Antigen binding proteins targeting ror1

Abstract

The present application relates to an isolated antigen binding protein capable of binding to ROR1 (Receptor Tyrosine Kinase Orphan Receptor 1), said isolated antigen protein comprising CDR1, CDR2 and CDR3. The present application also provides a method for preparing said isolated antigen binding protein and uses thereof.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an antigen-binding protein that targets ROR1. Background Technology

[0002] ROR1 is a transmembrane receptor tyrosine kinase protein. Human ROR1 consists of an extracellular immunoglobulin-like domain (Ig), two cysteine-rich domains (FZD), a juxtamembrane kringle domain, a single transmembrane structure, an intracellular tyrosine kinase domain (TKD), two serine / threonine-rich domains (S / TRD), and a proline-rich domain (PRD). It regulates cell division, proliferation, migration, and chemotaxis by mediating signal transduction in multiple signaling pathways. It is highly expressed during early embryonic development. ROR1 expression gradually decreases as the fetus develops.

[0003] ROR1 is expressed at low levels or not at all in normal human tissues, but it is highly expressed in various hematologic malignancies and solid tumors. Hematologic malignancies with high ROR1 expression include B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid hematologic cancers. In solid tumors, ROR1-expressing tumors include triple-negative breast cancer, colon cancer, lung cancer, pancreatic cancer, and ovarian cancer, and ROR1 expression is closely related to disease progression and treatment efficacy. Therefore, ROR1 can serve as a specific tumor marker and a potentially attractive target for cancer therapy. Summary of the Invention

[0004] This application provides an isolated antigen-binding protein targeting ROR1. In this application, the isolated antigen-binding protein specifically binds to the ROR1 antigen and exhibits good binding activity. The isolated antigen-binding protein described in this application can bind to ROR1 expressed on the surface of cells (e.g., A549 cells, MDA MB231 cells, MEC-ROR1 cells, JeKo-1 cells). The isolated antigen-binding protein described in this application can have a tumor-killing effect.

[0005] On one hand, this application provides an isolated antigen-binding protein capable of binding to ROR1 (orphan receptor tyrosine kinase 1), the isolated antigen protein comprising CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 being shown in SEQ ID NO:1, the amino acid sequence of CDR2 being shown in SEQ ID NO:2, and the amino acid sequence of CDR3 being shown in SEQ ID NO:3.

[0006] In some embodiments, the isolated antigen-binding protein is a single-domain antibody.

[0007] In some embodiments, the isolated antigen-binding protein comprises VHH.

[0008] In some embodiments, the amino acid sequence of the VHH is as shown in SEQ ID NO:8.

[0009] In some embodiments, the isolated antigen-binding protein includes an antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0011] In some embodiments, the isolated antigen-binding protein contains at least one VHH fragment.

[0012] In some embodiments, the isolated antigen-binding protein comprises two VHH fragments.

[0013] In some embodiments, the isolated antigen-binding protein further includes an Fc region.

[0014] In some implementations, the Fc region is derived from the IgG Fc region.

[0015] In some implementations, the Fc region is derived from the human IgG Fc region.

[0016] In some embodiments, the isolated antigen-binding protein has one or more of the following properties:

[0017] (1) It can specifically bind to ROR1; and

[0018] (2) It can bind to ROR1 expressed on the cell surface.

[0019] On the other hand, this application also provides a drug molecule comprising the isolated antigen-binding protein.

[0020] On the other hand, this application also provides a nucleic acid molecule that encodes the isolated antigen-binding protein.

[0021] On the other hand, this application also provides a vector containing the nucleic acid molecule.

[0022] On the other hand, this application also provides cells containing the nucleic acid molecules and / or the vector.

[0023] On the other hand, this application also provides pharmaceutical compositions comprising the isolated antigen-binding protein, the pharmaceutical molecule, the nucleic acid molecule, the carrier, and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0024] On the other hand, this application also provides a kit containing the isolated antigen-binding protein, the kit being capable of detecting the presence and / or content of ROR1 in a sample.

[0025] On the other hand, this application also provides the use of the isolated antigen-binding protein, the drug molecule, the nucleic acid molecule, the carrier, the cell, and / or the drug composition in the preparation of a drug for the prevention and / or treatment of ROR1-related diseases and / or conditions.

[0026] In some implementations, the disease and / or condition includes tumors.

[0027] In some implementations, the tumor includes solid tumors and / or hematomas.

[0028] In some embodiments, the tumor includes a ROR1-positive tumor.

[0029] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0030] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0031] Figure 1 The figure shows the ROR1-specific antibody titers in alpaca serum collected from days 1 to 5 post-immunization as described in this application, before and after immunization.

[0032] Figure 2 The displayed value is the size of the sequence amplified from the cDNA library as described in this application. Figure 2 A shows that the first PCR produced two different sets of amplicones, with molecular weights of approximately 0.7 kb and 0.9 kb. Figure 2 B shows that using a 0.7kb amplicon as a template for amplification yields a DNA fragment of approximately 400bp in size.

