Antigen binding proteins targeting ROR1

Abstract

Provided are an isolated antigen binding protein capable of binding ROR1 (receptor tyrosine kinase orphan receptor 1), the antigen protein comprising CDR1, CDR2 and CDR3, a preparation method and use thereof.

Description

Antigen binding proteins targeting ROR1 Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting ROR1. Background Art

[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 signaling through multiple signaling pathways. It is highly expressed during early embryonic development. However, ROR1 expression gradually decreases during fetal development.

[0003] ROR1 is lowly expressed or not expressed in normal human tissues, but is highly expressed in a variety of hematological tumors and solid tumors. Hematological tumors that highly express ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid blood cancers. Among solid tumors, tumors that express ROR1 include triple-negative breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, etc., and the expression of ROR1 is also closely related to the progression of the disease and the effect of treatment. Therefore, ROR1 can be used as a specific tumor marker and a potentially attractive target for tumor treatment.

[0004] Summary of the Invention

[0005] The present application provides an isolated antigen-binding protein targeting ROR1. In the present application, the isolated antigen-binding protein can specifically bind to the ROR1 antigen and has good binding activity. The isolated antigen-binding protein described in the present 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 the present application can have a tumor-killing effect.

[0006] On the one hand, the present application provides an isolated antigen-binding protein that can bind to ROR1 (receptor tyrosine kinase orphan receptor 1), wherein the isolated antigen protein comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 3; or the amino acid sequence of the CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 13; or the amino acid sequence of the CDR1 is shown in SEQ ID NO: 19, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 20, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 21.

[0007] In certain embodiments, the isolated antigen binding protein is a single domain antibody.

[0008] In certain embodiments, the isolated antigen binding protein comprises a VHH.

[0009] In certain embodiments, the amino acid sequence of the VHH is shown in SEQ ID NO:8, SEQ ID NO:18 or SEQ ID NO:25.

[0010] In certain embodiments, the isolated antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0011] 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.

[0012] In certain embodiments, the isolated antigen binding protein comprises at least one VHH fragment.

[0013] In certain embodiments, the isolated antigen binding protein comprises two VHH fragments.

[0014] In certain embodiments, the isolated antigen binding protein further comprises an Fc region.

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

[0016] In certain embodiments, the Fc region is derived from a human IgG Fc region.

[0017] In certain embodiments, the isolated antigen binding protein has one or more of the following properties:

[0018] (1) can specifically bind to ROR1; and

[0019] (2) Able to bind to ROR1 expressed on the cell surface.

[0020] In another aspect, the present application also provides a chimeric antigen receptor comprising the isolated antigen binding protein described herein.

[0021] In certain embodiments, the chimeric antigen receptor further comprises a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0022] In certain embodiments, the transmembrane domain comprises a transmembrane domain 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.

[0023] In certain embodiments, the transmembrane domain is derived from the transmembrane domain of CD28, and the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 28.

[0024] In certain embodiments, the costimulatory domain comprises a costimulatory domain selected from one or more of the following proteins: 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 of CD83, CD40, and MyD88.

[0025] In certain embodiments, the costimulatory domain is derived from a 4-1BB costimulatory domain, and the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:29.

[0026] In certain embodiments, the intracellular signaling domain comprises an intracellular signaling region 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 PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0027] In certain embodiments, the intracellular signaling domain is a signaling domain derived from CD3ζ, and the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:30.

[0028] In certain embodiments, the chimeric antigen receptor further comprises a hinge region.

[0029] In certain embodiments, the hinge region is derived from the CD28 hinge region.

[0030] In certain embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:27.

[0031] In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:31.

[0032] On the other hand, the present application also provides an immune cell comprising the chimeric antigen receptor described in the present application.

[0033] In certain embodiments, the immune cells are selected from the group consisting of: T cells, NK cells, iNKT cells, CIK cells, or γδT cells.

[0034] On the other hand, the present application also provides a drug molecule comprising the isolated antigen-binding protein or the chimeric antigen receptor.

[0035] On the other hand, the present application also provides a nucleic acid molecule encoding the isolated antigen-binding protein or the chimeric antigen receptor.

[0036] On the other hand, the present application also provides a vector comprising the nucleic acid molecule.

[0037] On the other hand, the present application also provides a cell comprising the nucleic acid molecule and / or the vector.

[0038] On the other hand, the present application also 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.

[0039] On the other hand, the present application also provides a kit comprising the isolated antigen binding protein or the chimeric antigen receptor, wherein the kit is capable of detecting the presence and / or content of ROR1 in a sample.

