Antigen-binding proteins targeting ROR1

Abstract

Provided are isolated antigen-binding proteins capable of binding to ROR1 (receptor tyrosine kinase-like orphan receptor 1), methods for preparing and uses thereof, wherein the antigen binding protein comprises CDR1, CDR2 and CDR3.

Description

[Technical Field]

[0001] This application relates to the biomedical field, and specifically to antigen binding proteins that target ROR1. [Background technology]

[0002] ROR1 is a transmembrane receptor tyrosine kinase protein. Human ROR1 consists of one extracellular immunoglobulin-like domain (Ig), two cysteine-rich domains (FZD), a membrane-proximal kringle domain, a single transmembrane domain, one intracellular tyrosine kinase domain (TKD), two silk / threonine-rich domains (S / TRD), and one proline-rich domain (PRD). Cell division, proliferation, migration, and cell chemotaxis are regulated by signal transduction that mediates various signaling pathways. ROR1 is highly expressed during early embryonic development. ROR1 expression gradually decreases during fetal development.

[0003] ROR1 is expressed at low or no expression in normal human tissues, but is highly expressed in various blood tumors and solid tumors. Hematological tumors with high ROR1 expression include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid hematologic tumors. Among solid tumors, ROR1 expression is observed in tumors such as triple-negative breast cancer, colon cancer, lung cancer, pancreatic cancer, and ovarian cancer. ROR1 expression is closely associated with disease progression and therapeutic efficacy. Therefore, ROR1 may be a specific tumor marker and a potentially attractive target for tumor therapy. Summary of the Invention

[0004] The present application provides an isolated antigen-binding protein that targets ROR1. The isolated antigen-binding protein described herein is capable of specifically binding to the ROR1 antigen and has good binding activity. The isolated antigen-binding protein described herein is capable of binding 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 herein can have a tumor-killing effect.

[0005] According to one aspect, the present application provides an isolated antigen binding protein capable of binding to ROR1 (receptor tyrosine kinase-like orphan receptor 1), said isolated antigen binding protein comprising CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 2 and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 3, or wherein the amino acid sequence of CDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 2 and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 13, or wherein the amino acid sequence of CDR1 is set forth in SEQ ID NO: 19, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 20 and the amino acid sequence of CDR3 is set forth in SEQ ID NO: 21.

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

[0007] In certain embodiments, the isolated antigen binding protein comprises a VHH.

[0008] In certain embodiments, the amino acid sequence of said VHH is set forth in SEQ ID NO:8, SEQ ID NO:18 or SEQ ID NO:25.

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

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

[0011] In certain embodiments, the isolated antigen binding protein comprises at least one VHH fragment.

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

[0013] In certain embodiments, the isolated antigen binding protein further comprises an Fc region.

[0014] In certain embodiments, the Fc region is derived from an IgG Fc region.

[0015] In certain embodiments, the Fc region is derived from a human IgG Fc region.

[0016] In certain embodiments, the isolated antigen binding protein has one or more of the following properties: (1) capable of specifically binding to ROR1; and (2) It can bind to ROR1 expressed on the cell surface.

[0017] According to another aspect, the present application further provides a chimeric antigen receptor comprising the isolated antigen binding protein described herein.

[0018] In certain embodiments, the chimeric antigen receptor further comprises a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0019] In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, or SLAM.

[0020] In certain embodiments, the transmembrane domain is derived from the transmembrane domain of CD28, and the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:28.

[0021] In certain embodiments, the costimulatory domain comprises a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 specific ligand, CD40, and MyD88.

[0022] In certain embodiments, the costimulatory domain is derived from a 4-1BB costimulatory domain, and the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:29.

[0023] In certain embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM.

[0024] In certain embodiments, the intracellular signaling domain is a signaling domain derived from CD3ζ, and the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:30.

[0025] In certain embodiments, the chimeric antigen receptor further comprises a hinge region.

[0026] In certain embodiments, the hinge region is derived from the CD28 hinge region.

[0027] In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO:27.

[0028] In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:31.

[0029] According to another aspect, the present application further provides an immune cell comprising a chimeric antigen receptor described herein.

[0030] In certain embodiments, the immune cells are selected from T cells, NK cells, iNKT cells, CIK cells, or γδT cells.

[0031] According to another aspect, the present application further provides a drug molecule comprising said isolated antigen binding protein or said chimeric antigen receptor.

[0032] According to another aspect, the present application further provides a nucleic acid molecule encoding said isolated antigen binding protein or said chimeric antigen receptor.

[0033] In another aspect, the present application further provides a vector comprising the nucleic acid molecule.

[0034] In another aspect, the present application further provides a cell comprising the nucleic acid molecule and / or the vector.

[0035] According to another aspect, the present application further provides a pharmaceutical composition comprising the isolated antigen-binding protein, the drug molecule, the immune cell, the nucleic acid molecule, the vector, and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0036] According to another aspect, the present application further provides a kit comprising said isolated antigen binding protein or said chimeric antigen receptor, which is capable of detecting the presence and / or content of ROR1 in a sample.

[0037] According to another aspect, the present application further provides a use of the isolated antigen binding protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a ROR1-associated disease and / or disorder.

[0038] In certain embodiments, the disease and / or disorder comprises a tumor.

[0039] In certain embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0040] In certain embodiments, the tumor comprises a ROR1-positive tumor.

[0041] Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. It will be apparent to those skilled in the art that, in light of the content of this application, modifications to the specific embodiments disclosed may be made by those skilled in the art without departing from the spirit and scope of the invention to which this disclosure pertains. Accordingly, the drawings and description in the present application are illustrative only and not restrictive. [Brief explanation of the drawings]

[0042] Particular features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below, the brief description of which follows.