[0033] Figure 3 The plate shown is the plate used in this application to calculate the number of colonies by serial dilution and then estimate the size of the library.

[0034] Figure 4 The diagram shown is a phylogenetic analysis of 90 sequences in the library as described in this application.

[0035] Figure 5 The plate shown is the plate used for three rounds of continuous screening of ROR1 specific antigen-binding proteins as described in this application.

[0036] Figure 6 shows the ELISA results described in this application. Figure 6A Displays the OD450 values ​​of the filtered sequences. Figure 6B Display the OD450 values ​​of VHH1, VHH2, and anti-ROR1 mAb.

[0037] Figure 7 The image shows the SDS-PAGE results of the purified antigen-binding protein described in this application.

[0038] Figure 8 shows the results of the purified antigen-binding protein specifically binding to recombinant ROR1 protein as described in this application. Figure 8A Displays antigen-binding proteins after screening. Figure 8B The combined effect of negative and positive controls is shown.

[0039] Figure 9 The results shown are the specificity verification results of the antigen-binding protein described in this application.

[0040] Figure 10 The results shown are flow cytometry results of A549 cells expressing ROR1, which are bound to the antigen-binding protein described in this application.

[0041] Figure 11 The results shown are flow cytometry results of MB231 cells expressing ROR1 bound to the antigen-binding protein described in this application.

[0042] Figure 12 The results shown are flow cytometry results of JeKo-1 cells expressing ROR1, which are bound to the antigen-binding protein described in this application. Detailed Implementation

[0043] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0044] Terminology Definition

[0045] In this application, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion, and optionally, a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. Antigen-binding proteins may include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, provided they exhibit the desired antigen-binding activity. The "isolated antigen-binding protein" of this application may comprise an antigen-binding portion and optionally, allow the antigen-binding portion to adopt a scaffold or backbone portion that promotes the binding of the antigen-binding portion to the antigen.

[0046] In this application, the term "single-domain antibody" generally refers to a fragment containing a single variable domain of an antibody that can selectively bind to a specific antigen. The single-domain antibody described in this application may include a heavy chain variable domain, and the single-domain antibody may include an antigen-binding fragment, which may include a VHH.

[0047] In this application, the term "peptide" generally refers to a polymer of amino acid residues. This term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term may also include amino acid polymers modified, for example, by adding sugar residues to form glycoproteins or by phosphorylation. Peptides can be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids with a natural sequence that have been deleted, added, and / or substituted. The term "peptide" can include antigen-binding fragments, antibodies, or sequences having one or more amino acids with a deletion, addition, and / or substitution.

[0048] In this application, the term "nucleic acid" generally refers to a polymer of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and may include naturally occurring (adenine, guanine, cytosine, uracil, and thymine), non-natural, and modified nucleic acids. The term "nucleic acid" may include genes, cDNA, or mRNA. For example, nucleic acid molecules may be synthetic (e.g., chemically synthesized) or recombinant. Nucleic acids may include nucleic acids containing analogs or derivatives of natural nucleotides, having similar binding properties to a reference nucleic acid and being metabolized in a manner similar to naturally occurring nucleotides. Nucleic acid sequences may also include variants of their conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly indicated sequences. The term is not limited by the length of the polymer. Nucleic acids may be single-stranded or double-stranded and generally contain 5'-3' phosphodiester bonds; nucleotide analogs may have other linkages.

[0049] In this application, the term "constant region" generally refers to the sum of the antibody's structural domains, excluding the variable region. Constant regions are not directly involved in antigen binding but exhibit different effector functions. Based on the amino acid sequence of their heavy chain constant regions, antibodies are classified into the following categories: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into categories such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different antibody classes are referred to as α, δ, ε, γ, and μ, respectively.

[0050] In this application, the term "antigen-binding fragment" generally refers to a polypeptide fragment of an immunoglobulin or antibody that competes with an intact antibody (i.e., an intact antibody derived from an antigen) for binding to or specifically binding to an antigen. The antigen-binding fragment may include, but is not limited to: Fab, Fab', F(ab')2, and Fv fragments, VHH, linear antibodies, single-chain antibodies, dimeric antibodies, and multispecific antibodies formed from antibody fragments.

[0051] In this application, the term "variable region" or "variable domain" generally refers to a portion of the antibody light chain and / or heavy chain, typically comprising approximately 120-130 amino acids at the amino terminus of the heavy chain and approximately 100-110 amino acids at the amino terminus of the light chain. Variable regions generally vary widely in their amino acid sequences, even within antibodies of the same species. The variable regions of an antibody can determine the binding and specificity of each specific antibody to a particular antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more conserved regions within the variable regions are called framework regions (FRs). The CDRs of both the light and heavy chains may contain amino acids that are largely responsible for the direct interaction between the antibody and the antigen.

[0052] In this application, the term "drug molecule" can generally encompass any form of drug known in the art. For example, the drug molecule can be a protein. For example, the drug molecule can be a polypeptide. For example, the drug molecule can be a combination of different forms of drugs. For example, the drug molecule may include small molecule drugs.

