQ ID NO: 201 (96) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1H8 v15 hFc(Y407T) SEQ ID NO:202 (97) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0177] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:167. P3A1 hFc(T366Y) SEQ ID NO:167: (98) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0178] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:188 or SEQ ID NO:189. G3CP hFc (T366Y) SEQ ID NO: 188 (99) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK G3CPG4 hFc(T366Y) SEQ ID NO: 189 (100) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0179] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:203, SEQ ID NO:204 or SEQ ID NO:205. 1H8 hFc(T366Y) SEQ ID NO:203 (chemical 101) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1H8 G4 hFc(T366Y) SEQ ID NO:204 (chemical 102) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1H8 v15 hFc(T366Y) SEQ ID NO:205 (C103) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0180] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:190. P3A1 hFc(Y407T) SEQ ID NO: 190 (C104) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0181] According to a fifth aspect, the present invention provides a method for producing a composition comprising the steps of: (a) a first recombinant fusion protein, the first recombinant fusion protein being a recombinant fusion protein according to the third or fourth aspect, and (b) a second recombinant fusion protein comprising a second antigen-binding molecule fused to a second fragment of an immunoglobulin Fc region that is engineered to dimerize with the first fragment of the immunoglobulin Fc region. The present invention provides a recombinant fusion protein dimer comprising:

[0182] In one embodiment, the second fragment of an immunoglobulin Fc region is selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[0183] In one embodiment, the second fragment of an immunoglobulin Fc region is an Fc heavy chain.

[0184] In one embodiment, the second fragment of an immunoglobulin Fc region is engineered to dimerize with a second fragment of an immunoglobulin Fc region by a method selected from the group consisting of knobs-into-holes (YT), 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.

[0185] In one embodiment, one or more residues of the fragment of an immunoglobulin Fc region contain one or more amino acid substitutions suitable for knobs-in-holes (KIH) dimerization with a second fragment of an immunoglobulin Fc region that contains one or more corresponding amino acid mutations.

[0186] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.

[0187] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0188] Any sequence of the recombinant fusion proteins disclosed herein may include any one or more amino acid substitutions selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V. Thus, SEQ ID NO: 145 (human Fc region) may be modified by the incorporation of any one or more amino acid substitutions selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V and incorporated into the recombinant fusion proteins described herein in place of the human Fc region sequence.

[0189] In one embodiment, the second antigen binding molecule is a ROR1-specific antigen binding molecule.

[0190] In one embodiment, the second specific antigen binding molecule is an immunoglobin, an immunoglobulin Fab region, a Fab', an Fv, an Fv-Fc, a single chain Fv (scFv), a scFv-Fc, an (scFv)2, a diabody, a triabody, a tetrabody, a bispecific t cell engager (BiTE), an intein, a VNAR domain, a single domain antibody (sdAb) or a VH domain.

[0191] In one embodiment, (a) the first recombinant fusion protein comprises a sequence according to SEQ ID NO: 146 or SEQ ID NO: 147, and (b) the second recombinant fusion protein comprises a sequence according to SEQ ID NO:148.

[0192] According to a sixth aspect, the present invention provides a ROR1-specific chimeric antigen receptor (CAR) comprising at least one ROR1-specific antigen binding molecule as defined by the first or second aspect of the invention, fused or conjugated to at least one transmembrane region and at least one intracellular domain.

[0193] The present invention also provides a cell comprising a chimeric antigen receptor according to the sixth aspect, which is preferably an engineered T cell.

[0194] In a seventh aspect of the invention there is provided a nucleic acid sequence comprising a polynucleotide sequence encoding a 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 aspect of the invention.

[0195] Also provided is a vector comprising a nucleic acid sequence according to the seventh aspect and a host cell comprising such nucleic acid.

[0196] Provided is a method for preparing the 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 culturing or maintaining a host cell comprising the polynucleotide or vector under conditions such that the host cell produces the specific antigen-binding molecule, recombinant fusion protein, or chimeric antigen receptor, and optionally further comprising isolating the specific antigen-binding molecule, recombinant fusion protein, or chimeric antigen receptor.

[0197] In an eighth aspect of the present invention, a pharmaceutical composition is provided comprising the 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. The pharmaceutical composition may contain various pharma- ceutical acceptable carriers. The pharmaceutical composition of the present 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. In a preferred embodiment, the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule or foam.

[0198] The specific antigen-binding molecule, recombinant fusion protein or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspect may be for use in a therapeutic method. More specifically, the 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 may be for use in the treatment of cancer. Preferably, the cancer is a ROR1-positive cancer type. More preferably, the cancer is selected from the group comprising hematological cancers such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML) and solid tumours including neuroblastoma, kidney 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, esophageal cancer, gastric cancer or liver cancer.

[0199] There is also provided herein the use of the 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 in the manufacture of a medicament for the treatment of a disease in a patient in need thereof.

[0200] Further according to the present invention there is provided a method of treating a disease in a patient in need of such treatment, comprising administering to said patient a therapeutically effective dose of the 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, or the pharmaceutical composition of the sixth aspect.

[0201] Preferably, the cancer is a ROR1 positive cancer type. More preferably, the cancer is selected from the group including blood cancers such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML) and solid tumors including neuroblastoma, kidney 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, esophageal cancer, gastric cancer or liver cancer.

[0202] Also provided herein is a method of assaying for the presence of a target analyte in a sample, comprising adding a detectably labeled specific antigen-binding molecule of the first or second aspect, or a recombinant fusion protein or recombinant fusion protein dimer of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect to a sample, and detecting binding of the molecule to the target analyte.

[0203] Further provided herein is a method of imaging a site of disease in a subject, the method comprising administering to the subject a detectably labeled specific antigen-binding molecule of the first or second aspect, or a detectably labeled recombinant fusion protein or recombinant fusion protein dimer of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect.

[0204] Also provided herein is a method of diagnosing a disease or medical condition in a subject, the method comprising administering a specific antigen-binding molecule of the first or second aspect, or a recombinant fusion protein or recombinant fusion protein dimer of the third or fourth aspect, or a recombinant fusion protein dimer of the fifth aspect.

[0205] Also contemplated herein are antibodies, antibody fragments or antigen-binding molecules that compete for binding to ROR1 with the ROR1-specific antigen-binding molecule of the first or second aspect. The term "compete" when used in relation to an antigen-binding protein (e.g., a neutralizing antigen-binding protein or neutralizing antibody) refers to competition between antigen-binding proteins as determined by an assay in which the antigen-binding protein under test (e.g., an antibody or functional fragment thereof) prevents or inhibits specific binding of an antigen-binding molecule as defined herein (e.g., the 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).

[0206] Also described herein is a kit for diagnosing a subject suffering from or susceptibility to cancer or for providing a prognosis of a condition in a subject, comprising a detection means for detecting the concentration of an antigen present in a sample from a test subject, the detection means comprising a ROR1-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein or a 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, each optionally derivatized, and the presence of the antigen in the sample indicates that the subject suffers from cancer. Preferably, the antigen comprises the ROR1 protein, more preferably the extracellular domain thereof. More preferably, the kit is used to identify the presence or absence of ROR1-positive cells in a sample or to determine their concentration in a sample. The kit may also comprise a positive control and / or a negative control to which the assay is compared and / or a label that can be detected.

[0207] The present invention also provides a method of diagnosing a subject suffering from or susceptibility to cancer or providing a prognosis of a condition in a subject, comprising detecting the concentration of an antigen present in a sample obtained from the subject, wherein detection is achieved using a ROR1-specific antigen binding molecule of the first or second aspect, a recombinant fusion protein or a 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, each optionally derivatized, and wherein the presence of the antigen in the sample is indicative that the subject is suffering from cancer.

[0208] Also contemplated herein is a method of killing or inhibiting the growth of a cell expressing ROR1 in vitro or in a patient, comprising administering to the cell a pharma- ceutical effective amount or dose of (i) a ROR1-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, or a nucleic acid sequence of the sixth aspect, or a CAR or cell according to the seventh aspect, or (ii) a pharmaceutical composition of the eighth aspect. Preferably, the cell expressing ROR1 is a cancer cell. More preferably, the ROR1 is human ROR1.

[0209] According to a ninth aspect, the present invention provides a compound of formula (II): X-FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4-Y (II) (In the formula, 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 specific antigen-binding molecule comprises an amino acid sequence represented by:

[0210] In certain preferred embodiments, the specific antigen-binding molecule according to this aspect of the invention may be further conjugated to a third, fourth or fifth moiety. Conjugation of additional moieties is also contemplated. In some cases, the third, fourth or fifth moiety may be conjugated to the second moiety. It is therefore understood that any of the moieties according to this aspect of the invention may have additional moieties conjugated thereto. The following description of preferred features of the second moiety also applies mutatis mutandis to the third, fourth, fifth or higher moieties.

[0211] Preferably, X or Y are individually either absent or selected from the group including 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, Fab', Fv, Fv-Fc, single chain Fv (scFv), scFv-Fc, (scFv)2, diabody, triabody, tetrabody, bispecific t cell engager, an intein, a VNAR domain, a single domain antibody (sdAb), a VH domain, a scaffold protein (such as an affibody, centyrin, darpin, etc.) or a toxin, including but not limited to Pseudomonas exotoxin PE38, diphtheria toxin.

[0212] Preferably, conjugation is via a cysteine ​​residue in the amino acid sequence of the specific antigen-binding molecule. The cysteine ​​residue may be present anywhere in the sequence, including in optional sequences X or Y (if present).

[0213] 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.

[0214] Preferably, the second moiety is selected from the group comprising a detectable label, a dye, a toxin, a drug, a prodrug, a radionuclide or a biologically active molecule.

[0215] More preferably, the second portion is Maytansinoids, Auristatins, anthracyclines, preferably PNU-derived anthracyclines, calicheamicin, amanitin derivatives, preferably α-amanitin derivatives, Tubulysin, Duocarmycins, radioisotopes, such as alpha-emitting radionuclides, e.g. 227Th or 225Ac; Liposomes containing toxic payloads, Protein toxins, Taxanes, Pyrrole benzodiazepines, Indolinobenzodiazepine pseudodimers, and / or Spliceosome inhibitors, CDK11 inhibitors, Pyridinobenzodiazepines, Irinotecan and its derivatives and at least one toxin selected from the group comprising:

[0216] In other preferred embodiments according to this aspect, the second moiety may be from the group including 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', an Fv, an 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 (affibody, centyrin, darpin, etc.) or a toxin, including but not limited to Pseudomonas exotoxin PE38, diphtheria toxin.

[0217] In a particularly preferred embodiment, the second portion is a VNAR domain that may be the same or different from the specific antigen-binding molecule according to this aspect.Thus, dimers, trimers or higher multimers of VNAR domains linked by chemical conjugation are expressly contemplated herein.In such an embodiment, each individual VNAR domain may have the same antigen specificity as the other VNAR domains or they may be different.

[0218] According to this embodiment, the specific antigen-binding molecule may comprise, for example, a biparatopic specific antigen-binding molecule as described in connection with the first to fifth embodiments, fused to a further biologically active molecule (including but not limited to a molecule for half-life extension, such as BA11), which is then further conjugated to a second moiety, including but not limited to a cytotoxic payload.

[0219] According to this aspect, the specific antigen-binding molecule may be 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. Thus, any of the preferred features described in relation to the first, second and third aspects apply mutatis mutandis to the sixth aspect.

[0220] The specific antigen-binding molecule of the ninth aspect may be for use in therapy. More specifically, the specific antigen-binding molecule of the ninth aspect may be for use in the treatment of cancer. Preferably, the cancer is a ROR1-positive cancer type. More preferably, the cancer is selected from the group including blood cancers, such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), and solid tumors, including neuroblastoma, kidney 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, esophageal cancer, gastric cancer, or liver cancer.

[0221] Also provided herein is the use of a specific antigen-binding molecule of the ninth aspect in the manufacture of a medicament for treating a disease in a patient in need thereof.

[0222] Pharmaceutical compositions comprising the ninth specific antigen-binding molecule are also provided. The pharmaceutical compositions may contain a variety of pharma- ceutical acceptable carriers.

[0223] Furthermore, the present invention provides a method of treating a disease in a patient in need of such treatment, comprising administering to the patient a therapeutically effective dose of the specific antigen-binding molecule of the ninth aspect or a pharmaceutical composition comprising the specific antigen-binding molecule of the ninth aspect.

[0224] Preferably, the cancer is a ROR1 positive cancer type. More preferably, the cancer is selected from the group including blood cancers such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML) and solid tumors including neuroblastoma, kidney 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, esophageal cancer, gastric cancer or liver cancer.

[0225] Also provided herein is a method for assaying the presence of a target analyte in a sample, comprising adding to the sample a detectably labeled specific antigen-binding molecule of the ninth aspect and detecting binding of the molecule to the target analyte.

[0226] Further provided herein is a method of imaging a site of disease in a subject, comprising administering to the subject a specific antigen-binding molecule of the ninth aspect, which is detectably labeled.

[0227] Also provided herein is a method of diagnosing a disease or medical condition in a subject, the method comprising administering a specific antigen-binding molecule of the ninth aspect.

[0228] Furthermore, any feature described in relation to any of the above aspects of the invention may be combined with other aspects of the invention mutatis mutandis.

[0229] In addition to the sequences listed, the following sequences are expressly disclosed, some of which relate to examples of molecules of the invention described herein.

[0230] [Table 4] JPEG2024500405000012.jpg218170JPEG2024500405000013.jpg220170JPEG2024500405000014.jpg220170JPEG2024500405000015.jpg204170

[0231] A highly attractive class of DNA intercalating toxins for use as payloads for drug conjugates are the anthracyclines because of their proven clinical validation as chemotherapeutic agents in cancer treatment.

[0232] The stability of chemical conjugate protein drug conjugate is an important consideration, because the unintended release of highly potent anthracycline toxins, such as PNU-159682, in the circulation of patients before targeting of tumor cells leads to off-target effects and undesirable side effects.Some example molecules released from PNU conjugate include the release of PNU159682 derivatives from different Val-Cit-PAB-containing drug linkers.

[0233] Therefore, there is a demand for a potent toxin that can be bound to the target protein with high stability to avoid or at least reduce undesirable side effects. Alternatively, the linker payload is designed so that extracellular cleavage releases the derivative of the payload with reduced potency. However, sufficient potency needs to be maintained to avoid any reduction in side effects being countered by the need to administer higher doses to achieve efficacy.

[0234] Ease of conjugation is an important factor in producing an easily manufacturable product. The payloads of the present disclosure may use maleimide groups that can react with any available thiol groups on the conjugation partner using simple and standard conditions. Furthermore, the use of maleimide / thiol chemistry for conjugation allows for site-specific conjugation to, for example, an introduced thiol group on the side chain of an engineered cysteine ​​residue in a protein sequence. In some cases described herein, cysteine ​​may be introduced via the introduction of an engineered cysteine-containing his-myc tag (example sequences include, but are not limited to, QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) or QACGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 99)) at the C- or N-terminus of the protein.