[0043] [Figure 1] 1 shows ROR1-specific antibody titers in pre- and post-immunization alpaca sera collected on days 1 to 5 after immunization as described herein. [Figure 2] FIG. 2A shows that the first PCR generated two sets of different amplicons with molecular weights of approximately 0.7 kb and 0.9 kb, and FIG. 2B shows that the 0.7 kb amplicon was amplified as a template to obtain a DNA fragment with a size of approximately 400 bp. [Figure 3] 1 shows the plates used to calculate the number of colonies in the plates and estimate library size by serial dilution as described in this application. [Figure 4] 1 shows a phylogenetic analysis of 90 sequences in the library described in this application. [Figure 5] 1 shows plates used in three consecutive rounds of screening of antigen-binding proteins specific to ROR1 described in this application. [Figure 6]The results of the ELISA described in this application are shown in Figure 6A, where OD450 values ​​of sequence 5-26 after screening are shown, Figure 6B, where OD450 values ​​of sequence 4-92 after screening are shown, and Figure 6C, where OD450 values ​​of VHH1, VHH2, and anti-ROR1 mAb are shown. [Figure 7] FIG. 7 shows the results of SDS-PAGE of the purified antigen-binding proteins described in this application. FIG. 7A shows the results of SDS-PAGE of 5-26, and FIG. 7B shows the results of SDS-PAGE of 4-92. [Figure 8A] ~ [Figure 8C] FIG. 8 shows the results of specific binding of purified antigen-binding proteins described in the present application to recombinant ROR1 protein. FIG. 8A shows the results of antigen-binding protein 5-26 after screening, FIG. 8B shows the results of antigen-binding protein 4-92 after screening, and FIG. 8C shows the binding effects of negative and positive controls. [Figure 9A] ~ [Figure 9B] The results of verifying the specificity of the antigen-binding proteins described in this application are shown in Figure 9A, which shows the results for 5-26, and Figure 9B, which shows the results for 4-92. [Figure 10A] ~ [Figure 10B] 10A and 10B show the results of flow cytometry of the antigen-binding proteins described in the present application bound to A549 cells expressing ROR1. FIG. 10A shows the results of 5-26, and FIG. 10B shows the results of 4-92. [Figure 11A] ~ [Figure 11B] 11A and 11B show the results of flow cytometry of the antigen-binding proteins described herein bound to MB231 cells expressing ROR1, with FIG. 11A showing the results for 5-26 and FIG. 11B showing the results for 4-92. [Figure 12A] ~ [Figure 12B] 12A and 12B show the results of flow cytometry of the antigen-binding proteins described herein bound to ROR1-expressing JeKo-1 cells, with FIG. 12A showing the results for 5-26 and FIG. 12B showing the results for 4-92. [Figure 13A] ~ [Figure 13D]Figure 13 shows the results of flow cytometry of humanized ROR1 single domain antibodies binding to ROR1-expressing MEC cells, JeKo-1 cells, A549 cells, and MB231 cells, where Figure 13A shows the results of flow cytometry of binding to ROR1-expressing MEC cells, Figure 13B shows the results of flow cytometry of binding to ROR1-expressing JeKo-1 cells, Figure 13C shows the results of flow cytometry of binding to ROR1-expressing A549 cells, and Figure 13D shows the results of flow cytometry of binding to ROR1-expressing MB231 cells. [Figure 14] 1 shows the structural design of the ROR1 nanochimeric antigen receptor. [Figure 15] 1 shows that activated T cells can use ROR1 protein to detect high levels of CAR construct expression. [Figure 16] The percentage of surviving target cells in the co-culture system after 24 hours is shown when the initial effector to target ratio is 1:1. [Figure 17] The secretion levels of γ-interferon and IL2 in the supernatant of the co-culture system are shown. [Figure 18A] ~ [Figure 18C] This shows that the ROR1 nanochimeric antigen receptor has strong specific killing ability. DETAILED DESCRIPTION OF THE INVENTION

[0044] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0045] Definition of Terms As used herein, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion and, optionally, a scaffold or backbone portion that enables the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to an 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, etc., as long as they exhibit the required antigen-binding activity. As used herein, an "isolated antigen-binding protein" may comprise an antigen-binding portion and, optionally, a scaffold or backbone portion that enables the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding portion to an antigen.

[0046] As used herein, the term "single domain antibody" generally refers to a fragment of an antibody comprising a single variable domain, which can selectively bind to a specific antigen. The single domain antibody of the present application may comprise a heavy chain variable domain, and the single domain antibody described herein may comprise an antigen-binding fragment, which may comprise a VHH.

[0047] As used herein, the term "polypeptide" generally refers to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The term also applies to amino acid polymers that have been modified, for example, by the addition of sugar residues to form glycoproteins or by phosphorylation. Polypeptides may be produced by naturally occurring non-recombinant cells or by genetically engineered or recombinant cells, and may include molecules having the amino acid sequence of a native protein or molecules with one or more amino acid deletions, additions, and / or substitutions of the native sequence. The term polypeptide also includes antigen-binding fragments, sequences of antibodies or antigen-binding fragments with one or more amino acid deletions, additions, and / or substitutions.

[0048] As used herein, 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-naturally occurring, and modified nucleic acids. The term "nucleic acid" may include genes, cDNAs, or mRNAs. For example, nucleic acid molecules may be synthetic (e.g., chemically synthesized) or recombinant. Nucleic acids may also contain analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Nucleic acid sequences may also include conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly depicted sequences. The term is not limited by the length of the polymer. Nucleic acids may be single- or double-stranded and generally contain 5'-3' phosphodiester linkages, although nucleotide analogs may have other linkages.

[0049] In this application, the term "constant region" generally refers to the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in antigen binding, but exhibits different effector functions. Depending on the amino acid sequence of the constant region of the heavy chain, antibodies are classified into classes IgA, IgD, IgE, IgG, and IgM, and some of these can be further classified into classes such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different types of antibodies are called α, δ, ε, γ, and μ, respectively.

[0050] As used herein, the term "antigen-binding fragment" generally refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with the intact antibody (i.e., the intact antibody from which it is derived) for binding (i.e., specifically binding) to an antigen. Such antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, VHHs, linear antibodies, single-chain antibodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0051] As used herein, the term "variable region" or "variable domain" generally refers to a portion of an antibody's light and / or heavy chain, generally comprising approximately the amino-terminal 120-130 amino acids of the heavy chain and approximately the amino-terminal 100-110 amino acids of the light chain. Variable regions generally vary widely in amino acid sequence, even within antibodies of the same species. The variable regions of antibodies can determine the binding and specificity of each particular antibody for its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more conserved regions of the variable region are called framework regions (FRs). The CDRs of the light and heavy chains may contain amino acids primarily involved in direct antibody-antigen interactions.

[0052] As used herein, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds to an antigen or target, a transmembrane region, and an intracellular domain. For example, a hinge region may be included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may also comprise a signal peptide. Binding of the extracellular domain of the CAR to a target antigen on the surface of a target cell results in CAR clustering, and an activating stimulus is delivered to the CAR-containing cell. CARs redirect the specificity of immune effector cells and induce proliferation, cytokine production, phagocytosis, and / or production of molecules that can mediate the death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner.

[0053] As used herein, the term "intracellular domain" refers to an intracellular domain, including any cleavage portion sufficient to transmit an activation signal. The intracellular domain may include an intracellular signaling domain and / or a costimulatory signaling domain. The term "intracellular signaling domain" refers to an intracellular region capable of generating a signal that promotes immune effector function of a CAR-containing cell (e.g., a CART cell or a CAR-expressing NK cell). For example, the intracellular signaling domain may include the intracellular signaling domain of one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM. For example, the intracellular signaling domain may be a signaling domain derived from CD3ζ. The term "co-stimulatory signaling domain" refers to a portion of a CAR that can transmit an effector signal in the intracellular signaling domain. For example, the costimulatory signaling domain may include an intracellular costimulatory signaling domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88. For example, the costimulatory signaling domain may be an intracellular costimulatory signaling domain derived from 4-1BB.

[0054] As used herein, the term "transmembrane domain" generally refers to a domain of a peptide, polypeptide, or protein that can span a cell membrane. These domains can be used to anchor an extracellular domain to the cell membrane. For example, the transmembrane region may include a transmembrane region of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane domain may be a transmembrane region derived from CD8.