[0053] In this application, the term "cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector including the nucleic acid molecules described in this application, or that is capable of expressing the fusion protein or its antigen-binding fragment described in this application. The cell may include progeny cells from a single cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they must be capable of expressing the antigen-binding protein described in this application. The cell can be obtained by in vitro transfection of cells using the vector described in this application. The cell can be a prokaryotic cell (e.g., *Escherichia coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells).

[0054] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into cells (e.g., host cells) and / or between cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell. Typically, by culturing suitable cells containing the vector, the vector can produce a desired expression product. In this application, the vector may be a plasmid.

[0055] In this application, the term "pharmaceutically acceptable" generally refers to a non-toxic material that does not interfere with the effectiveness of the bioactivity of the active ingredient. Such formulations may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, supplemental immunostimulants such as adjuvants and cytokines, and optionally other therapeutic agents such as chemotherapeutic agents.

[0056] In this application, the term "complementarity-determining region" or "CDR" generally refers to a complementarity-determining region within a variable sequence of an antibody. Three CDRs exist in each variable region of the heavy chain and / or light chain, and these CDRs are named CDR1, CDR2, and CDR3 for each variable region. As used herein, a CDR combination may refer to a group of three CDRs appearing in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only a definitive residue numbering system applicable to any variable region of an antibody but also precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) discovered that certain subregions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite significant diversity at the amino acid sequence level. These subregions were named L1, L2, and L3 or H1, H2, and H3, where “L” and “H” refer to the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5): 732-45 (1996)). Other CDR boundary definitions may not strictly follow one of the systems described above, but will still overlap with Kabat CDRs, although they may be shortened or lengthened according to specific residues or groups of residues or even the entire CDR without significantly affecting the prediction or experimental findings of antigen binding. The CDRs described in this article can be defined using the IMGT partitioning method.

[0057] In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, and may be a human patient. For example, a pharmaceutical composition described in this application may comprise the antigen-binding protein described in this application, the immunoconjugate described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are non-toxic to the recipient at the doses and concentrations used. The pharmaceutical compositions of the present invention may include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0058] In this application, the term "ROR1" generally refers to receptor tyrosine kinase orphan receptor 1. In this application, the term encompasses the full length of ROR1 and its functionally active fragments or variants thereof. For example, the term may include the Ig-like domain and / or coiled domain of ROR1. For example, the term may include amino acids 30-305 of ROR1. For example, the sequence of the full-length human ROR1 antigen may be described under GeneBank accession number Q01973.

[0059] The proteins described in this application may also include their functional variants, derivatives, analogs, homologs, and fragments.

[0060] The term "functional variant" refers to a polypeptide that has a substantially identical amino acid sequence or is encoded by a substantially identical nucleotide sequence to the naturally occurring sequence and is capable of possessing one or more activities of the naturally occurring sequence. In the context of this application, a variant of any given sequence refers to a sequence in which a specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function. Variant sequences can be obtained by adding, deleting, substituting, modifying, replacing, and / or mutating at least one amino acid residue and / or nucleotide residue present in naturally occurring proteins and / or polynucleotides, as long as the original functional activity is maintained.

[0061] In this application, the term "derivative" generally refers to any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues of the self / pair sequence for the polypeptide or polynucleotide of this application, provided that the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

[0062] In this application, the term "analyte" generally refers to a polypeptide or polynucleotide, including any mimic of a polypeptide or polynucleotide, i.e., a chemical compound having at least one endogenous function of the polypeptide or polynucleotide that the mimic mimics.

[0063] Typically, amino acid substitutions can be made, such as substitutions of at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20), as long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0064] The proteins or peptides used in this application may also have deletions, insertions, or substitutions of amino acid residues, which produce silent changes and result in functionally equivalent proteins. Intentional amino acid substitutions can be made based on the similarity of the residues' polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphoteric properties, as long as the endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids containing non-polarized head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine. Invention Details

[0066] Isolated antigen-binding proteins

[0067] On one hand, this application provides an isolated antigen-binding protein that may contain at least one CDR in the variable region VH of the antibody heavy chain, wherein the CDR may be HCDR1, HCDR2, and / or HCDR3. The heavy chain variable region VH may contain the amino acid sequence shown in SEQ ID NO:8. In this application, the HCDR sequence of the isolated antigen-binding protein may contain an HCDR sequence obtained by any method of partitioning, as long as the VH sequence is identical to the amino acid sequence shown in SEQ ID NO:8. Any HCDR sequence obtained by any method of partitioning is within the scope of protection of this application. In some specific embodiments, IMGT can be used to partition the CDR.

[0068] In this application, the isolated antigen-binding protein may contain HCDR3, which may contain the amino acid sequence shown in SEQ ID NO:3.

[0069] In this application, the isolated antigen-binding protein may include HCDR2, which may contain the amino acid sequence shown in SEQ ID NO:2.

[0070] In this application, the isolated antigen-binding protein may include HCDR1, which may contain the amino acid sequence shown in SEQ ID NO:1.