[0235] Antibody / protein drug conjugates produced using non-selective labeling methods, for example, through reaction with amino functional groups in proteins, deliver products containing multiple different species with different drug-antibody ratios. This affects the properties of the conjugates that affect in vivo efficacy and toxicity, such as potency and PK properties. Therefore, thiol-reactive payloads are of great importance, because they can be reacted with high yields in a single process with naturally occurring cysteine ​​residues in proteins or with cysteine ​​residues engineered into specific sites at any point in the sequence of a protein using molecular biology / recombinant protein expression or chemical synthesis or through expression, synthesis, or chemical modification of natural proteins. In some cases described herein, cysteines are engineered into the Fc region of Fc fusion proteins.

[0236] The present disclosure provides anthracycline (PNU) derivatives suitable for use in drug conjugates. Specifically, a derivative of PNU159682 is provided that lacks the C14 carbon and the attached carboxyl functional group and is functionalized with an ethylenediamino (EDA) group at the C13 carbonyl of PNU159682. This EDA-PNU159682 can then be functionalized with a maleimide-containing linker via the amino group of the EDA moiety. A maleimide group is present in the anthracycline (PNU) derivative of formula (V) and can also be present in the anthracycline (PNU) derivative of formula (VI). Such a payload can react with a free thiol group on another molecule. If a free thiol is present on a protein, a protein-drug conjugate (PDC) can be formed.

[0237] Surprisingly, derivatives of PNU159682 functionalized with ethylenediamino (EDA) groups and linked to thiol groups via maleimide groups show increased stability with slightly lower potency compared to non-EDA payloads or free payload derivatives. A more stable payload may be advantageous due to reduced off-target effects, which in turn may lead to reduced side effects and increased patient compliance.

[0238] PCT / EP 2020 / 067210 discloses a compound of formula (V): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, 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), peptide, -(CH2)n-, -(CH2CHO)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. This paper describes anthracycline (PNU) derivatives.

[0239] The anthracycline (PNU) derivative of formula (V) may contain [L1], [L2] or [L1] and [L2].

[0240] Preferably, when [L1] and / or [L2] are peptides, said peptides do not contain glycine.

[0241] It will be clear to one skilled in the art that in the absence of the optional spacer and / or the optional linker, a bond will remain in its place.

[0242] Preferably, [X] is polyethylene glycol, [ka] is selected from the group comprising: [ka] represents the point of attachment to the remainder of the molecule, and [R] is an optional spacer selected from the group including a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0243] Most preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0244] Preferably, [L2] is p-aminobenzyloxycarbonyl (PAB) or alanine.

[0245] Preferably, the anthracycline (PNU) derivative comprises [L1] and / or [L2], and [X] is optional. Thus, [L1] and / or [L2] may be a linker selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH2)n-, -(CH2CHO)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].

[0246] PCT / EP 2020 / 067210 discloses a compound of formula (V): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, 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), peptide, -(CH2)n-, -(CH2CHO)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 present invention describes an anthracycline (PNU) derivative of formula (V) which contains [L1], [L2] or [L1] and [L2].

[0247] Preferably, [X] is polyethylene glycol, [ka] is selected from the group comprising: [ka] represents the point of attachment to the remainder of the molecule, and [R] is an optional spacer selected from the group including a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0248] Most preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0249] Preferably, [L2] is p-aminobenzyloxycarbonyl (PAB) or alanine.

[0250] Preferably, the PNU derivative is [ka] The structure is selected from:

[0251] PCT / EP 2020 / 067210 discloses a compound of formula (VI): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [Z] is a reactive group. Also described are anthracycline (PNU) derivatives of the formula: The reactive group can be any reactive group suitable for use in conjugation reactions, particularly conjugation reactions to target binding molecules.

[0252] Thus, [Z] can be a moiety that contains a functional group for use in a bioconjugation reaction. Functional groups for use in a bioconjugation reaction include, but are not limited to, Maleimides or alkyl halides for reaction with thiol or selenol groups on proteins via thioether and seronoether reactions; · maleimides, alkyl halides or sulfhydryl groups for reaction with thiol-functionalized molecules, including the thiol groups of protein cysteine ​​residues; Activated disulfides for reaction with thiol groups to form disulfide bonds via thiol-disulfide exchange, such as pyridyldithiol (Npys thiol) or TNB thiol (5-thiol-2-nitrobenzoic acid); Amino groups for attachment to carboxyl groups on proteins and biomolecules via amide bond formation reactions; · Alkyne groups, in particular ring-constrained alkynes, such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (BCN), for reaction with azide-functionalized biomolecules via strain-promoted alkyne-azide cycloaddition copper-free chemistry. Azide functionality can be introduced into proteins, for example, via incorporation of the unnatural amino acid para-azidomethy-L-phenylalanine, or into protein glycans using enzyme-mediated glycoengineering to attach azide-containing sugar analogues; · Azide groups for reaction with alkyne-functionalized target binding molecules via strain-promoted alkyne-azide cycloaddition copper-free chemistry; · Aminoxy groups for reaction with aldehyde and ketone groups on biomolecules via oxime-forming ligation. Ketones can be introduced into proteins via the use of amber stop codon technology, e.g., incorporation of the unnatural amino acid para-acetylphenylalanine. Aldehydes can be found on biomolecules via the presence of reducing sugars and can be introduced into proteins via periodate oxidation of N-terminal serine residues or periodate oxidation of carbohydrate cis-glycol groups. Aldehyde groups can also be incorporated into proteins via conversion of protein cysteines within defined sequences to formylglycine by formylglycine generating enzyme. Additionally, formylglycine-containing proteins have been conjugated to payloads via hydrazino-Pictet-Spengler (HIPS) ligation; · Aldehyde or ketone groups for reaction with aminoxy or hydrazide or hydrazinyl functionalized biomolecules via oxime or hydrazine bond forming ligation reactions. Proteins Aminoxy and hydrazide functionalized proteins can be generated via cleavage of intein fusion proteins.

[0253] Thus, [Z] may be selected from the group consisting of maleimide, alkyl halide, sulfhydryl group, activated disulfide (e.g., pyridyldithiol (Npys thiol) or TNB thiol (5-thiol-2-nitrobenzoic acid)), amino group, alkyne group (e.g., ring-constrained alkynes such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (BCN)), azide group, aminoxy group, aldehyde group, and ketone group.

[0254] [Z] can be a moiety for enzyme-mediated bioconjugation reactions. Moieties for use in enzyme-mediated bioconjugation reactions include, but are not limited to, suitable primary amines for bacterial transglutaminase-mediated conjugation to glutamine γ-carboxyamide groups containing sequences such as polyGly[(Gly)n] or Lys-Lys-Gln-Gly and Lys-Pro-Glu-Thr-Gly for use in sortase enzyme-mediated antibody conjugation.

[0255] Thus, [Z] may be selected from the group consisting of polyGly and primary amines.

[0256] Thus, the PNU derivative according to formula (IV) may correspond to a PNU derivative of formula (V) in which L1 is Val-Cit-PAB, L2 is absent, and the maleimide group may be replaced by another reactive group as defined above.

[0257] Preferably, [X] is polyethylene glycol, [ka] is selected from the group comprising: [ka] represents the point of attachment to the remainder of the molecule, and [R] is an optional spacer selected from the group including a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0258] Most preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0259] 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 a recombinant fusion protein or recombinant fusion protein dimer of the present invention.

[0260] According to a tenth aspect, the present invention provides a method for producing a composition comprising: (a) a ROR1-specific antigen-binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein or a recombinant fusion protein dimer according to the third or fourth aspect, or a recombinant fusion protein dimer according to the fifth aspect, and (b) Anthracycline (PNU) derivatives A target binding molecule-drug conjugate comprising the formula (III): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, 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), peptide, -(CH2)n-, -(CH2CHO)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; and Y comprises a ROR1-specific antigen-binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein or a recombinant fusion protein dimer according to the third or fourth aspect, or a recombinant fusion protein dimer according to the fifth aspect. The present invention provides a target binding molecule-drug conjugate having the structure:

[0261] The target binding molecule-drug conjugate of formula (III) may comprise [L1], [L2] or [L1] and [L2].

[0262] Preferably, in the target binding molecule-drug conjugate in which [L1] and / or [L2] is a peptide, said peptide does not contain glycine.

[0263] It will be clear to one skilled in the art that in the absence of the optional spacer and / or the optional linker, a bond will remain in its place.

[0264] Preferably, the target binding molecule-drug conjugate comprises [ka] The structure is selected from:

[0265] According to an eleventh aspect, the present invention provides a method for producing a composition comprising the steps of: (a) a ROR1-specific antigen-binding molecule of the first, second or ninth aspect, or a recombinant fusion protein or a recombinant fusion protein dimer of the third, fourth or fifth aspect, and (b) Anthracycline (PNU) derivatives A target binding molecule-drug conjugate comprising the formula (IV): [ka] wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [Z] is a linker derived from a reactive group used to conjugate an anthracycline (PNU) derivative and a target binding molecule; and Y comprises a ROR1-specific antigen-binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein or a recombinant fusion protein dimer according to the third, fourth or fifth aspect. The present invention provides a target binding molecule-drug conjugate having the structure:

[0266] [Z] is typically a moiety derived from a reactive group used to conjugate an anthracycline (PNU) derivative and a target-binding molecule. [Z] may be a moiety derived from a reactive group selected from the group consisting of maleimide, alkyl halide, sulfhydryl group, activated disulfide, amino group, alkyne group, azide group, aminoxy group, aldehyde group, and ketone group.

[0267] Thus, [Z] may be selected from the group consisting of a disulfide bond, an amide bond, an oxime bond, a hydrazone bond, a thioether bond, a 1,2,3 triazole, and a polyGly.

[0268] Preferably, [X] is polyethylene glycol, [ka] is selected from the group comprising: [ka] represents the point of attachment to the remainder of the moiety, and [R] is an optional spacer selected from the group including a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof.

[0269] Most preferably, [X] is polyethylene glycol. The polyethylene glycol may be PEG4.

[0270] Preferably, the target binding molecule is a protein or a nucleic acid. Examples of target binding proteins (which may also be referred to as specific antigen binding proteins) include, but are not limited to, immunoglobulins or antibodies, immunoglobulin Fc regions, fragments of immunoglobulin Fc regions, Fc heavy chains, CH2 regions, CH3 regions, immunoglobulin Fab regions, Fab', Fv, Fv-Fc, single chain Fv (scFv), scFv-Fc, (scFv)2, diabodies, triabodies, tetrabodies, bispecific t cell engagers, inteins, VNAR domains, single domain antibodies (sdAbs), VH domains, scaffolding proteins (affibodies, centyrin, darpin, etc.). Examples of target binding nucleic acids include, but are not limited to, aptamers.

[0271] Preferably, 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 the protein or to a thiol group introduced by chemical modification of the protein, e.g., incorporated at the N-terminus or C-terminus of the amino acid sequence of a specific antigen-binding protein. Thiol groups can also be incorporated into other target-binding molecules, e.g., nucleic acids.

[0272] In one embodiment of the target binding molecule-drug conjugate of the tenth or eleventh aspect, Y comprises a ROR1-specific antigen-binding molecule according to the first or second aspect of the invention conjugated to a PNU derivative via a human immunoglobulin Fc region or a fragment thereof.

[0273] In one embodiment, the fragment of a human immunoglobulin Fc region may be selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[0274] There is also provided herein a target binding molecule-drug conjugate according to the above aspects for use in therapy.

[0275] There is also provided herein a target binding molecule-drug conjugate according to the above aspects for use in the treatment of cancer.

[0276] There is also provided herein the use of 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.

[0277] Also provided herein is a method of treating a disease in a patient in need of treatment, comprising administering to said patient a therapeutically effective dose of a target binding molecule-drug conjugate according to the above embodiment. The disease may be cancer.

[0278] Preferably, the cancer is a ROR1 positive cancer type. More preferably, the cancer is selected from the group including blood cancer, such as lymphoma and leukemia, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML) and solid tumors including neuroblastoma, kidney 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, esophageal cancer, gastric cancer or liver cancer. The cancer can be mesothelioma or triple negative breast cancer (TNBC). The mesothelioma can be pleural mesothelioma.

[0279] Also provided herein is a pharmaceutical composition comprising a target binding molecule-drug conjugate according to any of the above aspects and at least one other pharma- ceutically acceptable ingredient.

[0280] definition The antigen-specific binding molecules of the present invention comprise amino acid sequences derived from a synthetic library of VNAR molecules or a library derived from immunization of cartilaginous fish. The terms VNAR, IgNAR and NAR may also be used interchangeably.

[0281] Amino acids are represented herein as one-letter codes or three-letter codes or both.

[0282] The term "affinity purification" refers to the purification of a molecule based on specifically attracting or binding a molecule to a chemical or binding partner to form a combination or complex that allows the molecule to be bound or attracted to the partner moiety while being separated from impurities.

[0283] The term "complementarity determining region" or CDR (i.e., CDR1 and CDR3) refers to the amino acid residues of a VNAR domain whose presence is typically involved in antigen binding. Each VNAR typically has two CDR regions, identified as CDR1 and CDR3. In addition, each VNAR domain also contains amino acids from a "hypervariable loop" (HV) that may also be involved in antigen binding. In some cases, a complementarity determining region may contain amino acids from both the CDR region and the hypervariable loop. In other cases, antigen binding may only involve residues from a single CDR or HV. According to the generally accepted nomenclature for VNAR molecules, there is no CDR2 region.

[0284] "Framework regions" (FW) are those VNAR residues other than the CDR residues. Each VNAR typically has five framework regions identified as FW1, FW2, FW3a, FW3b and FW4.

[0285] The boundaries between the FW, CDR and HV regions in a VNAR are not intended to be fixed, and thus some variation in the length and composition of those regions should be expected. This will be understood by those of skill in the art, particularly with reference to the work that has been carried out in the analysis of those regions. (Anderson et al., PLoS ONE (2016) 11(8); Lui et al., Mol Immun (2014) 59, 194-199; Zielonka et al., Mar Biotechnol (2015). 17, (4) 386-392; Fennell et al., J Mol Biol (2010) 400. 155-170; Kovalenko et al., J Biol Chem (2013) 288. 17408-17419; Dooley et al., (2006) PNAS 103(6). 1846-1851). The molecules of the invention are defined herein by reference to FW, CDR and HV regions, but are not limited by their precise definitions, and thus variations in accordance with art-understood structure of VNAR domains are expressly contemplated herein.

[0286] "Codon set" refers to a set of different nucleotide triplet sequences used to code for desired variant amino acids. A set of oligonucleotides represents all possible combinations of nucleotide triplets provided by the codon set and contains sequences that code for the desired group of amino acids, and can be synthesized, for example, by solid phase synthesis. The standard format for codon designation is that of the IUB code, known in the art and described herein.

[0287] Codon sets are typically represented by three capital letters in italics, e.g., NNK, NNS, XYZ, DVK, etc. Thus, a "non-random codon set" refers to a codon set that codes for selected amino acids that partially, preferably completely, meet the amino acid selection criteria described herein. Synthesis of oligonucleotides with selected nucleotide "degeneracy" at certain positions is well known in the art, for example, the TRIM approach (Knappek et al.; J. Mol. Biol. (1999), 296, 57-86; Garrard & Henner, Gene (1993), 128, 103). Such a set of oligonucleotides with a certain codon set can be synthesized using commercially available nucleic acid synthesis agents (e.g., available from Applied Biosystems, Foster City, CA) or can be obtained commercially (e.g., from Life Technologies, Rockville, MD). A set of synthetic oligonucleotides with a particular codon set typically includes multiple oligonucleotides with different sequences, the differences being established by the codon sets within the overall sequence. Oligonucleotides used according to the invention have sequences that allow hybridization to a VNAR nucleic acid template and may also contain restriction enzyme sites, as appropriate.