[0055] As used herein, the term "signal peptide" generally refers to a leader sequence located at the amino-terminus (N-terminus) of a nascent CAR protein, which co- or post-translationally targets the nascent protein to the endoplasmic reticulum and subsequent surface expression. For example, the signal peptide may be derived from the signal peptide of the CD8 protein.

[0056] As used herein, the term "drug molecule" generally includes any form of drug known in the art. For example, the drug molecule may be a protein. For example, the drug molecule may be a polypeptide. For example, the drug molecule may be a conjugate of different forms of a drug. For example, the drug molecule may include a small molecule drug.

[0057] As used herein, the term "immune effector cell" generally refers to an immune cell that participates in an immune response and exerts an effector function. For example, exerting the effector function may include removing foreign antigens or promoting an immune effector response. For example, immune effector cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphocyte progenitor cells, and / or pluripotent stem cells. For example, immune effector cells may be T cells.

[0058] As used herein, the term "host cell" generally refers to an individual cell, cell line, or cell culture that can contain or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or that can express a fusion protein or antigen-binding fragment thereof described herein. Such a cell may include the progeny of a single cell. Due to natural, accidental, or deliberate mutation, progeny cells may not necessarily be completely morphologically or genomically identical to the original parent cell, but they may be capable of expressing the antigen-binding protein described herein. Such cells may be obtained by transfecting cells in vitro with a vector described herein. Such cells may be prokaryotic (e.g., Escherichia coli) or eukaryotic (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or bone marrow cells).

[0059] As used herein, the term "vector" generally refers to a nucleic acid molecule that can autonomously replicate in a suitable host and transfer an inserted nucleic acid molecule into and / or between cells (e.g., host cells). The vector may include vectors primarily used to insert DNA or RNA into cells, vectors primarily used to replicate DNA or RNA, and vectors primarily used for transcriptional and / or translational expression of DNA or RNA. The vector further includes vectors having more than one of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate cell. Generally, the vector can produce a desired expression product by culturing an appropriate cell containing the vector. As used herein, the vector may be a plasmid.

[0060] As used herein, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded. The term "promoter" generally refers to a DNA sequence that can regulate the expression of a selected DNA sequence operably linked to the promoter, thereby affecting the expression of the selected DNA sequence in a cell. For example, the nucleic acid molecule can encode an associated antigen-binding protein and / or the chimeric antigen receptor. For example, the nucleic acid molecule may include a promoter. For example, the promoter may be a constitutive promoter.

[0061] As used herein, the term "pharmaceutically acceptable" generally refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. Formulations of this type may typically contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants and supplementary immune-enhancing agents such as cytokines, and optionally other therapeutic agents such as chemotherapeutic agents.

[0062] As used herein, the term "complementarity-determining region" or "CDR" generally refers to a complementarity-determining region within an antibody variable sequence. Each heavy and / or light chain variable region contains three CDRs, designated CDR1, CDR2, and CDR3 for each variable region. As used herein, a CDR combination can refer to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are 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)) not only provides an unambiguous residue numbering system applicable to any antibody variable region, but also provides the exact residue boundaries of the three CDRs. These CDRs are also referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) discovered that certain subregions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These subregions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions are also called Chothia CDRs, and the Chothia CDRs have overlapping boundaries with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are 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 above systems, but still overlap with the Kabat CDRs, and they may be shortened or extended according to predictions or experimental findings that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding.The CDRs described herein can be defined using the IMGT numbering scheme.

[0063] As used herein, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, which may be a human patient. For example, a pharmaceutical composition described herein may include an antigen-binding protein described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and optionally a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition may further include an appropriate formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of the composition are non-toxic to the recipient at the dosages and concentrations used. Pharmaceutical compositions of the present invention may include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0064] As used herein, the term "ROR1" generally refers to receptor tyrosine kinase-like orphan receptor 1. As used herein, the term encompasses full-length ROR1 and functionally active fragments or variants thereof. For example, the term may include the Ig-like domain and / or the coiled-coil 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 found in GeneBank Accession No. Q01973.

[0065] The proteins described herein may include functional mutants, derivatives, analogs, homologs and fragments thereof.

[0066] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a naturally occurring sequence or is encoded by a substantially identical nucleotide sequence and that can possess 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 intrinsic function. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one amino acid residue and / or nucleotide residue present in the naturally occurring protein and / or polynucleotide, so long as the original functional activity is maintained.

[0067] As used herein, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application that contains any substitution, mutation, modification, replacement, deletion and / or addition of / to one (or more) amino acid residue(s) of the sequence, wherein the resulting polypeptide or polynucleotide substantially retains at least one endogenous function thereof.

[0068] As used herein, the term "analog", in general, with respect to a polypeptide or polynucleotide, includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that is mimicked by the mimetic.

[0069] Generally, amino acid substitutions, e.g., at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, can be made as long as the altered sequence substantially retains the required activity or capability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0070] Proteins or polypeptides used herein may have deletions, insertions, or substitutions of amino acid residues, which produce silent mutations and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, so long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine. [Mode for Carrying Out the Invention]

[0071] Isolated antigen-binding proteins According to one aspect, the present application provides an isolated antigen-binding protein capable of binding to ROR1. For example, the binding may be specific. In the present application, the isolated antigen-binding protein can be used to bind to ROR1 and can be used in various fields, such as antibody drug development, bispecific antibody drug development, and chimeric antigen receptor development.

[0072] In the present application, the isolated antigen-binding protein may comprise at least one CDR of an antibody heavy chain variable region VH, and the CDR may be HCDR1, HCDR2, and / or HCDR3. For example, the heavy chain variable region VH may comprise the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 18, or SEQ ID NO: 25. In the present application, the HCDR sequence of the isolated antigen-binding protein may comprise an HCDR sequence numbered by any method, and any HCDR sequence numbered by any method is within the scope of protection of the present application, as long as the VH sequence is the same as the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 18, or SEQ ID NO: 25. For example, CDRs may be numbered using methods such as CCG, Kabat, Chothia, IMGT, or a combination of Kabat and Chothia. In a specific embodiment, CDRs may be numbered using IMGT.

[0073] In the present application, the isolated antigen binding protein may comprise an HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:13 or SEQ ID NO:21.

[0074] In the present application, the isolated antigen binding protein may comprise an HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:20.

[0075] In the present application, the isolated antigen-binding protein may comprise an HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 19.

[0076] In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, and HCDR3. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 13. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 19, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 20, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 21.

[0077] In the present application, the VH of the isolated antigen binding protein may comprise framework regions H-FR1, H-FR2, H-FR3 and / or H-FR4.

[0078] In the present application, the isolated antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 may be directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 14.

[0079] In the present application, the isolated antigen-binding protein may comprise an H-FR2, which may be located between the HCDR1 and the HCDR2, and which may comprise the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:15, or SEQ ID NO:22.

[0080] In the present application, the isolated antigen-binding protein may comprise an H-FR3, which may be located between the HCDR2 and the HCDR3, and which may comprise the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:16, or SEQ ID NO:23.