[0040] On the other hand, the present application also provides the 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 preparation of a drug for preventing and / or treating ROR1-related diseases and / or conditions.

[0041] In certain embodiments, the disease and / or condition comprises a tumor.

[0042] In certain embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0043] In certain embodiments, the tumor comprises a ROR1 -positive tumor.

[0044] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0046] FIG1 shows the ROR1-specific antibody titers in pre- and post-immunization alpaca sera collected on days 1 to 5 after immunization as described in the present application.

[0047] Figure 2 shows the size of the sequences amplified from the cDNA library as described in this application. Figure 2A shows that the first PCR produces two different groups of amplicons with molecular weights of approximately 0.7 kb and 0.9 kb. Figure 2B shows that the 0.7 kb amplicon is used as a template for amplification to obtain a DNA fragment with a fragment size of approximately 400 bp.

[0048] FIG3 shows the plates used for counting colony numbers and estimating library size by serial dilution in the plates as described in the present application.

[0049] FIG4 shows the phylogenetic analysis of 90 sequences in the library described in this application.

[0050] FIG5 shows the plates used for three rounds of continuous screening of ROR1-specific antigen-binding proteins as described in the present application.

[0051] Figure 6 shows the ELISA results described in this application. Figure 6A shows the OD450 values ​​of the screened sequences 5-26, Figure 6B shows the OD450 values ​​of the screened sequences 4-92, and Figure 6C shows the OD450 values ​​of VHH1, VHH2, and anti-ROR1 mAb.

[0052] FIG7 shows the SDS-PAGE results of the purified antigen-binding protein described in the present application, FIG7A shows the SDS-PAGE results of 5-26, and FIG7B shows the SDS-PAGE results of 4-92.

[0053] Figures 8A-8C show the results of the purified antigen-binding proteins described in the present application specifically binding to the recombinant ROR1 protein. Figure 8A shows the results of the antigen-binding proteins 5-26 after screening, Figure 8B shows the results of the antigen-binding proteins 4-92 after screening, and Figure 8C shows the binding effects of the negative control and positive control.

[0054] Figures 9A-9B show the specificity verification results of the antigen binding proteins described in this application, Figure 9A shows the results of 5-26, and Figure 9B shows the results of 4-92.

[0055] Figures 10A-10B show the flow cytometry results of the antigen binding protein described in the present application binding to A549 cells expressing ROR1, Figure 10A shows the result of 5-26, and Figure 10B shows the result of 4-92.

[0056] Figures 11A-11B show the flow cytometry results of the antigen binding protein described in the present application binding to MB231 cells expressing ROR1, Figure 11A shows the results of 5-26, and Figure 11B shows the results of 4-92.

[0057] Figures 12A-12B show the flow cytometry results of the antigen binding protein described in the present application binding to JeKo-1 cells expressing ROR1, Figure 12A shows the results of 5-26, and Figure 12B shows the results of 4-92.

[0058] Figures 13A-13D show the flow cytometry results of humanized ROR1 single domain antibodies binding to MEC cells, JeKo-1 cells, A549 cells and MB231 cells expressing ROR1; Figure 13A shows the flow cytometry results of binding to MEC cells expressing ROR1, Figure 13B shows the flow cytometry results of binding to JeKo-1 cells expressing ROR1, Figure 13C shows the flow cytometry results of binding to A549 cells expressing ROR1, and Figure 13D shows the flow cytometry results of binding to MB231 cells expressing ROR1.

[0059] Figure 14 shows the structural design of the ROR1 nano-chimeric antigen receptor.

[0060] Figure 15 shows that activated T cells can express high levels of CAR constructs using ROR1 protein.

[0061] FIG16 shows the proportion of target cells surviving in the co-culture system after 24 hours when the initial effector-target ratio is 1:1.

[0062] FIG17 shows the secretion levels of γ-interferon and IL2 in the supernatant of the co-culture system.

[0063] Figures 18A-18C show that the ROR1 nano-chimeric antigen receptor has a strong specific killing ability. DETAILED DESCRIPTION

[0064] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0065] Definition of terms

[0066] In the present application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen 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, etc., as long as they exhibit the desired antigen binding activity. The "isolated antigen binding protein" of the present application may comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes the binding of the antigen binding portion to the antigen.