[0071] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2 and HCDR3, wherein HCDR1 may contain the amino acid sequence shown in SEQ ID NO:1, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:2 and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:3.

[0072] In this application, the VH of the isolated antigen-binding protein may include frame regions H-FR1, H-FR2, H-FR3 and / or H-FR4.

[0073] In this application, the isolated antigen-binding protein may include H-FR1, the C-terminus of which may be directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 may contain the amino acid sequence shown in SEQ ID NO:4.

[0074] In this application, the isolated antigen-binding protein may include H-FR2, which may be located between HCDR1 and HCDR2, and H-FR2 may contain the amino acid sequence shown in SEQ ID NO:5.

[0075] In this application, the isolated antigen-binding protein may contain H-FR3, which may be located between HCDR2 and HCDR3, and the H-FR3 may contain the amino acid sequence shown in SEQ ID NO:6.

[0076] In this application, the isolated antigen-binding protein may include H-FR4, the N-terminus of which may be directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 may contain the amino acid sequence shown in SEQ ID NO:7.

[0077] In this application, the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.

[0078] In this application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and / or H-FR4, wherein the C-terminus of H-FR1 is directly or indirectly connected to the N-terminus of HCDR1, H-FR2 is located between HCDR1 and HCDR2, H-FR3 is located between HCDR2 and HCDR3, and the N-terminus of H-FR4 is directly or indirectly connected to the C-terminus of HCDR3. In this application, the direct or indirect connection can be achieved through intermolecular forces or through linkers.

[0079] In this application, the isolated antigen-binding protein may contain a heavy chain variable region VH, which may contain the amino acid sequence shown in SEQ ID NO:8.

[0080] In this application, the isolated antigen-binding protein may include a heavy chain constant region, and the heavy chain constant region may include an Fc fragment. The Fc region may be derived from an IgG Fc region, for example, the human IgG Fc region; for example, the IgG1 Fc region; for example, the IgG4 Fc region. For example, the Fc fragment may include variants thereof, which may include substitutions, deletions, and / or additions of one or more amino acids to the amino acid sequence of the Fc fragment. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; or it may cover its homologs, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the Fc fragment.

[0081] In this application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof.

[0082] In some embodiments, the isolated antigen-binding protein, wherein the antigen-binding fragment may be selected from the group consisting of: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0083] In this application, the isolated antigen-binding protein, wherein the antibody may be selected from the group consisting of monoclonal antibodies, single-chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0084] Single-domain antibodies

[0085] In this application, the isolated antigen-binding protein may be a single-domain antibody.

[0086] In this application, the single-domain antibody may contain at least one CDR in the antibody heavy chain variable region VHH, wherein the CDR may be CDR3, CDR2, and / or CDR1. The heavy chain variable region VHH may contain the amino acid sequence shown in SEQ ID NO:8.

[0087] In this application, the single-domain antibody may contain CDR3, and the CDR3 may contain the amino acid sequence shown in SEQ ID NO:3.

[0088] In this application, the single-domain antibody may contain CDR2, and the CDR2 may contain the amino acid sequence shown in SEQ ID NO:2.

[0089] In this application, the single-domain antibody may contain CDR1, and the CDR1 may contain the amino acid sequence shown in SEQ ID NO:1.

[0090] In this application, the single-domain antibody may comprise CDR1, CDR2, and CDR3, wherein CDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, CDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0091] In this application, the single-domain antibody may contain FR1, the C-terminus of which may be directly or indirectly linked to the N-terminus of CDR1, and the FR1 may contain the amino acid sequence shown in SEQ ID NO:4.

[0092] In this application, the single-domain antibody may contain FR2, which may be located between CDR1 and CDR2, and FR2 may contain the amino acid sequence shown in SEQ ID NO:5.

[0093] In this application, the single-domain antibody may contain FR3, which may be located between CDR2 and CDR3, and the FR3 may contain the amino acid sequence shown in SEQ ID NO:6.

[0094] In this application, the single-domain antibody may contain FR4, the N-terminus of which may be directly or indirectly linked to the C-terminus of CDR3, and the FR4 may contain the amino acid sequence shown in SEQ ID NO:7.

[0095] In this application, the single-domain antibody may contain FR1, FR2, FR3 and FR4, wherein FR1 may contain the amino acid sequence shown in SEQ ID NO:4, FR2 may contain the amino acid sequence shown in SEQ ID NO:5, FR3 may contain the amino acid sequence shown in SEQ ID NO:6 and FR4 may contain the amino acid sequence shown in SEQ ID NO:7.

[0096] In this application, the single-domain antibody may comprise CDR1, CDR2, CDR3, FR1, FR2, FR3, and / or FR4, wherein the C-terminus of FR1 is directly or indirectly connected to the N-terminus of CDR1, FR2 is located between CDR1 and CDR2, FR3 is located between CDR2 and CDR3, and the N-terminus of FR4 is directly or indirectly connected to the C-terminus of CDR3. In this application, the direct or indirect connection can be achieved through intermolecular forces or through a linker.