[0081] In the present application, the isolated antigen-binding protein may comprise an H-FR4, the N-terminus of the H-FR4 may be directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:17, or SEQ ID NO:24.

[0082] In the present application, the isolated antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4. For example, the H-FR1 may include the amino acid sequence set forth in SEQ ID NO: 4, the H-FR2 may include the amino acid sequence set forth in SEQ ID NO: 5, the H-FR3 may include the amino acid sequence set forth in SEQ ID NO: 6, and the H-FR4 may include the amino acid sequence set forth in SEQ ID NO: 7. For example, the H-FR1 may include the amino acid sequence set forth in SEQ ID NO: 14, the H-FR2 may include the amino acid sequence set forth in SEQ ID NO: 15, the H-FR3 may include the amino acid sequence set forth in SEQ ID NO: 16, and the H-FR4 may include the amino acid sequence set forth in SEQ ID NO: 17. For example, the H-FR1 may include the amino acid sequence set forth in SEQ ID NO: 4, the H-FR2 may include the amino acid sequence set forth in SEQ ID NO: 22, the H-FR3 may include the amino acid sequence set forth in SEQ ID NO: 23, and the H-FR4 may include the amino acid sequence set forth in SEQ ID NO: 24.

[0083] In the present 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 linked to the N-terminus of HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3. In the present application, the direct or indirect linkage may be between molecules or may be via a linker.

[0084] In the present application, the isolated antigen-binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:18 or SEQ ID NO:25.

[0085] In the present application, the isolated antigen-binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise an Fc fragment. The Fc region may be derived from an IgG Fc region, for example, a human IgG Fc region, for example, an IgG1 Fc region, or an IgG4 Fc region. For example, the Fc fragment may comprise a variant thereof, and the variant may comprise substitution, deletion, and / or addition of one or more amino acids in the amino acid sequence of the Fc fragment. For example, it may include substitutions, deletions, and / or insertions of 1 to 30, 1 to 20, or 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, amino acids, or a homolog thereof, and the homolog may be an amino acid sequence that has at least about 85% sequence identity (e.g., 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 more) with the amino acid sequence of the Fc fragment.

[0086] In the present application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof.

[0087] In certain embodiments, in the isolated antigen-binding protein, the antigen-binding fragment may be selected from the group consisting of a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab')2, a scFv, a di-scFv, a VHH, and / or a dAb.

[0088] In the present application, in the isolated antigen-binding protein, the antibody may be selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0089] Single Domain Antibodies In the present application, the isolated antigen-binding protein may be a single domain antibody.

[0090] In the present application, the single domain antibody may comprise at least one CDR in an antibody heavy chain variable region VHH, and the CDR may be CDR3, CDR2 and / or CDR1. The heavy chain variable region VHH may comprise the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 18 or SEQ ID NO: 25.

[0091] In the present application, the single domain antibody may comprise a CDR3, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:13 or SEQ ID NO:21.

[0092] In the present application, the single domain antibody may comprise a CDR2, and the CDR2 may comprise the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:20.

[0093] In the present application, the single domain antibody may comprise a CDR1, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 19.

[0094] In the present application, the single domain antibody may comprise CDR1, CDR2, and CDR3. For example, the CDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the CDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3. For example, the CDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the CDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 13. For example, the CDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 19, the CDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 20, and the CDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 21.

[0095] In the present application, the single domain antibody may comprise an FR1, the C-terminus of which may be directly or indirectly linked to the N-terminus of the CDR1, and the FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14.

[0096] In the present application, the single domain antibody may comprise FR2, which may be located between the CDR1 and the CDR2, and which may comprise the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 15 or SEQ ID NO: 22.

[0097] In the present application, the single domain antibody may comprise an FR3, which may be located between the CDR2 and the CDR3, and which may comprise the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 23.

[0098] In the present application, the single domain antibody may comprise an FR4, the N-terminus of which may be directly or indirectly linked to the C-terminus of the CDR3, and the FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 17 or SEQ ID NO: 24.

[0099] In the present application, the single domain antibody may comprise FR1, FR2, FR3, and FR4. For example, the FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 4, the FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 5, the FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 6, and the FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 7. For example, the FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 14, the FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 15, the FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 16, and the FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 17. For example, the FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 4, the FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 22, the FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 23, and the FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 24.

[0100] In the present 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 linked to the N-terminus of CDR1, the FR2 is located between the CDR1 and the CDR2, the FR3 is located between the CDR2 and the CDR3, and the N-terminus of FR4 is directly or indirectly linked to the C-terminus of CDR3. In the present application, the direct or indirect linkage may be between molecules or may be via a linker.

[0101] In the present application, the single domain antibody may comprise a heavy chain variable region VHH, and the VHH may comprise the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:18 or SEQ ID NO:25.

[0102] In the present application, the single domain antibody may include a heavy chain constant region, and the heavy chain constant region may include an Fc fragment. For example, the Fc fragment may include a variant thereof, and the variant may include substitutions, deletions, and / or additions of one or more amino acids in the amino acid sequence of the Fc fragment. For example, the variant may include substitutions, deletions, and / or insertions of 1 to 30, 1 to 20, or 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, amino acids, or a homolog thereof. The homolog may be an amino acid sequence that has at least about 85% sequence identity (e.g., 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 more) with the amino acid sequence of the Fc fragment.

[0103] Chimeric antigen receptor and immune cells containing said chimeric antigen receptor In another aspect, the present application provides a chimeric antigen receptor comprising an antigen-binding domain. For example, the antigen-binding domain may comprise an isolated antigen-binding protein described herein.

[0104] In the present application, the antigen-binding domain of the chimeric antigen receptor may comprise CDR1, CDR2, and CDR3 of an antibody. For example, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3. For example, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13. For example, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0105] In the present application, the chimeric antigen receptor may include a costimulatory signaling domain capable of providing a stimulatory signal. For example, the costimulatory signaling domain may include an intracellular costimulatory signaling domain of one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88.

[0106] In the present application, the costimulatory signaling domain may be an intracellular costimulatory signaling domain derived from 4-1BB. For example, the costimulatory signaling domain may include the amino acid sequence set forth in SEQ ID NO: 29. In the present application, the CAR may include an intracellular signaling domain, which is capable of transmitting an activation signal to the inside of a cell. For example, the intracellular signaling domain may include an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and other domains containing at least one ITAM.

[0107] For example, the intracellular signaling domain may be a signaling domain derived from CD3ζ. For example, the intracellular signaling domain may be the amino acid sequence set forth in SEQ ID NO:30.

[0108] In the present application, the CAR may include a transmembrane domain, and the transmembrane region is a sequence that spans the cell membrane of a cell surface protein, for example, it may include a hydrophobic alpha helix. The transmembrane domain may be derived from any type I transmembrane protein. The transmembrane region may also be a synthetic sequence predicted to form a hydrophobic helix. For example, the transmembrane domain may include a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0109] For example, the transmembrane region may be a transmembrane domain derived from CD28. For example, the transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO:28.