[0288] "Cell," "cell line," and "cell culture" are used interchangeably (unless the context indicates otherwise), and such designations include all progeny of the cell or cell line. Thus, for example, terms such as "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom regardless of the number of transformations. 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.

[0289] "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 suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, a ribosome binding site, etc. Eukaryotic cells use control sequences such as promoters, polyadenylation signals, and enhancers.

[0290] The term "coat protein" refers to a protein, at least a portion of which is present on the surface of a virus particle. From a functional standpoint, a coat protein is any protein that associates with a virus particle during the virus assembly process in a host cell and remains associated with the assembled virus until it infects another host cell.

[0291] The "detection limit" for a chemical entity in a particular assay is the lowest concentration of that entity that can be detected above background levels for that assay. For example, in a phage ELISA, 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 that exceeds the signal produced by a control phage that does not display the antigen-binding fragment.

[0292] "Fusion protein" and "fusion polypeptide" refer to a polypeptide having two moieties covalently linked to each other, each of which is a polypeptide having a different property. The property can be a biological property, such as in vitro or in vivo activity. The property can be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two moieties can be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two moieties and the linker are in reading frame with each other. Preferably, the two moieties of the polypeptide are obtained from heterologous or different polypeptides.

[0293] The term "fusion protein" as used herein generally refers to one or more proteins linked together by chemical means, such as hydrogen bonds or salt bridges, or peptide bonds via protein synthesis, or both. Typically, fusion proteins are prepared by recombinant DNA techniques and may be referred to herein as recombinant fusion proteins.

[0294] "Heterologous DNA" is any DNA that is introduced into a host cell. The DNA can be from a variety of sources, such as genomic DNA, cDNA, synthetic DNA, and fusions or combinations thereof. The DNA can include DNA from the same cell or cell type as the host or recipient cell, or from a different cell type, such as an allogenic or xenogenic source. The DNA can optionally include a marker or selection gene, such as an antibiotic resistance gene, a temperature resistance gene, and the like.

[0295] "Highly diverse positions" refers to amino acid positions located in the variable regions of the light and heavy chains that have a large number of different amino acids represented at that position when known amino acid sequences and / or naturally occurring antibodies or antigen-binding fragments are compared. Highly diverse positions are typically located in the CDR or HV regions.

[0296] "Identity" describes a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. Identity also sometimes means the degree of sequence relatedness (homology) between polypeptide or polynucleotide sequences, as determined by the match between strings of polypeptide or polynucleotide sequences. While there are many methods to measure the identity between two polypeptide or two polynucleotide sequences, commonly used methods to determine identity are codified in computer programs. Preferred computer programs for determining identity between two sequences include, but are not limited to, the 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).

[0297] Preferably, the amino acid sequence of the protein has at least 45% identity at the amino acid level with the amino acid sequence disclosed herein using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. (1990) 215, 403-410) provided by the Human Genome Mapping Project (HGMP).

[0298] More preferably, the protein sequences may have at least 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, and even more preferably 95% (and even more preferably at least 96%, 97%, 98% or 99%) identity at the nucleic acid or amino acid level to the amino acid sequences presented herein.

[0299] Proteins may also include sequences having 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 to a sequence disclosed herein using the default parameters of the BLAST computer program provided by HGMP.

[0300] A "library" refers to a plurality of VNAR or VNAR fragment sequences (e.g., polypeptides of the invention) or nucleic acids encoding those sequences that differ in the combinations of variant amino acids introduced into them by the methods of the invention.

[0301] "Ligation" is the process of forming phosphodiester bonds between two nucleic acid fragments. For two fragments to be ligated, the ends of the fragments must be compatible with each other. In some cases, the ends become directly compatible after endonuclease digestion. However, it may first be necessary to convert the protruding ends that are commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation. To blunt the ends, the DNA is treated with about 10 units of Klenow fragment of DNA polymerase I or T4 DNA polymerase in a suitable buffer in the presence of four deoxyribonucleotide triphosphates at 15°C for at least 15 minutes. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation or silica purification. The DNA fragments to be ligated together are loaded into the solution in approximately equimolar amounts. The solution also contains ATP, ligase buffer, and about 10 units of ligase, e.g., T4 DNA ligase, for 0.5 μg of DNA. If the DNA is to be ligated into a vector, the vector is first linearized by digestion with the appropriate restriction endonuclease, and the linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.

[0302] A "mutation" is a deletion, insertion or substitution of a nucleotide relative to a reference nucleotide sequence, e.g., a wild-type sequence.

[0303] "Natural" or "naturally occurring" VNARs refer to VNARs identified from non-synthetic sources, such as tissue sources obtained ex vivo or serum from animals of the Elasmobranchii. These VNARs may include VNARs produced in any type of immune response, natural or otherwise induced. Natural VNARs include the amino acid sequences and nucleotide sequences that constitute or encode those antibodies. As used herein, natural VNARs differ from "synthetic VNARs," which refer to VNAR sequences that have been altered from a source or template sequence, for example, by substituting, deleting, or adding one or more amino acids at a position with a different amino acid, providing an antibody sequence in which the different amino acids differ from the source antibody sequence.

[0304] The term "nucleic acid construct" generally refers to a nucleic acid of any length, which may be DNA, cDNA or RNA, such as mRNA, obtained by cloning or produced by chemical synthesis. The DNA may be single-stranded or double-stranded. Single-stranded DNA may be the coding sense strand, or it may be the non-coding or antisense strand. For therapeutic use, the nucleic acid construct is preferably in a form that can be expressed in the subject to be treated.

[0305] "Operably linked," when referring to a nucleic acid, means that the nucleic acid is placed into a functional relationship with another nucleic acid sequence. For example, 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 promoter 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. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linkage is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0306] The term "protein" generally refers to multiple amino acid residues linked together by peptide bonds. It is used interchangeably with peptide, oligopeptide, oligomer or polypeptide to mean the same, including glycoproteins and derivatives thereof. The term "protein" is also intended to include fragments, analogs, variants and derivatives of proteins, which fragments, analogs, variants or derivatives retain essentially the same biological activity or function as the reference protein. Examples of protein analogs and derivatives include peptide nucleic acids and DARPins (designed ankyrin repeat proteins).

[0307] A protein fragment, analogue, variant or derivative may be at least 25, preferably 30 or 40 or up to 50 or 100 or 60-120 amino acids long, depending on the length of the original protein sequence from which it is derived, although lengths of 90-120, 100-110 amino acids may be convenient in some cases.

[0308] A protein fragment, derivative, variant, or analog is one in which (i) one or more of the amino acid residues are substituted with a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), which may or may not be an amino acid residue encoded by the genetic code, or (ii) one or more of the amino acid residues include a substituent group, or (iii) additional amino acids are fused to the mature polypeptide, for example, to a leader or accessory sequence used in purification of the polypeptide. Such fragments, derivatives, variants, and analogs are deemed to be within the scope of the skill of one of ordinary skill in the art from the teachings herein.

[0309] An "oligonucleotide" is a short single- or double-stranded polydeoxynucleotide that is chemically synthesized by known methods (e.g., phosphotriester, phosphorous, or phosphoramidite chemistry using solid-phase techniques). Further methods include the polymerase chain reaction (PCR), which is used when the entire nucleic acid sequence of a gene is known or the sequence of a nucleic acid complementary to the coding strand is available. Alternatively, when the target amino acid sequence is known, the known preferred coding residues for each amino acid residue can be used to deduce the potential nucleic acid sequence. Oligonucleotides can be purified on polyacrylamide gels or molecular sizing columns or by precipitation. DNA is "purified" when it is separated from non-nucleic acid impurities (which can be polar, non-polar, ionic, etc.).

[0310] As used herein, a "source" or "template" VNAR refers to a VNAR or VNAR antigen-binding fragment whose antigen-binding sequence serves as a template sequence when diversification is performed according to the criteria described herein. The antigen-binding sequence generally preferably includes at least one CDR and preferably includes framework regions within the VNAR.

[0311] A "transcriptional regulatory element" contains one or more of the following components: an enhancer element, a promoter, an operator sequence, a repressor gene, and a transcription termination sequence.

[0312] "Transformation" refers to the process by which a cell takes up DNA and becomes a "transformant." DNA uptake can be permanent or transient. A "transformant" is a cell that has taken up and retained 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).

[0313] A "variant" or "mutant" of a starting or reference polypeptide (e.g., a source VNAR or its CDR), such as a fusion protein (polypeptide) or heterologous polypeptide (heterologous to a phage), is a polypeptide that (1) has an amino acid sequence that differs from that of the starting or reference polypeptide and (2) is 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 a polypeptide of interest. For example, a fusion polypeptide of the invention generated using an oligonucleotide comprising a non-random codon set that encodes a sequence having a variant amino acid (with respect to the amino acid found at the corresponding position in the source VNAR or antigen-binding fragment) is a variant polypeptide with respect to the source VNAR or antigen-binding fragment. Thus, a variant CDR refers to a CDR that comprises a variant sequence with respect to the starting or reference polypeptide sequence (e.g., of the source VNAR or antigen-binding fragment). In this context, a variant amino acid refers to an amino acid that differs from the amino acid at the corresponding position in the starting or reference polypeptide sequence (e.g., of the source VNAR or antigen-binding fragment). Any combination of deletion, insertion and substitution can be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics. Amino acid changes can also alter post-translational processing of the polypeptide, for example, changing the number or position of glycosylation sites.

[0314] A "wild-type" or "reference" sequence or a sequence of a "wild-type" or "reference" protein / polypeptide, such as a coat protein or CDR of a source VNAR, may be a reference sequence from which a variant polypeptide is derived via the introduction of mutations. Generally, the "wild-type" sequence for a given protein is the sequence that is most common in nature. Similarly, a "wild-type" gene sequence is the sequence for that gene that is most commonly found in nature. Mutations can be introduced into a "wild-type" gene (and thus the protein it encodes) either through natural processes or through artificial means. The product of such a process is a "variant" or "mutant" form of the original "wild-type" protein or gene.

[0315] A "humanized" antigen-specific antigen-binding molecule can be modified at one or more amino acid sequence positions to reduce the potential for immunogenicity in vivo while retaining functional binding activity for a specific epitope on a specific antigen.

[0316] Humanization of antibody variable domains is a well-known technique in the art for modifying antibodies raised in non-human species against therapeutically useful targets so that the humanized forms can avoid undesired immunological responses when administered to human subjects. Methods for humanization are summarized in Almagro JC and William Strohl W. Antibody Engineering: Humanization, Affinity Maturation, and Selection Techniques in Therapeutic Monoclonal Antibodies: From Bench to Clinic.Edited by An J.2009 John Wiley&Sons,Inc and Strohl WR and Strohl LM, Therapeutic Antibody Engineering, Woodhead Publishing 2012.

[0317] Although IgNARs have a distinct origin compared to immunoglobulins and have 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 VNAR domains. For example, WO 2013 / 167883, incorporated by reference, provides a description of humanization of VNARs. See also Kovalenko OV, et al. J Biol Chem. 2013.288(24):p.17408-19.

[0318] A humanized antigen-specific binding molecule can differ from a wild-type antigen-specific binding molecule by replacing one or more framework amino acid residues with the corresponding framework amino acid residues of DPK-9. DPK-9 is a human germline VL scaffold that is a member of the variable kappa subgroup 1 (Vκ1). DPK-9 is (C120) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIK (SEQ ID NO:132) The sequence is as follows:

[0319] The term "chimeric antigen receptor (CAR)" as used herein may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and includes an artificial engineered receptor that grafts an artificial specificity onto a specific immune effector cell. CARs can be used to confer the specificity of an antigen-specific binding protein, such as a monoclonal antibody or a VNAR, onto a T cell, thereby allowing the generation of a large number of specific T cells, for example, for use in adoptive cell therapy. CARs can, for example, direct the specificity of a cell to a tumor-associated antigen. CARs can include an extracellular domain that includes an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen binding region. In certain embodiments, a CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane and endodomain. In other specific embodiments, a CAR comprises a fusion of a VNAR domain described herein with a CD3-zeta transmembrane and endodomain. The specificity of other CAR designs can be derived from the receptor's ligand (e.g., a peptide) or a pattern recognition receptor, such as dectin. In certain embodiments, malignant B cells can be targeted by redirecting the specificity of T cells by using a CAR specific for the B-lineage molecule CD19. In some cases, the spacing of the antigen recognition domain can be modified to reduce activation-induced cell death. In some cases, the CAR contains domains for additional costimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules such as costimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally eliminate T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors can be co-expressed with the CAR.

[0320] The term "conjugation" as used herein may refer to any method of chemically linking two or more chemical moieties. Typically, conjugation is via a covalent bond. In the context of the present invention, at least one of the chemical moieties is a polypeptide, and in some cases, the conjugation involves two or more polypeptides, one or more of which may be produced by recombinant DNA technology. Numerous systems for conjugating polypeptides are known in the art. For example, conjugation may be achieved through lysine residues present in the polypeptide molecule using N-hydroxy-succinimide, or through cysteine ​​residues present in the polypeptide molecule using maleimidobenzoylsulfosuccinimide ester. In some embodiments, conjugation occurs via short-acting degradable bonds, including but not limited to physiologically cleavable bonds, such as esters, carbonates, carbamates, sulfates, phosphates, acyloxyalkyl ethers, acetals and ketals, hydrazones, oximes, and disulfide bonds. In some embodiments, a linker is incorporated that is cleavable by an intracellular or extracellular enzyme, such as a member of the cathepsin family, cleavable under reducing conditions or acidic pH, to allow release of the conjugated moiety from the polypeptide or protein to which it is conjugated.

[0321] 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 in the protein-intein conjugate leads to a thioester bond intermediate that can be chemically cleaved with bis-aminoxyl agents or amino-thiols to yield the desired protein C-terminal aminoxy or thiol derivatives, respectively. These C-terminal aminoxy and thiol derivatives can be chemoselectively reacted with aldehyde / ketone and maleimide functionalized moieties, respectively, to yield site-specific C-terminal modified proteins.

[0322] In another preferred method of conjugation, the VNAR is directly expressed with an additional cysteine ​​incorporated at or near the C-terminal region of the VNAR or within a short C-terminal tag sequence, allowing conjugation with a thiol-reactive payload, such as a maleimide-functionalized moiety.

[0323] Conjugation as referred to herein is also intended to encompass the use of linker moieties that can confer 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. In some cases, the linker moiety can be selected to be cleavable under certain conditions, such as through the use of enzymes, nucleophilic / basic reagents, reducing agents, light irradiation, electrophilic / acidic reagents, organometallic and metallic reagents, or oxidizing reagents, or the linker can be specifically selected to resist cleavage under such conditions.

[0324] Polypeptides can be conjugated to various functional moieties to achieve a number of goals. Examples of functional moieties to reduce immunogenicity and antigenicity or improve solubility include, but are not limited to, polymers, such as polyethylene glycol. Further non-limiting examples include conjugation of the polypeptide to a therapeutic or cytotoxic agent.