[0097] In this application, the single-domain antibody may contain a heavy chain variable region VHH, wherein the VHH may contain the amino acid sequence shown in SEQ ID NO:8.

[0098] In this application, the single-domain antibody may comprise a heavy chain constant region, and the heavy chain constant region may comprise an Fc fragment. For example, the Fc fragment may comprise variants thereof, which may comprise an amino acid sequence of the Fc fragment with substitutions, deletions, and / or additions of one or more amino acids. For example, 1-30, 1-20, or 1-10, or for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; or it may encompass homologs thereof, which may be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology to the amino acid sequence of the Fc fragment.

[0099] Polypeptides, drug molecules, nucleic acids, carriers, cells, preparation methods

[0100] On the other hand, this application provides a polypeptide that may contain the isolated antigen-binding protein.

[0101] On the other hand, this application provides a drug molecule that may contain the isolated antigen-binding protein.

[0102] On the other hand, this application provides one or more isolated nucleic acid molecules that can encode the isolated antigen-binding protein or the polypeptide described in this application. For example, each of the one or more nucleic acid molecules can encode the complete isolated antigen-binding protein or the polypeptide, or it can encode a portion thereof. The nucleic acid molecules described in this application can be isolated. In this application, nucleic acids encoding the isolated antigen-binding protein or the polypeptide can be prepared using various methods known in the art.

[0103] On the other hand, this application provides one or more vectors containing one or more nucleic acid molecules described in this application. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in suitable host cells and under suitable conditions. Additionally, the vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, the vector may also contain a replication initiation site. Moreover, the vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering.

[0104] On the other hand, this application provides a cell that may contain one or more nucleic acid molecules and / or one or more vectors as described in this application. In some embodiments, each type or each cell may contain one or more nucleic acid molecules or vectors as described in this application. In some embodiments, each type or each cell may contain multiple (e.g., two or more) or more types (e.g., two or more) nucleic acid molecules or vectors as described in this application. For example, the vectors as described in this application may be introduced into the cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS / O cells, HEK293 cells, or other eukaryotic cells, such as cells from plants, fungi, or yeast cells. The vectors as described in this application may be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0105] On the other hand, this application provides a method for preparing the isolated antigen-binding protein or the polypeptide described in this application. The method may include culturing the cells described in this application under conditions that cause the isolated antigen-binding protein or the polypeptide to be expressed. For example, this can be achieved by using appropriate culture media, appropriate temperatures, and culture times, methods known to those skilled in the art.

[0106] Pharmaceutical composition, application

[0107] On the other hand, this application provides a pharmaceutical composition comprising the isolated antigen-binding protein, the polypeptide, the pharmaceutical molecule, the nucleic acid molecule, the carrier, the cell, and / or a pharmaceutically acceptable carrier.

[0108] For example, the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight peptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants.

[0109] In this application, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous storage depot. The pharmaceutical composition can be used to inhibit tumor growth. For example, the pharmaceutical composition of this application can inhibit or delay the development or progression of a disease, and / or alleviate and / or stabilize a disease state.

[0110] The pharmaceutical composition described in this application may contain a preventive and / or therapeutically effective amount of the isolated antigen-binding protein. The preventive and / or therapeutically effective amount is a dose required to prevent and / or treat (at least partially treat) a disease or condition and / or any complications thereof in a subject who has or is at risk of developing it.

[0111] On the other hand, this application provides a method for detecting or determining ROR1, the method including the use of the isolated antigen-binding protein or the polypeptide.

[0112] On the other hand, this application provides a kit for detecting or determining ROR1 in a sample, the kit including the isolated antigen-binding protein or the polypeptide.

[0113] On the other hand, this application provides the use of the isolated antigen-binding protein, the polypeptide, the drug molecule, the isolated nucleic acid molecule, the carrier, the cell, and / or the drug composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0114] On the other hand, this application provides the isolated antigen-binding protein, the polypeptide, the drug molecule, the isolated nucleic acid molecule, the carrier, the cell, and / or the drug composition thereof, for the preparation of prevention or treatment of diseases or conditions.

[0115] On the other hand, this application provides a method for preventing or treating a disease, comprising administering to a subject in need the isolated antigen-binding protein, the polypeptide, the drug molecule, the isolated nucleic acid molecule, the carrier, the cell, and / or the drug composition described in this application.

[0116] In this application, the diseases and / or conditions described in the intended use may be caused or mediated by abnormal expression of ROR1.

[0117] In this application, the disease and / or condition may include a tumor. For example, the tumor may include a solid tumor and / or a hematoma. For example, the tumor may be a ROR1-positive tumor.

[0118] This application also provides the following implementation schemes:

[0119] 1. An isolated antigen-binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said variable region comprising the amino acid sequence shown in SEQ ID NO:8.

[0120] 2. The antigen-binding protein isolated according to embodiment 1, comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, and HCDR2 comprises the amino acid sequence shown in SEQ ID NO:1.