[0110] In the present application, the chimeric antigen receptor may further comprise a hinge region, which may be located between the extracellular targeting moiety and the transmembrane region. For example, the hinge region may comprise a hinge region of one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0111] In the present application, the hinge region may be derived from CD28. For example, the hinge region may comprise the amino acid sequence set forth in SEQ ID NO: 27.

[0112] In the present application, the chimeric antigen receptor may further comprise a signal peptide at the N-terminus of the binding domain that binds to the ROR1 protein. For example, the signal peptide may be a signal peptide derived from the CD8 protein. For example, the signal peptide may comprise the amino acid sequence set forth in SEQ ID NO: 26.

[0113] In the present application, the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:31.

[0114] According to another aspect, the present application further provides immune cells comprising and / or expressing the chimeric antigen receptor described herein. For example, the immune cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells, and / or pluripotent stem cells.

[0115] In certain embodiments, the cell may be a T cell.

[0116] Polypeptides, drug molecules, nucleic acids, vectors, cells, and preparation methods In another aspect, the present application provides the isolated antigen-binding protein, or a polypeptide that may comprise the chimeric antigen receptor.

[0117] In another aspect, the present application provides a drug molecule that may comprise said isolated antigen-binding protein or said chimeric antigen receptor.

[0118] In another aspect, the present application provides one or more isolated nucleic acid molecules capable of encoding the isolated antigen-binding protein, chimeric antigen receptor, or polypeptide of the present application. For example, each nucleic acid molecule of the one or more nucleic acid molecules may encode the isolated antigen-binding protein, chimeric antigen receptor, or polypeptide in its entirety or in part. The nucleic acid molecules described in the present application may be isolated. In the present application, nucleic acids encoding the isolated antigen-binding protein, chimeric antigen receptor, or polypeptide can be prepared by various methods known in the art.

[0119] In another aspect, the present application provides one or more vectors containing one or more nucleic acid molecules described herein. Each vector may contain one or more of the nucleic acid molecules. The vector may also contain other genes, such as marker genes that allow the vector to be selected in a suitable host cell under appropriate conditions. The vector may also contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. The vector may also contain a replication origin. The vector may also be, for example, a plasmid, cosmid, virus, phage, or other vectors commonly used in genetic engineering.

[0120] In another aspect, the present application provides cells that may include one or more nucleic acid molecules and / or one or more vectors described herein. In certain embodiments, each cell may include one or more nucleic acid molecules or vectors described herein. In certain embodiments, each cell may include multiple (e.g., two or more) or multiple (e.g., two or more) types of nucleic acid molecules or vectors described herein. For example, a vector described herein can be introduced into the cell, such as a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / 0 cell, or an HEK293 cell, or into other eukaryotic cells, such as plant-derived cells, fungi, or yeast cells. The vector described herein can be introduced into the cell by methods known in the art, such as electroporation, lipofectine transfection, or lipofectamine transfection.

[0121] In another aspect, the present application provides a method for preparing the isolated antigen-binding protein, chimeric antigen receptor, or polypeptide of the present application. The method may include culturing a cell described herein under conditions that allow expression of the isolated antigen-binding protein, chimeric antigen receptor, or polypeptide. This can be achieved, for example, by using an appropriate medium, an appropriate temperature, an appropriate culture time, etc., and these methods will be understood by those skilled in the art.

[0122] Pharmaceutical Compositions, Uses In another aspect, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein, the immune cell, the polypeptide, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or a pharmaceutically acceptable carrier.

[0123] For example, the pharmaceutically acceptable carrier may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex, and / or a non-ionic surfactant.

[0124] In the present application, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation administration, enteral administration, vaginal administration, transdermal administration, or subcutaneous depot administration. The pharmaceutical composition can be used to inhibit tumor growth. For example, the pharmaceutical composition can inhibit or delay the onset or progression of a disease and / or alleviate and / or stabilize the disease state.

[0125] The pharmaceutical compositions described herein may comprise a prophylactically and / or therapeutically effective amount of the isolated antigen binding protein, which is the amount required to prevent and / or treat (at least partially treat) a disease or disorder and / or any complications thereof in a subject suffering from or at risk of developing the disease or disorder and / or any complications thereof.

[0126] According to another aspect, the present application provides a method that can be used to detect or measure ROR1, the method optionally comprising using the isolated antigen binding protein, the chimeric antigen receptor, or the polypeptide.

[0127] In another aspect, the present application provides a kit that can be used to detect or measure ROR1, the kit optionally including the isolated antigen binding protein, the chimeric antigen receptor, or the polypeptide.

[0128] In another aspect, the present application provides the use of said isolated antigen binding protein, said immune cell, said polypeptide, said drug molecule, said isolated nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of a disease and / or disorder.

[0129] In another aspect, the present application provides the isolated antigen-binding protein, the immune cell, the polypeptide, the drug molecule, the isolated nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition prepared for preventing or treating a disease or disorder.

[0130] According to another aspect, the present application provides a method of preventing or treating a disease, the method comprising administering to a subject in need thereof the isolated antigen binding protein, the immune cell, the polypeptide, the drug molecule, the isolated nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition of the present application.

[0131] In the present application, said disease and / or disorder in said use is caused or mediated by aberrant expression of ROR1.

[0132] In the present application, the disease and / or disorder may include a tumor. For example, the tumor may include a solid tumor and / or a hematological tumor. For example, the tumor may be a ROR1-positive tumor.

[0133] For example, the ROR1-positive tumor may be selected from breast cancer, ovarian cancer, melanoma, pancreatic cancer, lung cancer, leukemia, lymphoma, and / or thyroid cancer.

[0134] Without wishing to be bound by any theory, the following examples are only intended to illustrate the respective technical solutions of the present invention, and are not intended to limit the scope of the present invention.

[0135] Example Example 1 Immunization of Animals The experimental process is carried out according to the following steps.

[0136] (1) Immunize a 2.5-year-old unimmunized female alpaca five times every two weeks with freshly prepared immunogen (see Table 1, Immunization Schedule). Divide the immunogen into several portions (each portion is used for one immunization) and store at -80°C.

[0137] (2) For the first immunization, 300 μg of immunogen was mixed with an equal volume of Freund's complete adjuvant (CFA) to prepare an emulsion, and for subsequent immunizations (2nd to 5th), 300 μg of immunogen was mixed with an equal volume of Freund's incomplete adjuvant (CFA) to prepare an emulsion with adjuvant (IFA) (all vaccination experiments were approved by the local ethical committee).

[0138] (3) Before each administration of immunogen (1st to 5th), a 5 ml jugular vein blood sample is taken, and the serum is collected as a post-immunization IgG-containing sample to be used for antigen-specific antibody titer.

[0139] (4) Seven days after the last immunization, 50 ml of blood was collected and stained with Ficoll-Paque. TM Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using PLUS Media (GE Healthcare, USA), lysed in Trizol, and used for subsequent total RNA extraction.

[0140] (5) ELISA measured antigen-induced seroconversion by comparing the titers of pre- and post-immunization sera, and one experiment was performed for each diluted serum sample at each bleeding time point.