[0067] As used herein, the term "single-domain antibody" generally refers to a fragment comprising a single variable domain of an antibody that can selectively bind to a specific antigen. The single-domain antibody described herein may comprise a single heavy chain variable domain, and the single-domain antibody described herein may comprise an antigen-binding fragment, which may comprise a VHH.

[0068] In this application, the term "polypeptide" generally refers to a polymer of amino acid residues. The term is also applicable to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term can also include amino acid polymers modified, for example, by adding sugar residues to form glycoproteins or by phosphorylation. Polypeptides can be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and can include molecules with the amino acid sequence of a native protein or molecules with one or more amino acids deleted, added and / or substituted from the native sequence. The term "polypeptide" can include an antigen binding fragment, an antibody, or a sequence with one or more amino acids deleted, added and / or substituted from an antigen binding fragment.

[0069] In this application, the term "nucleic acid" generally refers to a polymer of nucleotides (such as ribonucleotides or deoxyribonucleotides) and can include naturally occurring (adenine, guanine, cytosine, uracil and thymine), non-naturally occurring and modified nucleic acids. The term "nucleic acid" can include genes, cDNA or mRNA. For example, nucleic acid molecules can be synthetic (such as chemically synthesized) or recombinant. Nucleic acid can include nucleic acids containing analogs or derivatives of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Nucleic acid sequences can also include conservatively modified variants thereof (such as degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences and sequences clearly indicated. The term is not limited by the length of the polymer. Nucleic acid can be single-stranded or double-stranded and generally contain a 5'-3' phosphodiester bond, and nucleotide analogs can have other connections.

[0070] In this application, the term "constant region" generally refers to the sum of the domains of an antibody excluding the variable region. The constant region is not directly involved in the binding of an antigen, but displays different effector functions. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies are divided into the following classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into classes such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of antibodies are referred to as α, δ, ε, γ, and μ, respectively.

[0071] As used herein, the term "antigen-binding fragment" generally refers to an immunoglobulin or antibody polypeptide fragment that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which it is derived) for antigen binding (i.e., specific binding). 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.

[0072] In this application, the term "variable region" or "variable domain" generally refers to a portion of an antibody light chain and / or heavy chain, generally including approximately 120-130 amino acid residues at the amino terminal end of the heavy chain and approximately 100-110 amino acid residues at the amino terminal end of the light chain. Variable regions generally differ widely in amino acid sequence, even among antibodies of the same species. The variable region of an antibody can determine the binding and specificity of each specific antibody for its specific antigen. The variability of the sequence is concentrated in regions called complementary determining regions (CDRs), while the more conserved regions in the variable region are called framework regions (FRs). The CDRs of the light and heavy chains may contain amino acids that are largely responsible for the direct interaction of the antibody with the antigen.

[0073] In this application, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds to an antigen or target. For example, a hinge region is included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may include a signal peptide. Binding of the extracellular domain of CAR to the target antigen on the surface of the target cell causes CAR clustering and transmits the activation stimulus to the CAR-containing cell. CAR redirects the specificity of the immune effector cell and triggers proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate cell death of cells expressing the target antigen in a manner independent of major histocompatibility (MHC).

[0074] In the present application, the term "intracellular domain" refers to an intracellular domain comprising any truncated portion sufficient to transduce an activation signal. The intracellular domain may include an intracellular signal region and / or a costimulatory signal region. The term "intracellular signal region" refers to an intracellular region that can produce a signal promoting the immune effector function of CAR cells (e.g., CART cells or NK cells expressing CAR). For example, the intracellular signal region may include an intracellular signal region 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 PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and at least one domain comprising an ITAM. For example, the intracellular signal region may be a signal transduction domain derived from CD3ζ. The term "costimulatory signal region" refers to a part of the CAR that can transduce effector signals in the intracellular signal region. For example, the costimulatory signal region can include an intracellular costimulatory signal region 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 of CD83, CD40, and MyD88. For example, the costimulatory signal region can be an intracellular costimulatory signal region derived from 4-1BB.

[0075] In this application, the term "transmembrane domain" generally refers to a domain of a peptide, polypeptide or protein that is capable of crossing the plasma membrane of a cell. These domains can be used to anchor the extracellular domain to the cell membrane. For example, the membrane-spanning region can include the membrane-spanning domain 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 membrane-spanning domain can be derived from the membrane-spanning domain of CD8.

[0076] In this application, the term "signal peptide" generally refers to a leader sequence at the amino terminus (N-terminus) of a nascent CAR protein, which guides the nascent protein to the endoplasmic reticulum and subsequent surface expression during or after translation. For example, the signal peptide is derived from the signal peptide of a CD8 protein.