[0121] The amino acid sequence shown in SEQ ID NO:3, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0122] 3. The isolated antigen-binding protein according to any one of embodiments 1-2, comprising H-FR1, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and wherein H-FR1 comprises SEQ ID NO:

[0123] The amino acid sequence shown in Figure 4.

[0124] 4. The isolated antigen-binding protein according to any one of embodiments 1-3, comprising H-FR2, wherein H-FR2 is located between HCDR1 and HCDR2, and wherein H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5.

[0125] 5. The isolated antigen-binding protein according to any one of embodiments 1-4, comprising H-FR3, said H-FR3 being located between said HCDR2 and said HCDR3, and said H-FR3 comprising the amino acid sequence shown in SEQ ID NO:6.

[0126] 6. The isolated antigen-binding protein according to any one of embodiments 1-5, comprising H-FR4, wherein the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and wherein H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0127] 7. The isolated antigen-binding protein according to any one of embodiments 1-6, comprising a heavy chain variable region VH, said VH comprising the amino acid sequence shown in SEQ ID NO:8.

[0128] 8. The isolated antigen-binding protein according to any one of embodiments 1-7 further comprises an antibody heavy chain constant region.

[0129] 9. The isolated antigen-binding protein according to embodiment 8, wherein the constant region of the antibody heavy chain comprises an Fc fragment.

[0130] 10. The isolated antigen-binding protein according to embodiment 9, comprising an antibody or an antigen-binding fragment thereof.

[0131] 11. The isolated antigen-binding protein according to embodiment 10, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0132] 12. The isolated antigen-binding protein according to any one of embodiments 10-11, wherein the antibody is selected from the group consisting of monoclonal antibodies, single-chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0133] 13. The isolated antigen-binding protein according to any one of embodiments 1-12 is a single-domain antibody.

[0134] 14. The isolated antigen-binding protein according to embodiment 13, wherein the single-domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:1, CDR2 comprises the amino acid sequence shown in SEQ ID NO:2 and CDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0135] 15. The isolated antigen-binding protein according to any one of embodiments 1-14, comprising VHH, wherein the VHH comprises the amino acid sequence shown in SEQ ID NO:8.

[0136] 16. The isolated antigen-binding protein according to any one of embodiments 1-15, having one or more of the following properties:

[0137] (1) It can specifically bind to ROR1; and

[0138] (2) It can bind to ROR1 expressed on the cell surface.

[0139] 17. The isolated antigen-binding protein according to embodiment 16, wherein the ROR1 comprises an Ig-like domain and a coiled domain of ROR1.

[0140] 18. The isolated antigen-binding protein according to any one of embodiments 16-17, wherein the ROR1 comprises the amino acid sequence at positions Q30-D305 of ROR1.

[0141] 19. A polypeptide comprising the isolated antigen-binding protein of any one of embodiments 1-18.

[0142] 20. A drug molecule comprising the isolated antigen-binding protein of any one of embodiments 1-18.

[0143] 21. A nucleic acid molecule encoding an isolated antigen-binding protein as described in any one of embodiments 1-18, or a polypeptide as described in embodiment 19.

[0144] 22. A vector comprising the nucleic acid molecule described in embodiment 21.

[0145] 23. A cell comprising the isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the drug molecule of embodiment 20, the nucleic acid molecule of embodiment 21, and / or the carrier of embodiment 22.

[0146] 24. A method for preparing the isolated antigen-binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19, the method comprising culturing the cells of embodiment 23 under conditions that cause expression of the isolated antigen-binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19.

[0147] 25. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the pharmaceutical molecule of embodiment 20, the nucleic acid molecule of embodiment 21, the carrier of embodiment 22, the cell of embodiment 23, and / or a pharmaceutically acceptable carrier.

[0148] 26. A method for detecting the presence and / or quantity of ROR1, the method comprising using the isolated antigen-binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19.

[0149] 27. A kit comprising the isolated antigen-binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19, the kit being used to detect or determine the presence and / or content of ROR1 in a sample.

[0150] 28. Use of the isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the drug molecule of embodiment 20, the nucleic acid molecule of embodiment 21, the carrier of embodiment 22, the cell of embodiment 23, and the drug composition of embodiment 25 in the preparation of a drug for the prevention and / or treatment of diseases and / or conditions related to ROR1 expression.

[0151] 29. The use according to embodiment 28, wherein the disease and / or condition includes tumors.

[0152] 30. The use according to embodiment 29, wherein the tumor includes solid tumors and / or hematomas.

[0153] 31. The use according to any one of embodiments 29-30, wherein the tumor includes a ROR1-positive tumor.

[0154] 32. A method for preventing and / or treating ROR1 expression-related diseases and / or conditions, comprising administering to a subject in need the isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the pharmaceutical molecule of embodiment 20, the nucleic acid molecule of embodiment 21, the carrier of embodiment 22, the cell of embodiment 23, and / or the pharmaceutical composition of embodiment 25.

[0155] 33. The method according to embodiment 32, wherein the disease and / or condition includes tumors.

[0156] 34. The method according to embodiment 33, wherein the tumor includes solid tumors and / or hematomas.