[0141] The experimental results are shown in Figure 1. The immune serum showed a clear reaction to the recombinant ROR1 protein, and the ROR1-specific antibody titers in the serum from the third to fifth rounds after immunization all reached 1 / 100,000 or higher.

[0142] [Table 1]

[0143] ( * The third immunization was delayed by two weeks due to COVID-19 quarantine on the alpaca farm.

[0144] Example 2: Creation of an immune phage library The experimental process is carried out according to the following steps.

[0145] (1) PBMCs were isolated from 50 ml of peripheral blood, and total RNA was extracted. The RNA was then used as a template to prepare cDNA using commercially available reagents. The preparation system is shown in Table 2.

[0146] (2) VHH sequences were amplified from the cDNA library and transformed into TGI cells, after which the library size was calculated based on the number of colonies in a series of dilutions of electroblotted E. coli cell suspensions on 90 mm plates.

[0147] (3) Colony sequencing and bioinformatics analysis were performed.

[0148] As can be seen from the experimental results, the initial PCR produced two sets of different amplicons with molecular weights of approximately 0.7 kb and 0.9 kb (Figure 2A). VHH was amplified using the 0.7 kb amplicon as a template to obtain DNA fragments with a fragment size of approximately 400 bp (Figure 2B). The size of the amplified library was 1.4 × 10 8 Phylogenetic analysis was performed on 90 sequences in the library, and 96 single colonies were randomly selected and subjected to colony sequencing. The percentage of phages with correctly inserted VHHs was 93.8% (90 / 96), and the percentage of specific VHH sequences was 96.7% (87 / 90) (Figure 4).

[0149] [Table 2]

[0150] Example 3 Screening for antigen-specific antigen-binding proteins By monitoring the number of colonies grown on the plates, three consecutive rounds of screening were performed to obtain ROR1-specific antigen-binding proteins (Figure 5). After each round of screening, the number of colonies in the ROR1-coated group increased, indicating enrichment of ROR1-specific antigen-binding proteins. R1, R2, and R3 represent the results of the first, second, and third rounds of screening, respectively, and NC represents the negative control.

[0151] Example 4 Recognition and validation of antigen-binding proteins (1) Specific antigen-binding proteins were verified through three rounds of screening, and antigen-positive clones were obtained through ELISA identification using periplasmic extracts.

[0152] (2) Next, the DNA sequences of the positive clones were obtained by colony sequencing, and the specific sequences were identified.

[0153] (3) Recombinant production and purification of the specific sequence is carried out to determine whether the detection purity meets the subsequent detection requirements.

[0154] (4) Further detecting the affinity and specificity of the antigen-binding protein for the ROR1 protein.

[0155] As can be seen from the experimental results, the OD450 values ​​of 5-26 (VHH sequence is as set forth in SEQ ID NO:8) and 4-92 (VHH sequence is as set forth in SEQ ID NO:25) are both higher than those of 5-09 (VHH sequence is as set forth in SEQ ID NO:11) and 5-95 (VHH sequence is as set forth in SEQ ID NO:12) (Figures 6A and 6B). 5-26 contains CDR1 set forth in SEQ ID NO:1, CDR2 set forth in SEQ ID NO:2, and CDR3 set forth in SEQ ID NO:3, while 4-92 contains CDR1 set forth in SEQ ID NO:19, CDR2 set forth in SEQ ID NO:20, and CDR3 set forth in SEQ ID NO:21. Two nonspecific VHHs (VHH1: sequence set forth in SEQ ID NO:9, VHH2: sequence set forth in SEQ ID NO:10) did not bind to the recombinant ROR1 protein, demonstrating good binding of anti-ROR1 mAb to the recombinant ROR1 protein (Figure 6C). The experimental results further demonstrate that 5-26, 4-92, 5-09 and 5-95 specifically bind to the recombinant ROR1 protein, but the binding ability of 5-26 and 4-92 is significantly superior to that of the other two groups.

[0156] As can be seen from the SDS-PAGE results, the purity of the purified antigen-binding proteins (approximately 0.5 mg each) was over 90%, meeting the requirements for subsequent functional testing (Figures 7A-7B). The purified antigen-binding proteins 5-26, 4-92, 5-9, and 5-95 specifically bound to recombinant ROR1 protein (Figures 8A-8B). The negative control VHH1 and VHH2 did not bind to ROR1 protein, while the positive control bound to ROR1 protein (Figure 8C). These experimental results demonstrate that 5-26 and 4-92 can bind to ROR1 protein at higher concentrations than 5-9 and 5-95 at multiple dilutions, demonstrating their excellent specificity.

[0157] Specificity assessment showed that purified antigen-binding proteins 5-26, 4-92, 5-9, and 5-95 bound exclusively to recombinant ROR1 protein and not to the other two His-tagged proteins (PD-L1 and RBD), indicating that the selected antigen-binding proteins did not recognize other tags. Anti-ROR1 mAb (positive control) bound exclusively to recombinant ROR1 protein. Anti-his mAb (Sinobiological, #105327-MM02T-H) bound to all three His-tagged proteins (PD-L1, RBD, and ROR1) (Figures 9A-9B). The concentration of purified antigen-binding proteins used in the experiments was 3 μg / ml. Each protein contained one His tag and one HA tag at its C-terminus. The secondary antibody used for recognition was an HRP anti-HA tag antibody (Abcam, #ab1190). The experimental results show that 5-26 and 4-92 have good specificity for the ROR1 protein and hardly bind to PD-L1 and RBD.

[0158] Anti-ROR 1 mAb: ROR1 mouse monoclonal antibody, from Proteintech (catalog No.: 66923-1-Ig), molecular weight 130 kDa, can be used in applications such as FC, IF, IHC, WB, and ELISA.

[0159] Example 5: Detection of the binding ability of antigen-binding proteins to cell lines expressing ROR1 ROR1 was expressed in A549, MB231, and JeKo-1 cells, and each well contained 1.5–2.0 × 10 5The cells were incubated with 1 μg / ml of antigen-binding protein at 4°C for 30 minutes and then stained with anti-HA APC. Flow cytometry analysis revealed that in A549 cells, the percentage of 5-26 binding cells reached 97.0%, 5-9 binding cells 18.5%, and 5-95 binding cells 11.8% (Figure 10A). The percentage of 4-92 binding cells reached 34.6%, 5-9 binding cells 18.5%, and 5-95 binding cells 11.8% (Figure 10B). In MB231 cells, the percentage of 5-26 binding cells reached 96.8%, 5-9 binding cells 61.7%, and 5-95 binding cells 53.3% (Figure 11A). The percentage of 4-92 binding cells reached 74.3%, 5-9 binding cells 61.7%, and 5-95 binding cells 53.3% (Figure 11B). Among JeKo-1 cells, the percentage of 5-26-binding cells reached 99.7%, 5-9 was 1.90%, and 5-95 was 1.18% (Figure 12A), while the percentage of 4-92-binding cells reached 2.96%, 5-9 was 1.90%, and 5-95 was 1.18% (Figure 12B). These experimental results indicate that 5-26 and 4-92 have higher binding ability to ROR1-expressing cell lines than 5-9 and 5-95.