[0077] In this application, the term "drug molecule" generally encompasses any drug form 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 combination of different drug forms. For example, the drug molecule may include a small molecule drug.

[0078] In this application, the term "immune effector cell" generally refers to an immune cell that participates in an immune response and exercises an effector function. For example, the exercise of effector function can include removing foreign antigens or promoting immune effector responses, etc. For example, immune effector cells can 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 can be T cells.

[0079] In this application, the term "host cell" generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector comprising a nucleic acid molecule as described herein, or that is capable of expressing a fusion protein or antigen-binding fragment thereof as described herein. The cell may include progeny of a single cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but may be capable of expressing the antigen-binding protein as described herein. The cell can be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell or a myeloma cell).

[0080] In the present application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, and the nucleic acid molecule to be inserted is transferred to a cell (e.g., a host cell) and / or between cells. The vector may include a vector primarily for inserting DNA or RNA into a cell, a vector primarily for replicating DNA or RNA, and a vector primarily for the expression of transcription and / or translation of DNA or RNA. The vector also includes a vector with multiple above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell. Typically, the vector may produce an expression product of interest by cultivating a suitable cell comprising the vector. In the present application, the vector may be a plasmid.

[0081] In this application, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule can 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 antigen-binding protein and / or a chimeric antigen receptor. For example, the nucleic acid molecule can include a promoter. For example, the promoter can be a constitutive promoter.

[0082] In this application, the term "pharmaceutically acceptable" generally refers to non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations may conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, supplementary immunopotentiators such as adjuvants and cytokines and optionally other therapeutic agents such as chemotherapeutic agents.

[0083] In this application, the term "complementarity determining region" or the term "CDR" generally refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy chain and / or light chain, and the 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 that appear in a single variable region that is capable of binding to an 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)) not only provides a clear residue numbering system that can be applied to any variable region of an antibody, but also provides the precise residue boundaries that define 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)) found that certain sub-segments within the Kabat CDRs adopt nearly identical peptide backbone conformations despite having great diversity at the amino acid sequence level. These sub-segments are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the 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 above systems but will still overlap with Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental findings that specific residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. For the CDRs described herein, the IMGT delineation scheme may be used.

[0084] In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, which may be a human patient. For example, the pharmaceutical composition described herein may include the antigen-binding proteins described herein, the immunoconjugates described herein, the nucleic acid molecules described herein, the vectors described herein and / or the cells described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may also include a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention may include, but is not limited to, liquid, frozen and lyophilized compositions.

[0085] In this application, the term "ROR1" generally refers to the receptor tyrosine kinase orphan receptor 1. In this application, the term encompasses full-length ROR1 and functionally active fragments or variants thereof. For example, the term can include the Ig-like domain and / or coiled-coil domain of ROR1. For example, the term can include amino acids 30-305 of ROR1. For example, the sequence of the full-length human ROR1 antigen can be found under GeneBank accession number Q01973.

[0086] The proteins described in this application may also include functional variants, derivatives, analogs, homologs and fragments thereof.

[0087] The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a naturally occurring sequence or encoded by a nucleotide sequence substantially identical to that of a naturally occurring sequence and 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 particular 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 addition, deletion, substitution, modification, replacement and / or variation of at least one amino acid residue and / or nucleotide residue present in a naturally occurring protein and / or polynucleotide, as long as the original functional activity is retained.

[0088] In the present application, the term "derivative" generally refers to any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues in the polypeptide or polynucleotide of the present application, as long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

[0089] In this application, the term "analog" generally refers to a polypeptide or polynucleotide and includes any mimetic of the polypeptide or polynucleotide, ie, a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.

[0090] Generally, amino acid substitutions can be made, such as at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions, as long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs.

[0091] The proteins or polypeptides used in the present application may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues, as 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 containing non-charged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0092] Detailed Description of the Invention

[0093] Isolated antigen binding protein

[0094] In one aspect, the present application provides an isolated antigen-binding protein that can bind to ROR1. For example, the binding can be specific. In the present application, the isolated antigen-binding protein can be used to bind to ROR1 and can be applied in various fields such as antibody drug development, bispecific antibody drug development, and chimeric antigen receptor development.

[0095] In the present application, the isolated antigen-binding protein may comprise at least one CDR in the 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 shown 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 a HCDR sequence obtained by dividing using any method. As long as the VH sequence is identical to the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:18, or SEQ ID NO:25, the HCDR sequence obtained by dividing using any method is within the scope of protection of the present application. For example, CDRs may be divided using CCG, Kabat, Chothia, IMGT, or a combination of Kabat / Chothia. In certain embodiments, CDRs may be divided using IMGT.