[0325] The term "detectable label" is used herein to define that an entity can be visualized or otherwise detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means. A detectable label can be selected such that it generates a signal that can be measured and whose intensity is proportional to the amount of bound entity. A wide variety of systems for labeling and / or detecting proteins and peptides are known in the art. A label can be directly detectable (i.e., does not require any further reaction or manipulation to be detectable, e.g., a fluorophore is directly detectable) or it can be indirectly detectable (i.e., becomes detectable through reaction or binding with another entity that is detectable, e.g., a hapten is detectable by immunostaining after reaction with a reporter, e.g., an appropriate antibody that contains 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.

[0326] Contemplated herein are methods of killing or inhibiting the growth of cells expressing ROR1 in vitro or in patients. In general, the term "killing" as used herein in reference to cells means causing cell death. This can be accomplished by a number of mechanisms, including induction of necrosis or other cell damage or apoptosis. The phrase "inhibiting growth" or "inhibiting proliferation" as used herein is intended to encompass prevention of cell development, more specifically, prevention of cell division.

[0327] As used herein, an alkyl group is a straight or branched chain substituted or unsubstituted (preferably unsubstituted) group containing 1 to 40 carbon atoms. The alkyl group may be optionally substituted at any position. As used herein, the term "alkenyl" 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 double bond. As used herein, the term "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.

[0328] Terms such as "alkyl", "aryl", "heteroaryl" and the like also include polyvalent species such as alkylene, arylene, "heteroarylene", etc. Examples of alkylene groups include ethylene (-CH-CH-) and propylene (-CH-CH-CH-). An exemplary arylene group is phenylene (-CH-), and an exemplary heteroarylene group is pyridinylene (-CHN-).

[0329] An aromatic ring is a cyclic aromatic group that may have 0, 1, 2 or more, preferably 0, 1 or 2, ring heteroatoms. The aromatic ring may be optionally substituted and / or 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.

[0330] Aromatic rings include both aryl and heteroaryl groups. Aryl and heteroaryl groups can be mononuclear, i.e., have only one aromatic ring (e.g., phenyl or phenylene), or polynuclear, i.e., have two or more aromatic rings that may be fused (e.g., naphthyl or naphthylene), individually covalently bonded (e.g., biphenyl), and / or have a combination of both fused and individually bonded aromatic rings. Preferably, the aryl or heteroaryl group is an aromatic group that is substantially conjugated throughout the group. Aryl groups can contain 5-40 ring carbon atoms, 5-25 carbon atoms, 5-20 carbon atoms, or 5-12 carbon atoms. Heteroaryl groups can be 5-40, 5-25, 5-20, or 5-12 membered rings containing one 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 aromatic rings, to form a polycyclic ring system.

[0331] Aryl and heteroaryl preferably denote mono-, bi- or tricyclic aromatic or heteroaromatic groups having up to 25 ring atoms, which may also include fused rings and which are optionally substituted.

[0332] Preferred aryl groups include, but are not limited to, 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, and the like.

[0333] Preferred heteroaryl groups include, but are not limited to, five-membered rings such as 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 such as 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-tetrazine, 1,2,3,5-tetrazine and condensed systems such as carbazole, indole, isoindole, indolizine, indazole, benzimidazole, benzotriazole, purine, naphthoimidazole, phenanthrimidazole, pyridoimidine, dazole, pyrazine imidazole, quinoxaline imidazole, benzoxazole, naphthoxazole, anthroxazole, phenanthroxazole, isoxazole, benzothiazole, benzofuran, isobenzofuran, dibenzofuran, quinoline, isoquinoline, pteridine, benzo-5,6-quinoline, benzo-6,7-quinoline, benzo-7,8-quinoline, benzoisoquinoline, acridine, phenothiazine, phenoxazine, benzopyridazine, benzopyrimidine, quinoxaline, phenanthroxazole, azine, naphthyridine, azacarbazole, benzocarboline, phenanthridine, phenanthroline, thieno[2,3b]thiophene, thieno[3,2b]thiophene, dithienothiophene, dithienopyridine, isobenzothiophene, dibenzothiophene, benzothiadiazothiophene, 2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (diketopyrrolopyrrole, DPP), 2-oxo-1H-indol-3-ylidene, [3,3'-bipyrrolo[2,3-b]pyridinylidene]-2,2'(1H,1'H)-dione (pyridine isoindigo) and (3E)-3-(2-oxo-1H-indol-3-ylidene)-1H-indol-2-one (isoindigo) or combinations thereof. The heteroaryl group may be substituted with alkyl, alkoxy, thioalkyl, fluoro, fluoroalkyl or further aryl or heteroaryl substituents. Preferably, the heteroaryl group is thiophene.

[0334] Particularly preferred heteroatoms are selected from O, S, N, P and Si. Typically, hydrogen completes the valence of the heteroatoms contained in the molecules of the invention, for example, -NH- or -NH2 may be present involving one or two other groups for N.

[0335] As used herein, the term "optionally substituted" means that one or more of the hydrogen atoms in the optionally substituted moiety are replaced by a suitable substituent. Unless otherwise indicated, 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 substituents may be the same or different at all positions. The combinations of substituents envisioned by the present invention are preferably those that result in the production of stable compounds. As used herein, the term "stable" refers to compounds that are chemically feasible and can exist at room temperature (i.e., 16-25°C) for a sufficient period of time to allow for their detection, isolation and / or use in chemical synthesis.

[0336] Any of the above groups (e.g., those referred to herein as "optionally substituted", e.g., alkyl, aryl and heteroaryl groups) may optionally include one or more substituents, which are preferably selected from the group consisting of silyl, sulfo, sulfonyl, formyl, amino, imino, nitrilo, mercapto, cyano, nitro, halogen, -NCO, -NCS, -OCN, -SCN, -C(=O)NR0R00, -C(=O)X0, C(=O)R0, -NR0R00, C1-12 alkyl, C1-12 alkenyl, C1-12 alkynyl, C6-12 aryl, C The aryl group is selected from 3-12 cycloalkyl, heterocycloalkyl having 4-12 ring atoms, heteroaryl having 5-12 ring atoms, C1-12 alkoxy, hydroxy, C1-12 alkylcarbonyl, C1-12 alkoxy-carbonyl, C1-12 alkylcarbonyloxy, or C1-12 alkoxycarbonyloxy, in which one or more H atoms are optionally replaced with F or Cl and / or combinations thereof; X0 is halogen, and R0 and R00 are independently H or an optionally substituted C1-12 alkyl. The optional substituents may include all chemically possible combinations of the same and / or multiple of the above groups (e.g., amino and sulfonyl represent a sulfamoyl group when directly attached to each other). In one embodiment, the substituent is not acyl. As used herein, acyl refers to an acyl group, which is a moiety derived by 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.

[0337] In some embodiments, the group can be unsubstituted. For example, an anthracycline (PNU) derivative can be represented by formula (V): [ka] wherein [X] is an optional spacer selected from the group comprising an unsubstituted alkyl group, an unsubstituted heteroalkyl group, an unsubstituted aryl group, an unsubstituted heteroaryl group, 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), peptide, -(CH2)n-, -(CH2CHO)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. It may be of the following type.

[0338] In embodiments where the group is unsubstituted, [X] is preferably polyethylene glycol, and [ka] is selected from the group comprising: [ka] represents the point of attachment to the remainder of the molecule, and [R] is an optional spacer selected from the group including an unsubstituted alkyl group, an unsubstituted heteroalkyl group, an unsubstituted aryl group, an unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or combinations thereof.

[0339] Generally, the term PAB is intended to mean p-aminobenzyloxycarbonyl. Sometimes in the literature, the term PAB may be used to indicate p-aminobenzyl. In this specification, PAB is intended to indicate p-aminobenzyloxycarbonyl.

[0340] The term "target binding molecule" refers to any molecule that binds to a given target. In this context, "target" and "antigen" may be used interchangeably. Examples of target binding molecules include natural or recombinant proteins, such as 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 (sdAbs), VH domains, scaffold proteins (affibodies, centyrin, darpin, etc.), and nucleic acids, such as aptamers or small molecules or natural products that are developed to bind to targets or that naturally bind to targets.

[0341] Chemical modification of proteins and biomolecules to introduce thiols is well established. 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.

[0342] The phrase "selected from the group comprising" whenever it appears in this specification may be replaced with the phrase "selected from the group consisting of" and vice versa.

[0343] The PNU derivatives described herein can be prepared according to standard synthetic methods. Mass spectrometry can be used to confirm that the correct molecule has been produced (Table 4).

[0344] [Table 5] JPEG2024500405000035.jpg94170

[0345] The invention will be further understood by reference to the following examples. EXAMPLES

[0346] Example 1 – Generation of Anti-ROR1 VNAR B1 Loop Library Sequences B1 protein library design. To gain a better understanding of the interaction between B1 and ROR1, we analyzed the crystal structure of B1 in complex with the ROR1 Ig domain (data not shown). This crystal structure provided which positions were changed in the protein library that was expressed and screened. We previously noted that mutation of B1 tryptophan residues at positions 88 and 94 to alanine (standard alanine scanning approach) caused loss of protein function or expression. From the crystal structure, it was observed that those residues in the CDR3 loop appear to be important for ROR1 binding. Therefore, a B1 loop library was designed to modify the biophysical properties of the protein through changes in selected positions within the CDR1 and CDR3 regions. A set of mutations made at each specific loop position was provided from the structural analysis of the B1:ROR1 complex with the aim of altering the biophysical properties while maintaining structural integrity and high affinity binding.

[0347] Library Construction The sequence of B1 and the loop library design are shown in Figure 1. The library was synthesized by controlled mutagenesis of CDR1 and CDR3. Residues 30, 32, 88, 94 and 95 located within the CDR loops were randomized.

[0348] Library construction. B1 loop library DNA was amplified by PCR using specific primers to introduce an SfiI restriction site for cloning into the pEDV1 phagemid vector. The library DNA ligated into pEDV1 was transformed into electrocompetent TG1 E. coli (Lucigen). The library size was calculated to be 8 x 104.

[0349] 84 single clones were picked and sequenced as library quality control. One sequence was found to be a WT B1 clone. A total of 70 unique clones based on CDR1 and CDR3 diversity were identified.

[0350] These sequences contain a C-terminal HisMyc tag to allow purification by IMAC chromatography and assessment of ROR1 binding by ELISA and flow cytometry.

[0351] Screening of libraries for ROR1-specific VNAR sequences Because B1 binds to both human and mouse ROR1, recombinant human and mouse ROR1-Fc proteins were used to screen the CDR loop library. A total of 928 clones were expressed in a 96-well format; periplasmic fractions were extracted and analyzed for binding to ROR1 by ELISA. In addition to the B1 WT sequence that bound to ROR1, 23 unique sequences were found.

[0352] Expression of ROR1 VNAR binders Twenty-three clones were expressed in TG1 E. coli bacteria and IMAC purified using Ni-NTA Sepharose. Proteins were dialyzed against PBS pH 7.4, and the absorbance Abs280 was measured and the concentration calculated. The yields obtained ranged from 1.5 to 9 mg / L. Protein purity was analyzed by SDS-PAGE.

[0353] Binding to human and mouse ROR1 by ELISA for the different loop variants is summarized in Table 5.

[0354] method Library synthesis The CDR loop library was synthesized according to the design provided by GeneArt Gene Synthesis.

[0355] Subcloning the library into pEDV1 PCR amplification of 11.4 ng (10 μl) of the synthetic library was carried out in a total reaction volume of 1 ml using Phusion High-Fidelity PCR Master Mix and the following primers: 280: 5'-CTACCGTGGCCCAGGCGGCC-3' (SEQ ID NO: 133) 287: 5'-GGTGATGGTGGGCCCCTGAGGCCT-3' (SEQ ID NO: 134)

[0356] The amplicon was purified with a Promega PCR purification kit, digested with SfiI, and ligated into the SfiI restriction enzyme-opened pEDV1 vector. The ligation was performed at a 1:3 ratio (0.54 μg of vector was ligated with 1.62 μg of library DNA).

[0357] Screening of CDR loop libraries: Periplasmic expression and ELISA binding of single clones in 96-well format Inoculate into Greiner 96 deep well plates containing 1.1 ml 2xTY / 0.1% glucose / 100 μg / μl Amp. Grow in incubation chamber at 37° C. for 5 hours at 180 rpm until slightly turbid. Induce with 110 μl / well of 1 mM IPTG (IPTG=100 μM final concentration) in 2.2×TY / Amp; overnight at 28° C. at the same shaking speed. 3. Spin the culture for 15 minutes at 4° C. and 3500 rpm. Decant the supernatant and tap dry on a paper towel. 4. Add 250 μl / well of ice-cold TES buffer (50 mM Tris / HCl, pH 8.0 / 1 mM EDTA, pH 8.0 / 20% sucrose) to the pellet. Vortex. 5. Add 250 μl TES buffer (ice cold) diluted 1:5 in water. Keep on ice (or on edge) for 30 minutes. Spin as above. Keep supernatant on ice until ready to use.

[0358] ELISA 1. Coat 96-well plates with 1 μg / ml huROR1-Fc, mouse ROR1-Fc, human ROR2-Fc or HSA and incubate overnight at 4° C. 2. Wash the plate 3x with PBST. 3. Block coated plates with 200 μl / well of 4% MPBS. Incubate at room temperature for 1 hour. 4. Wash with 3x PBST. 5. Incubate the plate with 100 μl / well of the periplasmic preparation for 1 hour at room temperature. 6. Add 100 μl of Anti-His-HRP (1:1000 in PBST) and incubate for 1 hour at room temperature. 7. Wash with 2x PBST and 2x PBS. 8. Add 100 μl / well TMB substrate. Stop reaction with 50 μl / well 1M H2SO4.

[0359] Large-scale expression and purification of ROR1 VNAR binders 1. Inoculate a clone from a glycerol stock into 20 ml of 2xTY / 0.1% glucose / 100 μg / μl Amp. Grow overnight at 37° C. in an incubation chamber shaking at 250 rpm. 2. Dilute the overnight culture 1:50 in TB+phosphate+1% glucose+100 μg / ml Amp media (10 ml overnight culture into 500 ml media; 450 ml TB+50 ml phosphate) and incubate at 37° C. with vigorous shaking (250 rpm) for the whole day or as long as possible. 3. Pellet the cells by centrifugation at 4,000 x g for 15 minutes at 20°C. 4. Resuspend the cells in the same volume of TB+phosphate+1% glucose+100 μg / ml Amp media and incubate overnight at 30° C. with shaking. 5. Pellet the cells by centrifugation at 4,000 x g for 15 minutes at 20°C and resuspend the cells in the same volume of TB + phosphate + 100 µg / ml Amp medium (no glucose). Add IPTG to a final concentration of 1 mM IPTG. Incubate with shaking at 30°C for 4-5 hours. 6. Cells are harvested by centrifugation at 4500×g for 20 minutes [pellets can be frozen at -20° C. at this point] 7. Resuspend the pellet in 10% culture volume of ice-cold TES buffer (50 ml for a 500 ml culture) and shake gently on ice for 15 minutes. 8. Add an equal volume of ice-cold 5 mM MgSO4 (for a final concentration of 2.5 mM MgSO4) and continue gentle shaking on ice for an additional 15 minutes. 9. Pellet the suspension by centrifugation at 8000×g for 30 minutes at 4° C. The supernatant contains the released periplasmic proteins. 10.10x PBS (pH 7.4) [final concentration of 1x PBS] is added to extracts of periplasmic preparations prior to IMAC purification.