[0160] Example 6: Detection of the binding ability of humanized antigen-binding proteins to cell lines expressing ROR1 The hu5-26 sequence was humanized to obtain the amino acid sequence hu5-26. The amino acid sequences of CDR1, CDR2, and CDR3 of hu5-26 are set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. The amino acid sequence of VHH is set forth in SEQ ID NO: 18. hu5-26 was then linked to IgG Fc to obtain the hu5-26 single-domain antibody. Using the method described in Example 5, the binding ability of hu5-26 to different cell lines (MECs) expressing ROR1 was examined. The experimental results are shown in Figures 13A-13D. The experimental results showed that the percentage of hu5-26-binding cells in MEC cells reached 99.6% (Figure 13A), in JeKo-1 cells, the percentage of hu5-26-binding cells reached 92.5% (Figure 13B), in A549 cells, the percentage of hu5-26-binding cells reached 80.1% (Figure 13C), and in MB231 cells, the percentage of hu5-26-binding cells reached 90.0% (Figure 13D).

[0161] In the present application, hu5-26 has stronger binding ability to ROR1 than other humanized antibodies and has stronger affinity to almost all ROR1-positive cells.

[0162] Example 7 Preparation and Expression of ROR1 Nanochimeric Antigen Receptors Cell culture HEK293T, MEC1, and MDA-MB-231 cell lines were obtained from ATCC, USA. The MEC1-ROR1 cell line is an in-house generated stable ROR1 expressing line. The HEK293T cell line was maintained in complete medium (DMEM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). The MDA-MB-231, MEC1, and MEC1-ROR1 cell lines were maintained in complete medium (IMDM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). PBMCs were isolated from whole blood of healthy donors using Ficoll-Paque and 2x10 7 1 mL of sample was dispensed at a concentration of 10 cells / mL and stored frozen in a liquid nitrogen tank. The medium was heat-inactivated FBS supplemented with 10% DMSO (vol / vol).

[0163] Building a clone All relevant gene constructs were constructed and synthesized using GeneWiz. The ROR1 nanochimeric antigen receptor used the second-generation CAR structure (Figure 14). The gene construct encoding the hu5-26 ROR1 VHH CAR (hereinafter referred to as ROR1VHH CAR) (CDR1 of the ROR1 VHH is set forth in SEQ ID NO: 1, CDR2 is set forth in SEQ ID NO: 2, CDR3 is set forth in SEQ ID NO: 13, VHH is set forth in SEQ ID NO: 18, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 26, the amino acid sequence of the hinge region is set forth in SEQ ID NO: 27, the amino acid sequence of the transmembrane domain is set forth in SEQ ID NO: 28, the amino acid sequence of the costimulatory domain is set forth in SEQ ID NO: 29, and the amino acid sequence of the intracellular signaling domain is set forth in SEQ ID NO: 30) was inserted into the pALD expression plasmid via the BamHI and SalI cloning sites. After double enzyme cleavage and sequencing verification, the expression plasmid was amplified in large quantities and used for lentiviral packaging.

[0164] Preparation of lentivirus All lentiviruses were prepared from HEK293T cells. Freshly resuspended HEK293T cells were washed with PBS buffer and seeded onto 10 cm culture dishes at a cell density of 90%–95% of the dish surface area. The lentiviral packaging plasmid and transfection plasmid were mixed and mixed with the transfection agent Lipofectamine-3000, and then transiently transfected into the seeded HEK293T cells. After 48 hours of culture, the supernatant was collected and filtered through a 0.45 μm filter. The sample was then dispensed into 1 ml aliquots and stored in a -80°C refrigerator.

[0165] T cell activation and transduction To transduce PBMCs with lentivirus encoding the corresponding constructs, cryopreserved PBMCs were resuscitated and then added to 24-well plates. They were activated for 24-72 hours with CD3- and CD28-conjugated magnetic beads and IL2 (500 IU / ml). The activated PBMCs were then distributed across multiple wells of the 24-well plate, and the corresponding lentivirus and polybrene (8 g / ml) were added. The cells were centrifuged at 2000 g in a tabletop centrifuge for 2 hours, then transferred to an incubator at 37°C and 5% CO2 for incubation. For the control group, the lentivirus was replaced with complete culture medium, and the remaining steps were the same. The transfected cells were then cultured for 48 hours in X-VIVO-15 cell culture medium containing IL2 (500 IU / ml) before further testing and analysis.

[0166] Flow cytometry measurement method Flow cytometry measurements were performed using a flow cytometer (Beckman Coulter) in semi-automated or platelet mode, and data were analyzed using FlowJo software. Cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0) and plated at 1–5 × 10 6The cells were resuspended at 37°C / mL and placed on ice before staining. A fluorophore-containing antibody was added and incubated in the dark in a refrigerator at 4°C for 40 minutes, followed by the addition of 7-AAD for 5 minutes. After staining was complete, a large amount of FACS buffer was added to wash away unbound antibody, followed by centrifugation at 300xg for 3 minutes and the supernatant removed. After washing and resuspending in FACS buffer, the cells were analyzed by flow cytometry.

[0167] Enzyme-linked immunosorbent assay The supernatant from the co-culture can be carefully aspirated after brief centrifugation and, after appropriate dilution, used in an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. TM Deluxe Set Human IFN-γ Kit (Biolegend; 430104) and ELISA MAX TM Cytokine secretion levels in the culture medium were measured using a Standard Set Human IL-2 (Biolegend; 431801) kit.

[0168] We successfully cloned the hu5-26 ROR1 CAR-containing construct and packaged it into lentivirus. As shown in Figure 15, 72 hours after lentivirus infection, activated human PBMCs expressed high levels of the CAR construct and were able to bind to PE-labeled ROR1 protein.

[0169] Example 8 In vitro functional experimental validation of ROR1 nanochimeric antigen receptor Tumor cell lines We engineered tumor cells, MEC1 (B-cell chronic lymphocytic leukemia), to generate cell lines that stably express the ROR1 antigen. The MDA-MB-231 cell line also expresses the ROR1 antigen. These three tumor cell lines expressed GFP after transfection and were used in subsequent studies.

[0170] CAR-T tumor short-acting killing experiment (flow cytometry detection method) The above construct (ROR1VHH CAR) was transfected into PBMC cells and cultured for 72 hours, after which CAR expression was detected by flow cytometry. Effector and target cells were counted separately, and then cultured at a cell density of 1 = 5 x 10 at an effector-to-target ratio of 1:1. 4 The cells were resuspended in 200 μl of cell culture medium and seeded in a 96-well plate for culture. After 24 hours, the 96-well plate was removed, centrifuged, and the supernatant was collected to measure the secretion of γ-interferon and IL-2 using an enzyme-linked immunosorbent assay.

[0171] At the same time, the co-cultured cells were washed with PBS, suspended, and then stained with 7-AAD for 5 minutes in the dark, and the proportion of target cells among viable cells was detected using flow cytometry.