[0096] In the present application, the isolated antigen-binding protein may comprise 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.

[0097] In the present application, the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 20.

[0098] In the present application, the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 19.

[0099] 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.

[0100] 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.

[0101] 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 connected 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.

[0102] In the present application, the isolated antigen-binding protein may comprise H-FR2, the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 15 or SEQ ID NO: 22.

[0103] In the present application, the isolated antigen-binding protein may comprise H-FR3, the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 23.

[0104] In the present application, the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 may be directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 17 or SEQ ID NO: 24.

[0105] In the present application, the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3, and H-FR4. For example, 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. For example, H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:14, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:15, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:16, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:17. For example, 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:22, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:23, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:24.

[0106] 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 the HCDR3. In the present application, the direct or indirect linkage may be through intermolecular forces or through a linker.

[0107] 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.

[0108] 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, e.g., a human IgG Fc region; e.g., an IgG1 Fc region; e.g., an IgG4 Fc region. For example, the Fc fragment may include a variant thereof, wherein the variant may include one or more amino acids in the amino acid sequence of the Fc fragment having been substituted, deleted, and / or added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; or homologs thereof can be encompassed, which can be amino acid sequences having at least about 85% (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) sequence identity with the amino acid sequence of the Fc fragment.

[0109] In the present application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof.

[0110] In certain embodiments, the isolated antigen-binding protein, wherein the antigen-binding fragment can be selected from the following group: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0111] In the present application, the isolated antigen-binding protein, wherein the antibody can be selected from the following group: monoclonal antibody, single-chain antibody, chimeric antibody, humanized antibody and fully human antibody.

[0112] Single domain antibodies

[0113] In the present application, the isolated antigen-binding protein may be a single domain antibody.

[0114] In the present application, the single-domain antibody may comprise 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 comprise the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 18 or SEQ ID NO: 25.

[0115] In the present application, the single-domain antibody may comprise 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.

[0116] In the present application, the single-domain antibody may comprise CDR2, and the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 20.

[0117] In the present application, the single-domain antibody may comprise CDR1, and the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 19.

[0118] In the present application, the single-domain antibody may comprise CDR1, CDR2, and CDR3. For example, the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3. For example, the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 20, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 21.

[0119] In the present application, the single-domain antibody may comprise FR1, the C-terminus of the FR1 may be directly or indirectly connected to the N-terminus of the CDR1, and the FR1 may comprise the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 14.

[0120] In the present application, the single-domain antibody may comprise FR2, the FR2 may be located between the CDR1 and the CDR2, and the FR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 15 or SEQ ID NO: 22.

[0121] In the present application, the single-domain antibody may comprise FR3, the FR3 may be located between the CDR2 and the CDR3, and the FR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 23.

[0122] In the present application, the single-domain antibody may comprise FR4, the N-terminus of the FR4 may be directly or indirectly connected to the C-terminus of the CDR3, and the FR4 may comprise the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 17 or SEQ ID NO: 24.

[0123] 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 shown in SEQ ID NO: 4, the FR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, the FR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, and the FR4 may comprise the amino acid sequence shown in SEQ ID NO: 7. For example, the FR1 may comprise the amino acid sequence shown in SEQ ID NO: 14, the FR2 may comprise the amino acid sequence shown in SEQ ID NO: 15, the FR3 may comprise the amino acid sequence shown in SEQ ID NO: 16, and the FR4 may comprise the amino acid sequence shown in SEQ ID NO: 17. For example, the FR1 may comprise the amino acid sequence shown in SEQ ID NO: 4, the FR2 may comprise the amino acid sequence shown in SEQ ID NO: 22, the FR3 may comprise the amino acid sequence shown in SEQ ID NO: 23, and the FR4 may comprise the amino acid sequence shown in SEQ ID NO: 24.

[0124] In the present application, the single-domain antibody may comprise CDR1, CDR2, CDR3, FR1, FR2, FR3 and / or FR4, wherein the C-terminus of the FR1 is directly or indirectly connected to the N-terminus of the 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 the FR4 is directly or indirectly connected to the C-terminus of the CDR3. In the present application, the direct or indirect connection may be connected by intermolecular forces, or may be connected by a linker.

[0125] 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.