[0360] Immobilized metal affinity chromatography (IMAC) purification Add 1.2-3 ml of nickel-resin (His Pur Ni-NTA resin, Thermo Fisher #88222) to 100 ml of osmotic shock solution (periplasmic extract) and incubate on a roller for 1 hour at room temperature. 2. Pass the periplasmic extract through a column (Polyprep chromatography column 10 ml, Bio-Rad #7321010) 3. Wash the resin with 50-100 ml of PBS. 4. Elute the protein with 5 x 1 ml of 500 mM Imidazole, pH 8. 5. Dialyze the eluate in 3 x 5 liters of PBS in a dialysis cassette (Slide A Lyzer dialysis cassette 7.000 MWCO, Thermo Fisher #66707) with stirring. 6. Analyze proteins by SDS-PAGE.

[0361] The concentration of purified proteins was determined from the absorbance at 280 nm using the theoretical extinction coefficient predicted from the amino acid sequence. All proteins were characterized by reducing and non-reducing SDC PAGE analysis and mass spectrometry. The formation of the desired disulfide bonds was confirmed by mass spectrometry.

[0362] Size Exclusion Chromatography The loop engineered variants were evaluated by size-exclusion chromatography. The monomericity and biophysical properties of the B1 loop variants were evaluated by size-exclusion chromatography (SEC) using an analytical SEC column (Superdex 75 10 / 300 GL). Chromatography was performed in PBS pH 7.4. The elution volume on SEC can be a measure of the relative hydrophobicity of different proteins. Increasing elution volume indicates increasing hydrophobicity as a result of interactions with the column matrix.

[0363] SEC elution volume runs under the same conditions are shown in Table 5.

[0364] Binding to human and mouse ROR1 by BLI Binding kinetics were determined using Biolayer Interference (BLI) Octet K2 system (ForteBio). Human or mouse ROR1-hFc fusion protein (extracellular domain) was immobilized using amine coupling on COOH2 chip or AR2G sensor in sodium acetate pH 5 buffer. VNARs were tested at various concentrations and Ka (M-1s-1), Kd (s-1) and KD (nM) values ​​were determined using Octet Data Analysis High Throughput software for Biolayer Interference (ForteBio).

[0365] Table 5 summarizes the BLI data for the affinity of these molecules for human and mouse ROR1.

[0366] [Table 6]

[0367] Binding of loop variant VNARs to cell surface ROR1 by flow cytometry Loop variant VNARs were re-expressed using intein technology. For expression as an intein fusion, DNA encoding the VNAR was optimized for E. coli expression (GeneArt, Thermo) and cloned in frame into an intein expression vector. This results in the gene encoding the VNAR protein of interest fused to an engineered intein domain that is in turn fused to a chitin binding domain (CBD) to allow purification on a chitin column.

[0368] Transformed E. coli cells were grown in 1 L shaker flasks to OD600nm=~0.6, cold shocked at 4°C for 2 hours, and then protein expression was induced with 0.5 mM IPTG overnight at 18°C. Cells were lysed by sonication in lysis buffer (50 mM sodium phosphate pH 7.4, 0.5 M NaCl, 15% glycerol, 0.5 mM EDTA, 0.1% Sarkosyl, 1 mM AEBSF) and centrifuged to remove cell debris. VNAR intein fusion proteins were purified from clarified cell lysates by immobilization on chitin beads (NEB, S6651). The beads were washed extensively with lysis buffer followed by cleavage buffer (50 mM sodium phosphate pH 6.9, 200 mM NaCl) and the VNARs were released from the beads by overnight chemical cleavage in 400 mM dioxyamine or O,O'-1,3-propanediylbishydroxylamine, or 100 mM cysteine ​​or cysteamine to generate the corresponding C-terminal aminoxy, C-terminal cysteine ​​or C-terminal thiol derivatives of the VNARs.

[0369] The cleaved VNAR supernatants were then further purified by SEC (Superdex75 26 / 60 GE healthcare) and / or IMAC (HisTrap HP, GE Healthcare). Concentrations were determined from absorbance at 280 nm using the theoretical extinction coefficient predicted from the amino acid sequence. All proteins were characterized by reducing and non-reducing SDC PAGE analysis and mass spectrometry. Formation of the desired disulfide bonds in the VNAR domains was confirmed by mass spectrometry. These C-terminal HisMyc-tagged proteins were then assessed for ROR1 cell surface binding by flow cytometry.

[0370] Cell surface binding of test agents to hROR1 was characterized in different cell lines (A549 and A427) and the resulting KDapp values ​​were determined. Adherent cancer cells were detached from tissue culture flasks by incubation with 0.1% EDTA / PBS solution at 37°C for approximately 10 min or until cells easily detached. Cells were resuspended in ice-cold PBS / 2% FCS in 15 ml tubes and centrifuged at 1500 rpm for 5 min at 4°C. The supernatant was removed and the cell pellet was resuspended in PBS / 2% FCS. Cell counts were performed using a Z1 Coulter Particle Counter (Beckman Coulter) or a Chemometec Nucleocounter NC-202 and 5x10^5 cells were aliquoted into 96-well plates for each test sample. Cells were incubated with 100 μl of test agents at a range of concentrations and controls on ice for 1 h. Sample plates were centrifuged at 2000 rpm for 5 min. Washes were performed by removing the supernatant and resuspending the cell pellet in 0.25 mL ice-cold PBS / 2% FCS using a multichannel pipette. Samples were centrifuged again at 2000 rpm for 5 min at 4° C. The supernatant was removed and two further washes were performed as above. After the final wash and centrifugation step, excess liquid was removed by blotting the plate on tissue paper. VNAR binding was determined by adding 100 μl of anti-×6His tag Ab (Abcam) per cell pellet sample as appropriate and incubating on ice for 30 min. Wash steps were performed as above. PE-anti-mouse antibody (JIR) was used to detect binding of VNAR (His6 tagged) drugs and corresponding drug-conjugates by incubating with the appropriate samples for 30 min on ice in the dark. Wash steps were performed as above. All cell pellets were finally resuspended in 0.3 ml ice-cold PBS / 2% FCS and kept on ice in the dark before analysis on a Merck-Millipore Guava EasyCyte HT or Thermo Fisher Attune NxT flow cytometer.

[0371] As shown in Figures 2 and 3, the loop library variants bind to the ROR1hi human cancer cell line A549 but not to the ROR1low human cancer cell line A427. 2V is a control VNAR sequence derived from a naive VNAR library and is therefore representative of this protein class but has no known target.

[0372] Example 2 – Humanization and further engineering of B1 loop library variants Two different strategies were used to generate humanized sequence derivatives of the three lead ROR1-binding B1 loop library VNARs.

[0373] The humanized sequences were designed based on the human germline Vκ1 sequence, DPK-9. For example, in P3A1 V1, framework regions 1, 3 and 4 of the VNAR were mutated to align with the framework regions of DPK-9.

[0374] The second approach involved grafting the binding loops of ROR1-binding VNARs onto a previously humanized VNAR framework (Kovalenko et al JBC 2013 288(24)17408-17419; WO 2013 / 167883), but engineered additional positions based on the structure of VNAR B1 in complex with the ROR1 Ig domain.

[0375] Additional sites for engineering include amino acid changes in the CDR1, HV2 and HV4 regions of the protein.

[0376] Similarly, this approach was used to develop a humanized variant of B1, which therefore contains amino acid changes in its CDR1, HV2 and HV4 regions as well as in the framework regions. Since B1G4 is effectively a loop library derivative of B1 or a loop library variant of a humanized variant of B1, the CDR1, HV2, HV4 and CDR3 sequences are the same as in the parent protein.

[0377] Examples of humanized / grafted loop library VNAR sequences are as follows: G3CP G4 (C124) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) G3CP V15 (C125) ASVTQSPRSASKETGESLITITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK (SEQ ID NO: 72) 1H8 G4 (C126) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIK (SEQ ID NO: 73) 1H8 V15 (C127) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVK (SEQ ID NO: 74) C3CP G4 (C128) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPRQVQWYDGAGTKVEIK (SEQ ID NO: 75) C3CPV15 (C129) ASVTQSPRSASKETGESLITITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSSLRISSLTVEDSATYYCKAYPWGAGAPRQVQWYDGQGTKLEVK (SEQ ID NO: 76) B1G4 (C130) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (sequence number 51).

[0378] DNA encoding the humanized constructs was codon-optimized for expression in E. coli and synthesized by GeneArt (Thermo). All humanized sequences were generated with the following C-terminal His6 tags: QASGAHHHHHH (SEQ ID NO: 102)

[0379] The G4 sequence was generated without an additional C-terminal tag.

[0380] The DNA encoding these proteins was subcloned into an intein expression vector, expressed in E. coli, and purified as previously described in the "Typical Methods for Expression of VNAR Intein Fusion Proteins" section.

[0381] The humanized ROR1-binding VNAR variants demonstrated high affinity binding to human ROR1 by BLI, good thermal stability and evidence of little aggregation by SEC. BLI was performed as described above using human ROR1 ECD-Fc immobilized on the chip surface. SEC was performed as described above. Thermal stability assays used the Applied Biosystems StepOne Real Time PCR system with the Protein Thermal Shift™ dye kit (Thermo). The assay mix was set up to a final concentration of 20 μM protein in 20 μL. 5 μL of Thermal Shift™ buffer was added in parallel with 2.5 uL of 8× Thermal Shift™ dye. The assay was run using StepOne software and the data was analyzed using Protein Thermal Shift™ software. All data are from first derivative analysis. The BLI data for hROR1 binding and thermal stability by protein thermal shift are shown in Table 6.

[0382] [Table 7]

[0383] Grafting the HV and CDR loops of G3CP, 1H8 and C3CP onto the humanized VNAR framework with additional mutations in the CDR1, HV2 and HV4 regions or replacing the VNAR framework sequences with regions from the human DPK-9 sequence resulted in essentially engineered proteins that were stable, monomeric and maintained high affinity binding to hROR1.

[0384] Example 3 – Generation of anti-ROR1 VNAR P3A1 G1 loop library sequences Library Design P3A1 G1 is a humanized version of the ROR1-binding VNAR P3A1. The P3A1 G1 loop library was designed to improve the ROR1-binding affinity of this humanized variant through randomization of the CDR1, HV2 and HV4 regions without any changes in the framework. Mutation selection was based on data analysis of the VNAR sequence from the spiny dogfish (Squalus acanthus). The sequence and library design of P3A1 G1 are shown in Figure 4.

[0385] Libraries were synthesized by controlled mutagenesis of CDR1, HV2 and HV4. Residues 26-33, 44-52 and 61-65 located within the CDR1, HV2 and HV4 loops, respectively, were changed to selected amino acids as defined in Figure 4, resulting in a total library diversity of 8.2 x 106 combinations.

[0386] Library construction. P3A1 G1 library DNA was amplified by PCR using specific primers to introduce an SfiI restriction site for cloning into the 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 2×108. 192 single clones were picked and sequenced as library quality control.

[0387] Screening the P3A1 G1 library for antigen-specific VNAR sequences Recombinant human ROR1 protein was used for selection and screening of the P3A1 G1 library. Two strategies were used to isolate ROR1-specific binders: selection on biotinylated antigen immobilized on pre-decorated streptavidin-coated beads and selection with antigen directly immobilized on immunotubes. Selection on pre-decorated biotinylated antigen beads included three rounds of panning, with low stringency for the first and second rounds (3x PBST and 3x PBS washes for both rounds, 100 nM and 10 nM biotinylated huROR1 for rounds 1 and 2, respectively), but high stringency for the third round (10x PBST and 10x PBS washes, 0.5 nM biotinylated huROR1). Selection on immunotubes consisted of two rounds of panning with a constant antigen concentration of 2 ng / ml. After the selection process, the output was screened for antigen-specific binding by monoclonal phage against human or mouse ROR1 and periplasmic extract ELISA. From selections using antigen directly immobilized on immunotubes, 95% of monoclonal phages displaying VNARs were specific for human and mouse ROR1, compared with 4% for selection on biotinylated antigen immobilized on pre-decorated streptavidin-coated beads.

[0388] A total of nine unique sequences were expressed from each selection round and analyzed for binding and selectivity (Tables 7 and 8).

[0389] [Table 8]

[0390] [Table 9] JPEG2024500405000040.jpg48170

[0391] Expression of ROR1 P3A1 G1 loop variants Clones were expressed in TG1 E. coli bacteria and the resulting C-terminally HisMyc-tagged proteins were purified by IMAC using Ni-NTA Sepharose. Proteins were dialyzed against PBS pH 7.4, absorbance Abs280 was measured and concentrations calculated. The yields obtained ranged from 0.5 to 6.5 mg / L. Protein purity was analyzed by SDS-PAGE.

[0392] All proteins were characterized by reducing and non-reducing SDC PAGE analysis and mass spectrometry. The formation of the desired disulfide bonds was confirmed by mass spectrometry.

[0393] Binding of P3A1 G1 loop variants to hROR1 by ELISA Binding of P3A1 G1 loop variants to human ROR1 was first assessed by ELISA. Briefly, the ELISA method is as follows: Wells were coated with 100 ng of ROR1-hFc antigen, covered and incubated at room temperature for 2 hours. Plates were washed 3x400ul per well with PBST (PBS + 0.05% Tween 20 (v / v)) and then blocked with 4% skim milk powder (w / v) in PBST for 1 hour at 37°C. Plates were washed as above and further washed with PBS alone. HisMyc-tagged binding proteins were diluted in 4% milk PBST and incubated overnight at 4°C. Plates were washed 3x with PBST, 3x with PBS, and binding was detected using the appropriate secondary detection antibody in 4% milk PBST for 1 hour at room temperature. Secondary antibodies used include anti-c-Myc, HRP (Invitrogen #R951-25) mouse anti-polyHis, HRP (Sigma #A7058).

[0394] Plates were washed 3 times with PBST. 100 μL of TMB substrate (Thermo #34029) was added and the reaction was allowed to proceed for 10 minutes at room temperature. The reaction was quenched by adding 100 μL of 2M H2SO4. Plates were centrifuged briefly before reading the absorbance at 450 nm on a CLARIOstar plate reader (BMG Labtech).

[0395] FIG. 5 shows the relative binding of the different variants to human ROR1, with sequences NAG8.S, AF7.S, NAC6.S and AE3.S showing the greatest signals for binding.

[0396] The same ELISA 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.

[0397] Dose response data shown in FIG. 6 indicates that these loop library sequences bind more strongly to human ROR1 than the parental P3A1 G1 protein.

[0398] Characterization of mouse ROR1 and ROR2 binding of P3A1 G1 loop variants by ELISA A selection of P3A1 G1 loop variants were further characterized for binding to mouse ROR1 and human ROR2 by ELISA. The same ELISA procedure was used as above, but with either mROR1-hFc or hROR2-hFc coated onto the plate. The tested variants did not bind to human ROR2. Of the variants tested, NAC6.S and AE3.S bound to mouse ROR1.

[0399] Expression of P3A1 G1 Loop Library Variants as Intein Fusion Proteins The P3A1 G1 loop variants VNAR NAG8.S, NAC6.S and AE3.S were re-expressed using intein technology but with Ser deleted from the CDR1 loop. Expression as intein fusions was performed as above with either a His-tag QACKAHHHHHHG (SEQ ID NO: 163) or a HisMyc-tag QACKAHHHHHHGAEFEQKLISEEDLG (SEQ ID NO: 164) incorporated at the C-terminus of the VNAR domain.