[0172] Long-lasting CAR-T tumor killing experiment (detected by Incucyte real-time imaging method) The above construct (ROR1VHH CAR) was transfected into PBMC cells and cultured for 72 hours, after which CAR expression was detected by flow cytometry. Effector and target cells were counted separately, and then cultured at a cell density of 1 x 10 cells at an effector-to-target ratio of 1:1. 4 The cells were resuspended in 200 μl of cell culture medium and seeded into a flat-bottom 96-well plate. The 96-well plate was placed in an IncuCyte for real-time imaging, allowing for monitoring of the 48-hour growth curve of GFP-labeled target cells.

[0173] For short-term killing detection by flow cytometry, the GFP-labeled target cells allowed counting of surviving target cells throughout the co-culture system. As shown in Figure 16, after 24 hours, the ROR1VHH CAR group exhibited potent killing ability against ROR1-expressing target cells, with the percentage of target cells decreasing from 50% at time 0 to 1.925% (MEC-ROR1) and 2.955% (MB231), respectively. Target cells that did not express ROR1 showed no significant difference from the control group, demonstrating good specificity.

[0174] At the same time, during the process of killing ROR1-positive target cells, the hu5-26 ROR1 VHH CAR group was detected to secrete higher levels of gamma interferon and IL-2 (Figure 17). As shown in Figures 18A-18C, in IncuCyte real-time imaging experiments, the ROR1 VHH CAR group was able to effectively inhibit the in vitro growth of ROR1-positive tumor cells and nearly eliminate target cells within 48 hours (MB231 killed 80% and MEC-ROR killed 90%). Similarly, this tumor-killing ability also showed good specificity and was safe for practical application.

[0175] The foregoing detailed description has been provided by way of illustration and example only and is not intended to limit the scope of the appended claims. Many variations of the embodiments described herein will be apparent to those skilled in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims

1. 1. An isolated antigen binding protein capable of binding to ROR1 (receptor tyrosine kinase-like orphan receptor 1), comprising: the isolated antigen protein comprises CDR1, CDR2 and CDR3; The CDR1, CDR2 and CDR3 are Group 1, wherein the amino acid sequence of the CDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the CDR2 is set forth in SEQ ID NO: 2, and the amino acid sequence of the CDR3 is set forth in SEQ ID NO: 3; Group 2, wherein the amino acid sequence of the CDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the CDR2 is set forth in SEQ ID NO: 2, and the amino acid sequence of the CDR3 is set forth in SEQ ID NO: 13; Group 3, wherein the amino acid sequence of the CDR1 is set forth in SEQ ID NO: 19, the amino acid sequence of the CDR2 is set forth in SEQ ID NO: 20, and the amino acid sequence of the CDR3 is set forth in SEQ ID NO: 21; The amino acid sequence of any one of the following groups is selected from: the isolated antigen binding protein is a VHH; Isolated antigen-binding proteins.

2. The isolated antigen-binding protein of claim 1, wherein the amino acid sequence of the VHH is set forth in SEQ ID NO: 8, SEQ ID NO: 18 or SEQ ID NO:

25.

3. 3. The isolated antigen-binding protein of any one of claims 1 to 2, comprising an antibody or an antigen-binding fragment thereof.

4. 4. The isolated antigen-binding protein of claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

5. 5. The isolated antigen-binding protein of any one of claims 1 to 4, comprising at least one VHH fragment.

6. 6. The isolated antigen-binding protein of any one of claims 1 to 5, comprising two VHH fragments.

7. The isolated antigen binding protein of any one of claims 1 to 6, further comprising an Fc region.

8. 8. The isolated antigen binding protein of claim 7, wherein the Fc region is derived from an IgG Fc region.

9. 9. The isolated antigen binding protein of claim 8, wherein the Fc region is derived from a human IgG Fc region.

10. 10. The isolated antigen-binding protein of any one of claims 1 to 9, having at least one of the following properties: capable of specifically binding to ROR1; and It can bind to ROR1 expressed on the cell surface.

11. A chimeric antigen receptor comprising the isolated antigen-binding protein of any one of claims 1 to 10.

12. The chimeric antigen receptor of claim 11, further comprising a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

13. The chimeric antigen receptor of claim 12, wherein the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, or SLAM.

14. The chimeric antigen receptor of claim 13, wherein the transmembrane domain is derived from the transmembrane domain of CD28, and the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

28.

15. The chimeric antigen receptor of any one of claims 12 to 14, wherein the costimulatory domain comprises a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88.

16. The chimeric antigen receptor of claim 15, wherein the costimulatory domain is derived from a 4-1BB costimulatory domain, and the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:

29.

17. The chimeric antigen receptor according to any one of claims 12 to 16, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM.

18. The chimeric antigen receptor of claim 17, wherein the intracellular signaling domain is a signaling domain derived from CD3ζ, and the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

30.

19. The chimeric antigen receptor of any one of claims 11 to 18, further comprising a hinge region.

20. The chimeric antigen receptor of claim 19, wherein the hinge region is a hinge region derived from CD28.

21. The chimeric antigen receptor of claim 20, wherein the hinge region comprises the amino acid sequence set forth in SEQ ID NO:

27.

22. The chimeric antigen receptor of any one of claims 11 to 21, comprising the amino acid sequence shown in SEQ ID NO:

31.

23. An immune cell comprising the chimeric antigen receptor of any one of claims 11 to 22.

24. The immune cell of claim 23, selected from a T cell, an NK cell, an iNKT cell, a CIK cell, or a γδT cell.

25. A drug molecule comprising the isolated antigen-binding protein of any one of claims 1 to 10 or the chimeric antigen receptor of any one of claims 11 to 22.

26. A nucleic acid molecule encoding the isolated antigen-binding protein of any one of claims 1 to 10 or the chimeric antigen receptor of any one of claims 11 to 22.

27. A vector comprising the nucleic acid molecule of claim 26.

28. A host cell comprising the nucleic acid molecule of claim 26 and / or the vector of claim 27.

29. 29. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1 to 10, the immune cell of any one of claims 23 to 24, the drug molecule of claim 25, the nucleic acid molecule of claim 26, the vector of claim 27, and / or the host cell of claim 28, and optionally a pharmaceutically acceptable carrier.

30. 23. A kit capable of detecting the presence and / or content of ROR1 in a sample, comprising the isolated antigen-binding protein of any one of claims 1 to 10 or the chimeric antigen receptor of any one of claims 11 to 22.

31. Use of the isolated antigen-binding protein of any one of claims 1 to 10, the chimeric antigen receptor of any one of claims 11 to 22, the immune cell of any one of claims 23 to 24, the drug molecule of claim 25, the nucleic acid molecule of claim 26, the vector of claim 27, the host cell of claim 28, or the pharmaceutical composition of claim 29 in the manufacture of a medicament for preventing and / or treating a ROR1-related disease and / or disorder.

32. 32. The use according to claim 31 , wherein the disease and / or disorder comprises a tumor.

33. 33. The use according to claim 32, wherein the tumor is a solid tumor and / or a blood tumor.

34. The use according to any one of claims 32 to 33, wherein the tumor is a ROR1-positive tumor.

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