[0157] 35. The method according to any one of embodiments 33-34, wherein the tumor is a ROR1 positive tumor.

[0158] 36. The isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the drug molecule of embodiment 20, the nucleic acid molecule of embodiment 21, the carrier of embodiment 22, the cell of embodiment 23, and / or the drug composition of embodiment 25, for the prevention and / or treatment of diseases and / or conditions related to ROR1 expression.

[0159] 37. The isolated antigen-binding protein, the polypeptide, the drug molecule, the nucleic acid molecule, the carrier, the cell, and / or the drug composition according to embodiment 36, wherein the disease and / or condition includes tumors.

[0160] 38. The isolated antigen-binding protein according to embodiment 37, wherein the tumor is a solid tumor and / or a hematologic malignancy.

[0161] 39. The isolated antigen-binding protein according to any one of embodiments 37-38, wherein the tumor is a ROR1-positive tumor.

[0162] Without being limited by any theory, the embodiments described below are merely for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0163] Example

[0164] Example 1: Animal Immunization

[0165] The experiment was conducted according to the following steps:

[0166] (1) Five immunizations were performed on a 2.5-year-old unimmunized female alpaca every two weeks using freshly prepared immunogen (see Table 1. Immunization schedule). The immunogen was aliquoted into several portions (each portion was used for one immunization) and stored at -80°C.

[0167] (2) For the first immunization, 300 μg of immunogen was mixed with an equal volume of Freund's complete adjuvant (CFA) to form an emulsion. For subsequent immunizations (the second to the fifth), 300 μg of immunogen was mixed with an equal volume of Freund's incomplete adjuvant (CFA) to form an adjuvant (IFA) to form an emulsion (all vaccination experiments were approved by the local ethics committee).

[0168] (3) Before each administration of the immunogen (1st to 5th times), take 5 ml of blood from the jugular vein and recover the serum as a post-immunization IgG sample for antigen-specific antibody titer.

[0169] (4) Seven days after the last immunization, collect 50 ml of blood and administer Ficoll-Paque. TM PLUS Media (GE Healthcare, USA) isolates peripheral blood mononuclear cells (PBMCs) from whole blood, lyses the PBMCs in Trizol, and uses them for subsequent total RNA extraction.

[0170] (5) ELISA compares the titers of serum before and after immunization to measure antigen-induced seroconversion, with one experiment performed on each diluted serum sample at each bleeding time point.

[0171] Experimental results are as follows Figure 1 As shown, the immune serum showed a significant response to the recombinant ROR1 protein, and the ROR1-specific antibody titers in the serum from the 3rd to the 5th immunizations all reached more than 1 / 100,000.

[0172] Table 1. Immunization Schedule

[0173]

[0174] (*The third immunization was delayed by two weeks due to COVID-19 quarantine at the alpaca farm.)

[0175] Example 2: Establishment of an Immunophage Bank

[0176] The experiment was conducted according to the following steps:

[0177] (1) After isolating PBMCs and extracting total RNA from 50 ml of peripheral blood, cDNA was prepared using RNA as a template with commercially available reagents. The preparation system is shown in Table 2.

[0178] (2) After amplifying the VHH sequence from the cDNA library and transforming it into TGI cells, the library size was calculated based on the number of colonies in a series of dilutions of E. coli cell suspension electroporated on a 90 mm plate.

[0179] (3) Perform colony sequencing and bioinformatics analysis.

[0180] The experimental results showed that the first PCR produced two different sets of amplicones with molecular weights of approximately 0.7 kb and 0.9 kb. Figure 2 A) Using a 0.7kb amplicon as a template, VHH was amplified, resulting in a DNA fragment of approximately 400bp in size. Figure 2 B), the amplified library size is 1.4 × 10⁻⁶. 8 ( Figure 3 Phylogenetic analysis was performed on 90 sequences in the library, and 96 single colonies were randomly selected for sequencing. The proportion of VHH correctly inserted phages was 93.8% (90 / 96), and the proportion of specific VHH sequences was 96.7% (87 / 90). Figure 4 ).

[0181] Table 2. Yields of total RNA, cDNA, and PCR products

[0182]

[0183]

[0184] Example 3: Screening of antigen-specific antigen-binding proteins

[0185] By monitoring the number of colonies growing on the plates and screening three times consecutively, the specific antigen-binding protein of ROR1 was obtained. Figure 5 After each round of screening, the number of colonies in the ROR1-coated group increased, indicating that ROR1-specific antigen-binding proteins were enriched. R1, R2, and R3 represent the results of the first, second, and third rounds of screening, respectively, and NC represents the negative control.

[0186] Example 4: Recognition and Validation of Antigen-Binding Proteins

[0187] (1) The specific antigen-binding proteins after three rounds of screening were verified and identified by ELISA using periplasmic extracts to obtain antigen-positive clones.