[0400] After expression and capture on chitin beads, the intein VNARs were released from the beads by overnight chemical cleavage in 400 mM dioxyamine or O,O'-1,3-propanediylbishydroxylamine, or 100 mM cysteine ​​or cysteamine to generate the corresponding C-terminal aminoxy, C-terminal cysteine, or C-terminal thiol derivatives of the VNAR.

[0401] The cleaved VNAR supernatant was then further purified by SEC (Superdex75 26 / 60 GE healthcare) and / or IMAC (HisTrap HP, GE Healthcare) to obtain proteins NAG8, NAC6 and AE3. Concentrations were determined from absorbance at 280 nm using theoretical extinction coefficients predicted from the amino acid sequences. All proteins were characterized by reducing and non-reducing SDC PAGE analysis and mass spectrometry. Formation of the desired disulfide bonds in the VNAR domains was confirmed by mass spectrometry.

[0402] These C-terminal HisMyc- or His-tagged proteins were then further evaluated for ROR1 binding by BLI, thermal stability and biophysical properties by SEC.

[0403] Binding to human and mouse ROR1 by BLI Binding kinetics were determined using Biolayer Interference (BLI) Octet K2 system (ForteBio). Human or mouse ROR1-hFc fusion protein (extracellular domain) was immobilized on COOH2 chip or AR2G sensor using amine coupling in sodium acetate pH 5 buffer. VNARs were tested at various concentrations and Ka (M-1s-1), Kd (s-1) and KD (nM) values ​​were determined using Octet Data Analysis High Throughput software for Biolayer Interference (ForteBio). Binding parameters are shown in Table 9.

[0404] Thermostability assay Thermostability assays used an Applied Biosystems StepOne Real Time PCR system using the Protein Thermal Shift™ dye kit (Thermo). The assay mix was set up to a final concentration of 20 μM protein in 20 μL of PBS pH 7.4. 2.5 μL of 8× Thermal Shift™ dye was added. Assays were run using StepOne software and data were analyzed using Protein Thermal Shift™ software. All data are from first derivative analysis and Tm values ​​are detailed in Table 9.

[0405] Size Exclusion Chromatography The monomericity and biophysical properties of the P3A1 loop variants were assessed by size-exclusion chromatography (SEC) using an analytical SEC column (Superdex 75 10 / 300 GL). Chromatography was performed in PBS pH 7.4.

[0406] The % monomeric and SEC elution volume runs under the same conditions are shown in Table 9.

[0407] [Table 10]

[0408] Example 4 – VNAR reformatting as multimers The ROR1 binding loop variant VNARs were successfully reformatted into heterodimers and trimers by gene fusion using different GlySer-based linkers to generate dual specific binders, ROR1 biparatopic binders and ROR1 biparatopic dual specific binders.

[0409] Bispecific binders Several bispecific VNAR-based binders were developed by combining a ROR1 loop variant VNAR binder with the humanized VNAR BA11, which binds serum albumin with high affinity, using the PGVQPSPGGGGGS (SEQ ID NO:96) linker.

[0410] The proteins were expressed with a C-terminal tag, QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) or QACKAHHHHHH (SEQ ID NO: 104), to aid in purification and characterization. This tag also contains a single cysteine ​​residue to facilitate site-selective bioconjugation of payloads to the protein using a thiol-mediated chemical coupling strategy.

[0411] Binding kinetics were determined using Biolayer Interferometry (K2 Octet instrument / Pall ForteBio) as described above. For BLI experiments, ROR1-hFc (extracellular domain) and HSA were immobilized on AR2G sensors in sodium acetate pH 5 buffer using amine coupling. VNAR-based molecules were tested at various concentrations and Ka (M-1s-1), Kd (s-1) and KD (nM) values ​​were determined using Octet data analysis HT software (Pall ForteBio).

[0412] Binding kinetics for hROR1 binding was also performed using saturating levels of HSA (200 nM) at baseline, association and dissociation conditions.

[0413] Binding to ROR1hi A549 cancer cell lines was determined by flow cytometry. A dose response was performed and the change in mean fluorescence intensity (background corrected) as a function of VNAR concentration was used to determine the KDapp for cell surface ROR1 binders.

[0414] The characterization of these dual specific VNARs is shown in Table 10.

[0415] [Table 11]

[0416] The dual specific VNAR binder was further modified via conjugation to a single cysteine ​​residue in the C-terminal tag.

[0417] Biparatopic Binder Different ROR1 loop variant VNAR binders were combined with or without the additional insertion of serum albumin-binding humanized VNAR BA11 to develop several ROR1 biparatopic binders. The VNAR domains were linked together using a PGVQPAPGGGGS (SEQ ID NO: 90) linker, and the proteins were expressed with a C-terminal tag QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) or QACKAHHHHHH (SEQ ID NO: 104) to aid in purification and characterization. This tag also contains a single cysteine ​​residue to facilitate site-selective bioconjugation of payloads to the protein using a thiol-mediated chemical coupling strategy.

[0418] Binding kinetics were determined using Biolayer Interferometry (K2 Octet instrument / Pall ForteBio) as described above. For BLI experiments, ROR1-hFc (extracellular domain) was immobilized on an AR2G sensor in sodium acetate pH 5 buffer using amine coupling. VNAR-based molecules were tested at various concentrations and Ka (M-1s-1), Kd (s-1) and KD (nM) values ​​were determined using Octet data analysis HT software (Pall ForteBio).

[0419] The characterization of these dual specific VNARs is shown in Table 11.

[0420] [Table 12]

[0421] Biparatopic binders exhibit increased affinity for binding ROR1 compared to individual ROR1-binding monomers.

[0422] Constructs containing BA11 are examples of biparatopic bispecific protein binders.

[0423] Additionally, several bispecific and biparatopic VNAR-based binders were developed by combining ROR1 loop variant VNAR binders with the humanized VNAR BA11 or by combining different ROR1 loop variant VNAR binders using the PGVQPCPGGGGGS (SEQ ID NO: 177) linker, which also contains a single cysteine ​​residue to facilitate site-selective bioconjugation of payloads to proteins in the linker using a thiol-mediated chemical coupling strategy.

[0424] The proteins were expressed with a C-terminal tag, QASGAHHHHHH (SEQ ID NO: 102) or QACKAHHHHHH (SEQ ID NO: 104), to aid in purification and characterization.

[0425] [Table 13]

[0426] The bispecific VNAR binders were further modified via conjugation to a single cysteine ​​residue in the linker sequence.

[0427] Prior to conjugation, 20 equivalents of TCEP were added to the bispecific protein to remove cysteine / glutathione capping of the linker thiols. After 1 hour incubation at room temperature, TCEP was removed by purification on a HiTrap SP cation exchange chromatography column (Cytiva). To load onto the column, the protein was diluted 3-fold in 50 mM Na phosphate buffer pH 6.0. The protein was then eluted with an increasing gradient of elution buffer consisting of 50 nM Na phosphate pH 6.0, 1 M NaCl. To the conjugate, 4 equivalents of maleimide-containing payload were added and allowed to incubate for 1 hour at room temperature. Free payload was then removed by cation exchange using the same protocol as above.

[0428] [Table 14]

[0429] Without being bound to any particular theory, it is predicted that the conjugate yield can be improved by increasing the scale of production and by using optimized purification processes.

[0430] Example 5 – VNAR reformatting as Fc fusion proteins VNAR Fc fusion proteins Fusion of proteins to the Fc domain can improve protein solubility and stability, significantly increase plasma half-life, and improve overall therapeutic efficacy. The human IgG1 Fc sequence is shown below, and further examples are shown in Figure 7. Human IgG1 Fc (hFc) (C131) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 145)

[0431] The VNAR loop variants were genetically fused to an engineered hIgG1 Fc domain containing the cysteine ​​substitution S239C (EU numbering) in the hIgG1 Fc sequence via a standard [G4S]3 linker. The VNAR Fc fusion proteins were transiently expressed as secreted proteins in CHO K1 cells and purified from the medium using MabSelect™ SuRe™ (Evitria, Switzerland). The purified proteins were exchanged into PBS pH 7.4 or PBS + 100 mM Arg pH 7.4 and analyzed by SEC (AdvanceBio, Agilent, running buffer DPBS pH 7.4), SDS PAGE and mass spectrometry to confirm sequence and protein integrity.

[0432] Binding kinetics were determined using a Pioneer surface plasmon resonance (SPR) instrument (SensiQ / Pall ForteBio) or a Biolayer Interference (BLI) Octet K2 system (ForteBio). ROR1-hFc fusion protein (extracellular domain) was immobilized using amine coupling on a COOH2 chip or AR2G sensor in sodium acetate pH 5 buffer. VNAR-Fc molecules were tested at various concentrations and Ka (M-1s-1), Kd (s-1) and KDapp (nM) values ​​were determined using Octet Data Analysis High Throughput software for Biolayer Interference (ForteBio). Alternatively, kinetic parameters for binding were determined by immobilizing VNAR-hFc fusions on an AHC chip. Human ROR1 (ECD) was tested at various concentrations and the Ka (M-1s-1), Kd (s-1) and KD (nM) values ​​for 1:1 binding were determined using Octet Data Analysis High Throughput software for Biolayer Interference (ForteBio). ROR1 2A2 mAb (Biolegend) was included as a control for positive / negative binding to ROR1. 2V is a control VNAR sequence derived from a naive VNAR library and thus is representative of this protein class but has no known target. SEC analysis was performed as described above. The data are summarized in Table 14, which demonstrates the advantageous properties of the loop library hFc variants relative to the parent B1-hFc protein.

[0433] Binding of VNAR-Fc fusions to ROR1 on the surface of cancer cell lines was measured by flow cytometry using the method described above, and binding of VNAR-hFc fusion molecules was determined by addition of 100 μL of PE-anti-human antibody (JIR) and incubation on ice for 30 minutes. 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 ROR1hiA549 lung adenocarcinoma cells.

[0434] Table 14 summarizes the expression yields (based on final purified buffer exchanged protein), SEC analysis of the hFc fusions, the affinity of these molecules for ROR1 by BLI and the KDapp for binding of ROR1hiA549 cells.

[0435] [Table 15]

[0436] The relative stability of VNAR-hFc fusion proteins in PBS buffer was evaluated. G3CP-hFc, G3CPG4-hFc and B1G4-hFc as well as parental B1-hFc were incubated at 2 mg / mL in sterile PBS buffer pH 7.4 containing 0.05% sodium azide for 96 hours at 37° C. UV absorbance was measured at 280 nm and 320 nm at t=0 and t=96 hours. Monomeric nature of the proteins at t=0 and t=96 hours was assessed by size exclusion chromatography (S200 Increase 10 / 300GL with PBS pH 7.4 running buffer). As shown in FIG. 16, UV absorbance at both 280 nm and 320 nm increased after 96 hours of incubation for B1-hFc but not for the loop library variants (i.e., G3CP-hFc and G3CPG4-hFc). The absorbance, especially at 320 nm, is due to light scattering by aggregate particles. The protein concentrations for the loop library variants (i.e., G3CP-hFc and G3CPG4-hFc), calculated from the absorbance at 280 nm, remain constant throughout the experiment. SEC analysis at t=0 vs. t=96 hours (FIG. 17) shows that the loop library variants (i.e., G3CP-hFc and G3CPG4-hFc) have good stability, more stable than the parent protein B1-hFc.

[0437] Biparatopic VNAR Fc fusion proteins The VNAR loop variants were genetically fused via a standard [G4S]3 linker to hIgG1 Fc engineered for heterodimerization (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). Using this approach, a biparatopic ROR1 binder was generated in which one arm contains the VNAR loop variant and the other arm contains a second ROR1-binding VNAR. Additionally, a cysteine ​​substitution was incorporated in the hIgG1 Fc sequence [S239C (EU numbering)] of both the knob and hole variants to facilitate bioconjugation with different payloads.

[0438] VNAR Fc fusion proteins were transiently co-expressed as secreted proteins in CHO K1 cells and purified from the medium using MabSelect™ SuRe™ (Evitria, Switzerland). Purified proteins were exchanged into PBS pH 7.4 and analyzed by SEC (AdvanceBio, Agilent, running buffer DPBS), SDS PAGE and mass spectrometry to confirm sequence and protein integrity. G3CP hFc(S239C+Y407T) (C132) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 146) G3CPG4 hFc(S239C+Y407T) (Chemical 133) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 147) P3A1 hFc(S239C+T366Y) (Chemical 134) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 148)

[0439] All proteins were characterized by reducing and non-reducing SDS PAGE analysis (Figure 9) and mass spectrometry (Table 15).

[0440] Binding to ROR1 was determined using Biolayer Interferometry (K2 Octet instrument / Pall ForteBio) as described above. For BLI experiments, ROR1-hFc, (extracellular domain) was immobilized on the sensor. Data are shown in Table 15.

[0441] [Table 16]

[0442] Binding of biparatopic VNAR-Fc fusions to ROR1 on the surface of cancer cell lines was measured by flow cytometry using the method described above. Binding of VNAR-hFc fusion molecules was determined by adding 100 μL of PE-anti-human antibody (JIR) and incubating on ice for 30 minutes. KDapp values ​​were calculated from the increase in fluorescence intensity as a function of VNAR-hFc concentration. Figure 10 shows the binding of biparatopic VNAR-Fc fusions to ROR1hiA549 lung adenocarcinoma cells and ROR1lowA427 cells. G3CP-P3A1 hFc(S239C+KIH) and G3CPG4-P3A1 hFc(S239C+KIH) bind strongly to A549 cells with KDapp of 0.06 nM and 0.20 nM, respectively, but show little binding to A427 cells.

[0443] Example 6 – Loop variant VNAR drug conjugates VNAR-hFc drug conjugates Another approach to generating ADCs is to engineer cysteine ​​substitutions or additions at positions on the light and heavy chains of antibodies that provide reactive thiol groups for site-specific labeling (Junutula 2008 Nature Biotechnology 26,925-932, Jeffrey 2013, Sutherland 2016).

[0444] Anti-ROR1 loop library VNAR-hFc fusions were generated as described above with an additional cysteine ​​engineered into the Fc region that allowed for site-specific labeling 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).

[0445] Generation of VNAR-hFc-drug conjugates These proteins were site-specifically labeled with maleimide PNU derivatives using a partial reduction, refolding and labeling method adapted from the literature [Junutula et al, 2008 Nat Biotech, Jeffrey et al, 2013 Bioconj Chem]. Briefly, a 1 mg / ml solution of VNAR hFc was prepared in PBS + 100 mM L-arginine pH 7.4 with 1 mM EDTA. 20 molar equivalents of TCEP were added and incubated at 4°C for a minimum of 48 hours. 30 molar equivalents of DHAA were added, the pH adjusted to 6.5 and incubated at room temperature for 1 hour. The refolded VNAR Fc S239C was extensively dialyzed or buffer exchanged into PBS + 50 mM L-arginine and quantified by UV before reacting with 4 or 5 molar equivalents of maleimide PNU solution at room temperature overnight. The conjugates were purified by SEC and analyzed by analytical HIC, analytical SEC and LC-MS. Table 16 summarizes the conjugates that were prepared.