[0188] (2) Then, the DNA sequence of the positive clones was obtained by colony sequencing, and the specific sequence was identified;

[0189] (3) The specific sequence is recombinantly produced and purified, and the purity is tested to determine whether it meets the requirements for subsequent detection;

[0190] (4) Further test the affinity and specificity of antigen-binding protein for ROR1 protein.

[0191] Experimental results showed that the OD450 value of 5-26 (VHH sequence as shown in SEQ ID NO:8) was higher than that of 5-09 (VHH sequence as shown in SEQ ID NO:11) and 5-95 (VHH sequence as shown in SEQ ID NO:12). Figure 6A Among them, 5-26 contain CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3. The two nonspecific VHHs (VHH1: sequence as shown in SEQ ID NO:9; VHH2: sequence as shown in SEQ ID NO:10) do not bind to recombinant ROR1 protein, while the anti-ROR1 mAb binds well to recombinant ROR1 protein. Figure 6B The results of this experiment further demonstrate that 5-26, 5-09, and 5-95 specifically bind to recombinant ROR1 protein, but the binding ability of 5-26 is significantly better than that of the other two groups.

[0192] The purified SDS-PAGE results showed that the purity of the antigen-binding proteins (approximately 0.5 mg of each) exceeded 90%, meeting the requirements for subsequent functional testing. Figure 7 The purified antigen-binding proteins 5-26, 5-9, and 5-95 specifically bind to recombinant ROR1 protein. Figure 8A The negative controls VHH1 and VHH2 do not bind to ROR1 protein, while the positive control binds to ROR1 protein. Figure 8BThe experimental results show that, compared with 5-9 and 5-95, 5-26 can bind to ROR1 protein at higher concentrations at multiple dilutions with good specificity.

[0193] Meanwhile, specificity evaluation showed that the purified antigen-binding proteins 5-26, 5-9, and 5-95 bound only to recombinant ROR1 protein and not to the other two his-tagged proteins (PD-L1 and RBD), indicating that the selected antigen-binding proteins do not recognize other tags. Anti-ROR1 mAb (positive control) bound only to recombinant ROR1 protein. Anti-his mAb (Sinobiological, #105327-MM02T-H) could bind to all three his-tagged proteins (PD-L1, RBD, and ROR1). Figure 9 The purified antigen-binding proteins used in the experiment were all at a concentration of 3 μg / ml. The C-terminus of each protein contained a his tag and an HA tag. The secondary antibody used for recognition was an HRP anti-HA tag antibody (Abcam, #ab1190). The experimental results indicate that 5-26 exhibits good specificity for ROR1 protein and does not bind significantly to PD-L1 and RBD.

[0194] anti-ROR 1mAb: ROR1 mouse monoclonal antibody, from Proteintech (catalog No.: 66923-1-Ig), molecular weight 130kDa, can be used in FC, IF, IHC, WB, ELISA and other applications.

[0195] Example 5: Antigen-binding protein binds to cell lines expressing ROR1

[0196] ROR1 was expressed in A549, MB231, and JeKo-1 cells at a density of 1.5–2.0 × 10⁶ cells per well. 5 Cells were incubated with antigen-binding protein at a concentration of 1 μg / ml at 4°C for 30 minutes. Anti-HA APC staining was performed. Flow cytometry analysis showed that in A549 cells, the binding rate of antigen-binding protein 5-26 reached 97.0%, 5-9 was 18.5%, and 5-95 was 11.8%. Figure 10 In MB231 cells, the percentage of cells bound to 5-26 reached 96.8%, 5-9 was 61.7%, and 5-95 was 53.3%. Figure 11 In JeKo-1 cells, the binding rate of 5-26 cells reached 99.7%, 5-9 cells 1.90%, and 5-95 cells 1.18%. Figure 12 The results of this experiment show that 5-26 has a stronger ability to bind to cell lines expressing ROR1 compared to 5-9 and 5-95.

[0197] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

Claims

1. An isolated antigen-binding protein capable of binding ROR1 (orphan receptor tyrosine kinase 1), said isolated antigen-binding protein comprising CDR1, CDR2 and CDR3, the amino acid sequence of said CDR1 being as shown in SEQ ID NO: 1, the amino acid sequence of said CDR2 being as shown in SEQ ID NO: 2, and the amino acid sequence of said CDR3 being as shown in SEQ ID NO: 3, said isolated antigen-binding protein being VHH.

2. The isolated antigen-binding protein according to claim 1, wherein the amino acid sequence of the VHH is as shown in SEQ ID NO:

8.

3. The isolated antigen-binding protein according to any one of claims 1-2, further comprising an Fc region.

4. The isolated antigen-binding protein according to claim 3, wherein the Fc region is derived from the IgG Fc region.

5. The isolated antigen-binding protein according to claim 4, wherein the Fc region is derived from the human IgG Fc region.

6. A nucleic acid molecule encoding the isolated antigen-binding protein of any one of claims 1-5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A cell comprising the nucleic acid molecule of claim 6 and / or the vector of claim 7.

9. A kit comprising the isolated antigen-binding protein of any one of claims 1-5, the kit being capable of detecting the presence and / or quantity of ROR1 in a sample.

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