[0446] [Table 17]

[0447] Analysis of the final conjugate by SDS-PAGE and mass spectrometry determined that the labeling proceeded quantitatively to yield a highly pure, homogenous protein drug conjugate with a drug-to-antibody ratio (DAR) of 2.

[0448] Binding of VNAR-hFc-drug conjugates to hROR1 by ELISA Binding of G3CP hFc and G3CPG4 hFc and their respective drug conjugates to human ROR1 was assessed by ELISA. Briefly, the ELISA method is as follows: Wells were coated with 100 ng of ROR1-his antigen, covered and incubated at room temperature for 2 hours. Plates were washed 3x400ul per well with PBST (PBS + 0.05% Tween 20 (v / v)) and then blocked with 4% skim milk powder (w / v) in PBST for 1 hour at 37°C. Plates were washed as above and further washed with PBS alone. B1 loop variant (VNAR-hFc fusion) binding protein was diluted in 4% milk PBST and incubated overnight at 4°C. Plates were washed 3x with PBST, 3x PBS, and binding was detected using appropriate secondary detection antibody in 4% milk PBST for 1 hour at room temperature. The secondary antibody used for detection was rabbit anti-human IgG H&L (HRP), Abcam catalogue number ab6759. Plates were washed 3 times with PBST, then 100 μL of TMB substrate (Thermo #34029) was added and the reaction was allowed to proceed for 10 minutes at room temperature. The reaction was then quenched by adding 100 μL of 2M H2SO4. Plates were centrifuged briefly before reading the absorbance at 450 nm on a CLARIOstar plate reader (BMG Labtech).

[0449] FIG. 12 shows that G3CP hFc and G3CPG4 hFc PNU conjugates bind potently to human ROR1, with no loss of binding activity following conjugation of different PNU linker payloads to the parent protein.

[0450] In vitro cell viability assay for cancer cells treated with anti-ROR1 VNAR drug conjugates Cells were seeded in white clear bottom 96 well plates (Costar) and incubated at 37°C, 5% CO2 for 24 hours. The next day, a dilution series was set up with a x10 working stock for each test agent. Dose response x10 stocks were 10000, 5000, 1000, 500, 100, 50, 10, 5, 1, 0.5 nM etc. 10 μL of the x10 stock solution was added to the cell plate using a multichannel pipette (90 μl per well). This resulted in a 1:10 dilution into the wells and a dose response ranging from 1000 nM (column 1) to 0.05 nM (column 10) or down to 0.5 fM as appropriate for the most sensitive cell line. 10 μl of vehicle control (PBS) was added to the control wells (columns 11 and 12). Plates were incubated at 37°C, 5% CO2 for 72-96 hours. Cell viability was assessed using Promega Cell Titre Glo reagent according to the manufacturer's instructions. Briefly, assay plates were removed from the incubator and equilibrated to room temperature before adding 100 μl of room temperature Cell Titre Glo reagent to each 100 μl assay well. Plates were placed on a plate shaker at 600 rpm for 2 minutes. Plates were allowed to sit at room temperature for an additional 10 minutes before measuring luminescence readouts using a Clariostar plate reader (BMG). Data was analyzed by calculating the average for untreated (vehicle only) control wells and determining the % of control for each treated well. The % of control data was then plotted against the Log[treated] concentration and IC50 values ​​were derived using nonlinear regression fitting in GraphPad Prism software.

[0451] The following cell lines were used: PA-1-Human ovarian cancer cell line: EMEM, 10% hiFCS PA-1 ROR1 ko - ROR1 knockout human ovarian cancer cell line: EMEM, 10% hiFCS HEK293-Human Embryonic Kidney Cell Line: EMEM, 10% FCS HEK293 stably transfected with human ROR1 (HEK293.hROR1) - a human embryonic kidney cell line stably expressing hROR1: EMEM, 10% FCS

[0452] Figure 13 shows dose response curves and corresponding IC50 values ​​(Table 17) for cell killing of 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 conjugates (PEG4-vc PAB EDA PNU159682). PA-1 ROR1 ko is a PA-1 cancer cell line in which ROR1 expression has been knocked out.

[0453] [Table 18]

[0454] The ROR1-targeted VNAR-hFc conjugate showed potent killing of the PA-1 cell line, which was suppressed upon knockdown of the ROR1 receptor. There is a >100-fold window in IC50 values ​​for both the G3CP-hFc PNU conjugate.

[0455] Figure 18 shows dose response curves and corresponding IC50 values ​​(Table 18) for cell killing of ROR1low 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 naive VNAR library and is therefore representative of this protein class but has no known target.

[0456] [Table 19]

[0457] ROR1-targeted VNAR-hFc conjugates show potent killing of the HEK293.hROR1 cell line stably transfected with the ROR1 receptor, but not ROR1low wild-type HEK293 cells. There is a >2000-fold window of IC50 values ​​for both G3CP-hFc PNU and G3CPG4-hFc-PNU conjugates for killing of HEK293 vs. HEK293.hROR1 cells, but no window exists for the 2V-hFc-PNU non-binding control conjugate.

[0458] Example 7 – In vivo efficacy of protein-drug conjugates in patient-derived xenograft models of triple-negative breast cancer (TNBC) Efficacy studies in the ROR1+HBCx-28 patient-derived TNBC xenograft model were performed by XenTech (Paris).

[0459] Outbred athymic (nu / nu) female mice (HSD: athymic nude-Foxn1nu) were implanted subcutaneously with tumors of the same in vivo passage. Mice were monitored until tumor grafts reached the test volume acceptance criteria of 60-200 mm3, preferably 75-196 mm3, in a sufficient number of animals. Mice were randomized into treatment groups such that there was no statistical difference between the tumor volumes of each group. Randomization was designated as day 0 of the experiment. Mice were treated with vehicle or 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 at a single dose of 0.3 mg / kg iv injection on day 2. All mice were pre-primed with mouse IgG for 20 hours prior to the first PDC administration. Tumor volumes were assessed by measuring the perpendicular tumor diameter with calipers three times a week during the experimental period. Absolute tumor volume (ATV) was calculated using the formula TV (mm3) = [length (mm) x width (mm)2] x 0.5, where length and width are the longest and shortest perpendicular diameters of the tumor measured perpendicularly, respectively. All animals were weighed at the same time as tumor size measurements. Mice were observed and reported daily for changes in physical appearance, behavior, adverse clinical signs, and general welfare in accordance with local welfare and best veterinary practice guidelines.

[0460] Figure 14 shows the effect of protein-drug conjugates on tumor 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 a level of in vivo efficacy comparable 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 showed improved efficacy compared to the parent B1 fusion, with complete and sustained regressions observed for both loop library variants at a single 0.3 mg / kg dose regimen.

[0461] Efficacy studies were also performed in a ROR1+HBCx-10 patient-derived TNBC xenograft model by XenTech (Paris).

[0462] Outbred athymic (nu / nu) female mice (HSD: athymic nude-Foxn1nu) were implanted subcutaneously with tumors of the same in vivo passage. Mice were monitored until tumor grafts reached the test volume inclusion criteria of 75-196 mm3 in a sufficient number of animals. Mice were randomized into treatment groups such that there was no statistical difference between the tumor volumes of each group. Randomization was designated as day 0 of the experiment. Mice were treated with vehicle or 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, iv injection three times, 4 days apart (3xQ4D, days 2, 6 and 10). All mice were pre-primed with mouse IgG for 20 hours prior to the first PDC dose. Tumor volumes were assessed by measuring the perpendicular tumor diameter with a caliper three times a week for the duration of the experiment. Absolute tumor volume (ATV) was calculated using the formula TV (mm3) = [length (mm) x width (mm)2] x 0.5, where length and width are the longest and shortest perpendicular diameters of the tumor measured perpendicularly, respectively. All animals were weighed at the same time as tumor size measurements. Mice were observed and reported daily for changes in physical appearance, behavior, adverse clinical signs, and general welfare in line with local welfare and best veterinary practice guidelines.

[0463] Figure 19 shows the effect of protein-drug conjugates on tumor growth compared to vehicle controls. All protein-drug conjugates were well tolerated and demonstrated highly statistically significant in vivo efficacy in this ROR1+TNBC PDX model, with complete and sustained regressions observed for this dosing regimen.

[0464] Example 8 – Biparatopic Loop Variant VNAR Drug Conjugates Biparatopic VNAR-hFc drug conjugates Biparatopic anti-ROR1 loop library VNAR-hFc fusions were generated as described in Example 5 with an additional cysteine ​​engineered into the Fc region as described above, which allowed for site-specific labeling of labels and cytotoxic drugs with maleimide derivatives.

[0465] Generation of biparatopic VNAR-hFc-drug conjugates The biparatopic ROR1 binding proteins G3CP-P3A1 hFc (S239C+KIH) and G3CPG4-P3A1 hFc (S239C+KIH) were conjugated with MC-vc-PAB-MMAE or MA-PEG4-vc-PAB-EDA-PNU159682 using the partial reduction, refolding and labeling method described in Example 6. The conjugates were purified by SEC and analyzed by analytical HIC, analytical SEC and LC-MS. Table 19 summarizes the conjugates prepared.

[0466] [Table 20]

[0467] Binding of biparatopic VNAR-Fc-PNU conjugates to ROR1 on the surface of cancer cell lines was measured by flow cytometry using the method described above. Binding of VNAR-hFc-PNU molecules was determined by adding 100 μL of PE-anti-human antibody (JIR) and incubating on ice for 30 minutes. KDapp values ​​were calculated from the increase in fluorescence intensity as a function of VNAR-hFc concentration. Figures 20a and b show the binding of biparatopic VNAR-Fc-PNU conjugates (PEG4-vc PAB EDA PNU159682) to ROR1hiA549 lung adenocarcinoma cells and ROR1lowA427 cells, along with the corresponding monoparatopic PNU conjugates. G3CP-P3A1 hFc(S239C+KIH)-PNU and G3CPG4-P3A1 hFc(S239C+KIH)-PNU bind strongly to A549 cells, with KDapp of 0.92nM and 1.83nM, respectively, but show little binding to A427 cells. G3CP-P3A1 hFc(S239C+KIH)-PNU demonstrates a high level of saturable binding to A549 cells compared to the corresponding G3CP-hFc-PNU and P3A1-hFc-PNU conjugates (Figure 20a). Similarly, G3CPG4-P3A1 hFc(S239C+KIH)-PNU demonstrates a high level of saturable binding to A549 cells compared to the corresponding G3CPG4-hFc-PNU and P3A1-hFc-PNU conjugates (Figure 20b).

[0468] In vitro cell viability assay for cancer cells treated with anti-ROR1 biparatopic VNAR drug conjugates The Cell Titre Glo assay was performed as described in Example 6. Cells were incubated with VNAR-hFc conjugates for 96 hours at 37°C, 5% CO2, and the % of cell viability was determined as a dose response. The % control data was plotted against the Log[treatment] concentration, and IC50 values ​​were derived using nonlinear regression fitting in GraphPad Prism software.

[0469] The following cell lines were used: PA-1-Human ovarian cancer cell line: EMEM, 10% hiFCS PA-1 ROR1 ko - ROR1 knockout human ovarian cancer cell line: EMEM, 10% hiFCS Kasumi-2 - Human pre-B cell leukemia cell line: RPMI 1640, 10% hiFCS MHH-ES1-Human Ewing's Sarcoma cell line: RPMI 1640, 10% hiFCS

[0470] Figure 21 shows dose response curves for cell killing of ROR1 positive PA-1 ovarian cancer cells 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). PA-1 ROR1 ko is a PA-1 cancer cell line in which ROR1 expression has been knocked out.

[0471] 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). Cell surface ROR1 receptor numbers were determined by flow cytometry using BD Biosciences Quantibrite beads for each cell line.

[0472] [Table 21]

[0473] ROR1-targeted biparatopic VNAR-hFc conjugates show potent killing of ROR1+ cancer cell lines but not the ROR1-negative PA1.ROR1ko cell line.

[0474] Example 9 –In vivo efficacy of biparatopic protein-drug conjugates in patient-derived xenograft models of triple-negative breast cancer (TNBC) Efficacy studies in the ROR1+HBCx-28 patient-derived TNBC xenograft model were performed by XenTech (Paris).

[0475] Outbred athymic (nu / nu) female mice (HSD: athymic nude-Foxn1nu) were implanted subcutaneously with tumors of the same in vivo passage. Mice were monitored until tumor grafts reached the test volume inclusion criterion of 100-200 mm3 in a sufficient number of animals. Mice were randomized into treatment groups such that there was no statistical difference between the tumor volumes of each group. Randomization was designated as day 0 of the experiment. Mice were treated with vehicle or biparatopic protein-drug conjugates G3CP-P3A1 hFc(S239C+KIH)-vc-PAB-EDA-PNU and G3CPG4-P3A1 hFc(S239C+KIH)vc-PAB-EDA-PNU by either a single dose of 0.3 mg / kg iv injection on day 2 or 3 x 0.1 mg / kg iv injections at 4-day intervals (3 x Q4D, days 2, 6 and 10). All mice were pre-primed with mouse IgG for 20 hours prior to the first PDC dose. Tumor volumes were assessed by measuring the perpendicular tumor diameter with a caliper three times a week for the duration of the experiment. Absolute tumor volume (ATV) was calculated using the formula TV (mm3) = [length (mm) x width (mm)2] x 0.5, where length and width are the longest and shortest perpendicular diameters of the tumor measured perpendicularly, respectively. All animals were weighed at the same time as tumor size measurements. Mice were observed and reported daily for changes in physical appearance, behavior, adverse clinical signs, and general welfare in line with local welfare and best veterinary practice guidelines.

[0476] Figure 22 shows the effect of protein-drug conjugates on tumor growth versus vehicle control. All protein-drug conjugates were well tolerated and both biparatopic loop library variants G3CP-P3A1-hFc-vc-PAB-EDA-PNU and G3CPG4-P3A1-hFc-vc-PAB-EDA-PNU showed excellent in vivo efficacy in this ROR1+TNBC PDX model, with tumor regression observed for both agents.

[0477] Example 10 –ROR1 VNAR bispecific Dual specific targeting combinations for ROR1 binding VNARs include, for example: Dual specific engagement of HSA;ROR1 and serum albumin for half-life extension RTKs, e.g. EGFR, Her3; dual specific targeting of both EGFR and ROR1 or both HER3 and ROR1 on the surface of cells Includes.

[0478] VNAR BA11, previously discussed and exemplified herein, is an example of an HSA-binding VNAR. Bispecific molecules comprising HSA-binding VNARs (e.g., BA11) and other specific binding molecules are contemplated.

[0479] ROR1xCD3 bispecific sequences combining the N-terminal ROR1 VNAR with the C-terminal anti-CD3 scFv (clone OKT3) via two different lengths of 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). Similarly, biparatopic ROR1xCD3 bispecific sequences combining the N-terminal biparatopic ROR1 VNAR with the C-terminal anti-CD3 scFv were also expressed in CHO (Evitria).

[0480] Examples and orientation of CD3 binding sequences for use as a CD3 BiTE-like approach; ROR1 VNAR bispecific anti-CD3 scFv clone OKT3 (WO2014028776 Zyngenia) and its humanized derivatives. VH-[G4S]3-VL (C135) DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS (SEQ ID NO: 149) Humanized anti-CD3 scFv UCHT1 (Arnett et al PNAS 2004 101(46)16268-16273) and its derivatives VL-[G4S]3-VH (C136) MDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 150)

[0481] Example 11 – ROR1 CAR-T approach Chimeric antigen receptors (CARs) based on the ROR1-specific antigen binding molecules described in this application can be generated. Furthermore, engineered T cells expressing such CARs can also be generated, which can then be used, for example, in adoptive cell therapy.

[0482] Briefly, a nucleic acid construct encoding a ROR1-specific CAR can be produced. The ROR1-specific CAR can include an intracellular activation domain, a transmembrane domain, and an extracellular domain that includes a ROR1-specific antigen binding molecule as described herein. The nucleic acid construct can then be incorporated into a viral vector, such as a retroviral vector (e.g., a lentiviral vector).

[0483] T cells can be isolated from a patient in need of treatment and then engineered to express a nucleic acid construct encoding a CAR, for example by retroviral transfection or gene editing using approaches such as CRISPR-CAS-9.

[0484] The engineered T cells can then be re-infused into the patient to treat the condition, e.g., to treat cancer.

Claims

1. Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, CDR3 is 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), YPWGAGAPPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), Y a CDR sequence having an amino acid sequence selected from the group consisting of PWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22) and YPWGAGAPWLVQWY (SEQ ID NO: 10); CDR1 is 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); 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 SSNQERISIS (SEQ ID NO:6) and SSNKERISIS (SEQ ID NO:7); FW3a is a framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO:8) and NKGTM (SEQ ID NO:9); FW3b is a framework region; FW4 is a framework region; When CDR3 is YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLSA (SEQ ID NO: 3) and GANYDLAA (SEQ ID NO: 4). A receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising an amino acid sequence represented by:

2. FW1 is a framework region of 20-28 amino acids; FW2 is a framework region of 6-14 amino acids; FW3a is a framework region of 6-10 amino acids; FW3b is a framework region of 17 to 24 amino acids; and / or 2. The ROR1-specific antigen-binding molecule of claim 1, wherein FW4 is a framework region of 7 to 14 amino acids, Optionally, 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 thereof having at least 45% sequence identity; FW2 has an amino acid sequence according to TYWYRKNPG (SEQ ID NO: 43) or a functional variant thereof having at least 45% sequence identity; 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 thereof having at least 45% sequence identity; FW3b has an amino acid sequence selected from the group consisting of SFSLRIKDLTVADSATYYCKA (SEQ ID NO: 84), SFTLTISSLQPEDSATYYCRA (SEQ ID NO: 46) and SFSLRISSSLTVEDSATYYCKA (SEQ ID NO: 47), or a functional variant thereof having at least 45% sequence identity; and / or FW4 is the ROR1-specific antigen-binding molecule having an amino acid sequence selected from the group consisting of DGAGTVLTVN (sequence number 48), DGAGTKVEIK (sequence number 49) or DGQGTKLEVK (sequence number 85), or a functional variant thereof having at least 45% sequence identity. (a) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO:50); (b) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO:51); (c) ASVNQTPRTATKETGESLTINCVVTGANYDLSATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPSGAGAPRPVQWYDGAGTVLTVN (SEQ ID NO:52); (d) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPCLVQWYDGAGTVLTVN (SEQ ID NO:53); (e) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRLVQWYDGAGTVLTVN (SEQ ID NO:54); (f) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRQVQWYDGAGTVLTVN (SEQ ID NO:55); (g) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRSVQWYDGAGTVLTVN (SEQ ID NO:56); (h) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRSVQWYDGAGTVLTVN (SEQ ID NO:57); (i) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSLVQWYDGAGTVLTVN (SEQ ID NO:58); (j) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSNVQWYDGAGTVLTVN (SEQ ID NO:59); (k) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSQVQWYDGAGTVLTVN (SEQ ID NO: 60); (l) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN (SEQ ID NO: 61); (m) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 62); (n) ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 63); (o) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWNVQWYDGAGTVLTVN (SEQ ID NO: 64); (p) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 65); (q) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 66); (r) ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 67); (s) ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 68); (t) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 69); (u) ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISSISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 70); (v) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71); (w) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISSISGRYSESVNKRTMSFSLRISSSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK (SEQ ID NO: 72); (x) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIK (SEQ ID NO: 73); (y) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISSISGRYSESVNKRTMSFSLRISSSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVK (SEQ ID NO: 74); (z) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPRQVQWYDGAGTKVEIK (SEQ ID NO: 75); and (aa) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISSISGRYSESVNKRTMSFSLRISSSLTVEDSATYYCKAYPWGAGAPRQVQWYDGQGTKLEVK (SEQ ID NO: 76) or any of the amino acid sequences selected from the group consisting of: and having FW1, FW2, FW3a, FW3b and FW4 sequences having at least 45% combined sequence identity with any of the combined FW1, FW2, FW3a, FW3b and FW4 sequences.

4. The ROR1-specific antigen-binding molecule according to any one of claims 1 to 3, The ROR1-specific antigen-binding molecule comprises: (a) does not bind to receptor tyrosine kinase-like orphan receptor 2 (ROR2); (b) binds to both human ROR1 and mouse ROR1 (mROR1); (c) binds to deglycosylated ROR1; (d) is humanized; (e) has been deimmunized; (f) is conjugated to a detectable label, dye, toxin, drug, prodrug, radionuclide or biologically active molecule; (g) selectively interacts with the ROR1 protein with an affinity constant of approximately 0.01-50 nM, preferably 0.1-30 nM, and even more preferably 0.1-10 nM; and / or (h) the ROR1-specific antigen-binding molecule, which is capable of mediating killing of ROR1-expressing tumor cells, or inhibiting cancer cell proliferation, or is capable of being endocytosed upon binding to ROR1.

5. A recombinant fusion protein comprising the ROR1-specific antigen-binding molecule of any one of claims 1 to 4.

6. Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (In the formula, FW1 is a framework region, CDR1 is a CDR sequence, FW2 is a framework region, HV2 is a hypervariable sequence, FW3a is a framework region, HV4 is a hypervariable sequence, FW3b is a framework region, CDR3 is a CDR sequence, FW4 is the framework region. or a functional variant thereof, wherein the antigen-binding molecule is fused to a fragment of an immunoglobulin Fc region, and the fragment of the immunoglobulin Fc region is modified to dimerize with a second fragment of an immunoglobulin Fc region, Optionally, (a) the fragment of an immunoglobulin Fc region is selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region, and further optionally the fragment of an immunoglobulin Fc region is an Fc heavy chain; or (b) the fragment of an immunoglobulin Fc region has been modified 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, the ZW1 approach, the Biclonic approach, EW-RVT, and Triomab; and / or (c) one or more residues of said fragment of said immunoglobulin Fc region comprise one or more amino acid substitutions suitable for knobs-in-holes (KIH) dimerization with a second fragment of an immunoglobulin Fc region comprising one or more corresponding amino acid mutations, and further optionally (i) the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V; and / or (ii) the recombinant fusion protein, wherein said one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

7. 6. The recombinant fusion protein of claim 5, comprising a sequence as set forth in SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:167, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:203, SEQ ID NO:204, or SEQ ID NO:

205.

8. (a) a first recombinant fusion protein, the first recombinant fusion protein being a recombinant fusion protein according to any one of claims 5 to 7; and (b) a second recombinant fusion protein comprising a second antigen-binding molecule fused to a second fragment of an immunoglobulin Fc region that is modified to dimerize with the first fragment of the immunoglobulin Fc region. A recombinant fusion protein dimer comprising:

9. 9. The recombinant fusion protein of claim 5 or the recombinant fusion protein dimer of claim 8, wherein the specific antigen-binding molecule is fused to one or more biologically active proteins, Optionally, (a) the specific antigen binding molecule is fused to one or more biologically active proteins via one or more linker domains, optionally at least one biologically active protein being an immunoglobin, 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', an Fv, an Fv-Fc, a single chain Fv (scFv), a scFv-Fc, an (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; (b) the Fc heavy chain has been modified to contain one or more cysteine ​​residues suitable for bioconjugation; (c) the fragment of an immunoglobulin Fc region is modified to dimerize with a second fragment of an immunoglobulin Fc region; (d) one or more residues of said fragment of said immunoglobulin Fc region comprise one or more amino acid substitutions suitable for heterodimerization with a second fragment of an immunoglobulin Fc region comprising one or more corresponding amino acid mutations, optionally wherein said one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V; and / or (e) the recombinant fusion protein or recombinant fusion protein dimer, wherein the fragment of an immunoglobulin Fc region has been 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, the ZW1 approach, the Biclonic approach, EW-RVT, and Triomab.

10. A ROR1-specific chimeric antigen receptor (CAR) comprising at least one ROR1-specific antigen binding molecule according to any one of claims 1 to 4, fused or conjugated to at least one transmembrane region and at least one intracellular domain.

11. A cell comprising the chimeric antigen receptor of claim 10, preferably a modified T cell.

12. A pharmaceutical composition comprising the specific antigen-binding molecule of any one of claims 1 to 4, the recombinant fusion protein of any one of claims 5 to 7, the recombinant fusion protein dimer of claim 8, or the chimeric antigen receptor of claim 10.

13. A specific antigen-binding molecule according to any one of claims 1 to 4, a recombinant fusion protein according to any one of claims 5 to 7, a recombinant fusion protein dimer according to claim 8, a chimeric antigen receptor according to claim 10 or a pharmaceutical composition according to claim 12 for use in the treatment of cancer, Optionally, the cancer is a ROR1 positive cancer; Optionally, the cancer is selected from the group consisting of blood cancers, such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), and solid tumors, including neuroblastoma, kidney 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, esophageal cancer, gastric cancer, or liver cancer; The specific antigen-binding molecule, the recombinant fusion protein, the recombinant fusion protein dimer, the chimeric antigen receptor, or the pharmaceutical composition.

14. Formula (II): X-FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4-Y (II) (In the formula, FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 is a ROR1-specific antigen-binding molecule according to any one of claims 1 to 4, X and Y are optional amino acid sequences.

5. The specific antigen-binding molecule according to any one of claims 1 to 4, comprising an amino acid sequence represented by: Optionally, (a) X or Y, individually, are either absent or selected from the group comprising an immunoglobulin, an immunoglobulin Fc region, an immunoglobulin Fab region, Fab', Fv, Fv-Fc, single chain Fv (scFv), scFv-Fc, (scFv)2, diabody, triabody, tetrabody, bispecific t cell engager, intein, VNAR domain, single domain antibody (sdAb), VH domain or scaffold protein; (b) the conjugation is via a cysteine ​​residue in the amino acid sequence of the specific antigen-binding molecule; and / or (c)(i) the second portion is selected from the group comprising an immunoglobulin or antibody, an immunoglobulin Fc region, an immunoglobulin Fab region, a Fab', an Fv, an Fv-Fc, a single chain Fv (scFv), a scFv-Fc, an (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; or (ii) the second moiety is selected from the group comprising a detectable label, a dye, a toxin, a drug, a prodrug, a radionuclide or a biologically active molecule; Optionally, The second portion is Maytansinoids, Auristatins, anthracyclines, preferably PNU-derived anthracyclines - amanitin derivatives, preferably α-amanitin derivatives, calicheamicin, - Tubulyshyn, Duocarmycin, radioisotopes, such as alpha-emitting radionuclides, e.g. 227Th or 225Ac labels; Liposomes containing a toxic payload, - Protein toxins, Taxanes, pyrrole benzodiazepines, and / or indolinobenzodiazepine pseudodimers, and / or - spliceosome inhibitors, CDK11 inhibitors, -pyridinobenzodiazepines, Irinotecan and its derivatives The specific antigen-binding molecule is at least one toxin selected from the group comprising:

15. (a) a ROR1-specific antigen-binding molecule according to any one of claims 1 to 4, or a recombinant fusion protein according to any one of claims 5 to 7, or a recombinant fusion protein dimer according to claim 8, and (b) Anthracycline (PNU) derivatives A target binding molecule-drug conjugate comprising the formula (III): 【Chemical 38】 wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are each independently selected from valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptide, -(CH 2 ) n-, -(CH 2 CH 2 O) an optional linker selected from the group consisting of 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; and Y comprises a ROR1-specific antigen-binding molecule according to any one of claims 1 to 4, or a recombinant fusion protein according to any one of claims 5 to 7, or a recombinant fusion protein dimer according to claim 8. A target binding molecule-drug conjugate having the structure: Optionally, (a) the target binding molecule-drug conjugate of formula (III) comprises [L1], [L2] or [L1] and [L2]; Further optionally, [L2] is p-aminobenzyloxycarbonyl (PAB) or alanine, or (b) 【Chemical 39】 A target binding molecule-drug conjugate having a structure selected from:

16. (a) a ROR1-specific antigen-binding molecule according to any one of claims 1 to 4, or a recombinant fusion protein according to any one of claims 5 to 7, or a recombinant fusion protein dimer according to claim 8, and (b) Anthracycline (PNU) derivatives A target binding molecule-drug conjugate comprising the formula (IV): 【Chemistry 40】 wherein [X] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, 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 ROR1-specific antigen-binding molecule according to any one of claims 1 to 4, or a recombinant fusion protein according to any one of claims 5 to 7, or a recombinant fusion protein dimer according to claim 8. A target binding molecule-drug conjugate having the structure: Optionally, [Z] is a target-binding molecule-drug conjugate selected from the group consisting of a disulfide bond, an amide bond, an oxime bond, a hydrazone bond, a thioether bond, a 1,2,3 triazole and a polyGly.

17. The target-binding molecule-drug conjugate of claim 15 or 16, (a) [X] is polyethylene glycol, 【Chemistry 41】 is selected from the group comprising: 【Chemistry 42】 represents a point of attachment to the remainder of the molecule, [R] is an optional spacer selected from the group comprising a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, one or more heteroatoms, polyethylene glycol, or a combination thereof; (b) [X] is polyethylene glycol, (c) the target binding molecule is a protein and the anthracycline (PNU) derivative is conjugated to a thiol-containing amino acid residue in the amino acid sequence of the protein, and / or the PNU derivative is conjugated via a thiol moiety incorporated by chemical modification at the N-terminus or C-terminus of the amino acid sequence of the protein, and / or (d) The target binding molecule-drug conjugate comprising the ROR1-specific antigen-binding molecule of any one of claims 1 to 7, wherein Y is conjugated to the PNU derivative via a human immunoglobulin Fc region or a fragment thereof.

18. The specific antigen-binding molecule of claim 17, (a) comprises SEQ ID NO: 178 and / or SEQ ID NO: 179; (b) X or Y, individually, are 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, Fab', Fv, Fv-Fc, 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; (c) the fragment of an immunoglobulin Fc region is an Fc heavy chain that has been modified to contain one or more cysteine ​​residues suitable for bioconjugation; (d) X or Y, individually, are either absent or are a fragment of an immunoglobulin Fc region that has been engineered to dimerize with a second fragment of an immunoglobulin Fc region, and optionally the method is 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, the ZW1 approach, the Biclonic approach, EW-RVT, and Triomab; and / or (e) X or Y, individually, are either absent or a fragment of an immunoglobulin Fc region comprising one or more amino acid substitutions suitable for heterodimerization with a second fragment of an immunoglobulin Fc region comprising one or more corresponding amino acid mutations, optionally wherein the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V; The specific antigen-binding molecule.

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