[0126] 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 the amino acid sequence of the Fc fragment after substitution, deletion and / or addition of one or more amino acids. For example, 1-30, 1-20 or 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; or it may encompass a homolog thereof, which may be an amino acid sequence having at least about 85% (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) sequence homology with the amino acid sequence of the Fc fragment.

[0127] Chimeric antigen receptor and immune cell containing the same

[0128] In another aspect, the present application provides a chimeric antigen receptor comprising an antigen binding domain. For example, the antigen binding domain may comprise the isolated antigen binding protein described herein.

[0129] 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 shown in SEQ ID NO: 1, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3. For example, the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 13. For example, the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 21.

[0130] In the present application, the chimeric antigen receptor may include a costimulatory signal region that may provide a stimulation signal. For example, the costimulatory signal region may include an intracellular costimulatory signal region 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 of CD83, CD40, and MyD88.

[0131] In the present application, the costimulatory signal region can be an intracellular costimulatory signal region derived from 4-1BB. For example, the costimulatory signal region can include the amino acid sequence shown in SEQ ID NO:29. In the present application, the CAR may include an intracellular signal region, and the intracellular signal transduction domain can transmit the activation signal to the interior of the cell. For example, the intracellular signal region can include an intracellular signal region 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 PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and other domains comprising at least one ITAM.

[0132] For example, the intracellular signaling region can be a signaling domain derived from CD3ζ. For example, the intracellular signaling region can comprise the amino acid sequence shown in SEQ ID NO:30.

[0133] In the present application, the CAR may include a transmembrane domain, which is a sequence in a cell surface protein that spans the cell membrane, for example, it may include a hydrophobic alpha helix. The transmembrane domain may be derived from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix. For example, the transmembrane domain can comprise 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.

[0134] For example, the transmembrane region can be a transmembrane domain derived from CD28. For example, the transmembrane domain can comprise the amino acid sequence shown in SEQ ID NO: 28.

[0135] In the present application, the chimeric antigen receptor may further comprise a hinge region, which may be located between the extracellular targeting portion and the transmembrane domain. 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.

[0136] In the present application, the hinge region may be a hinge region derived from CD28. For example, the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0137] 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 shown in SEQ ID NO: 26.

[0138] In the present application, the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO: 31.

[0139] On the other hand, the present application also provides an immune cell comprising and / or expressing the chimeric antigen receptor described in the present application. For example, the immune cell 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.

[0140] In certain embodiments, the cell may be a T cell.

[0141] Polypeptides, drug molecules, nucleic acids, vectors, cells, and preparation methods

[0142] On the other hand, the present application provides a polypeptide, which may comprise the isolated antigen-binding protein or the chimeric antigen receptor.

[0143] On the other hand, the present application provides a drug molecule, which may comprise the isolated antigen-binding protein or the chimeric antigen receptor.

[0144] In another aspect, the present application provides one or more isolated nucleic acid molecules that can encode the isolated antigen-binding protein, the chimeric antigen receptor, or the polypeptide described herein. For example, each of the one or more nucleic acid molecules can encode the entire isolated antigen-binding protein, the chimeric antigen receptor, or the polypeptide, or a portion thereof. The nucleic acid molecules described herein can be isolated. In the present application, nucleic acids encoding the isolated antigen-binding protein, the chimeric antigen receptor, or the polypeptide can be prepared by various methods known in the art.

[0145] On the other hand, the application provides one or more vectors, which comprise one or more nucleic acid molecules described herein. One or more nucleic acid molecules can be included in each vector. In addition, other genes can also be included in the vector, such as marker genes that allow the selection of the vector in a suitable host cell and under appropriate conditions. In addition, the vector can contain multiple elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector can also contain a replication initiation site. In addition, the vector can include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.

[0146] On the other hand, the application provides a kind of cell, and the cell can comprise one or more nucleic acid molecules and / or one or more vectors described in the application.In certain embodiments, every kind or each cell can comprise one or a nucleic acid molecule or vector described in the application.In certain embodiments, every kind or each cell can comprise multiple (for example, 2 or more) or multiple (for example, 2 kinds or more) nucleic acid molecules or vector described in the application.For example, the vector described in the application can be introduced into the cell, for example prokaryotic cell (for example, bacterial cell), CHO cell, NS / 0 cell, HEK293 cell, or other eukaryotic cells, such as cell, fungus or yeast cell etc. from plant.The vector described in the application can be introduced into the cell by methods known in the art, for example electroporation, lipofectine transfection, lipofectamin transfection etc.

[0147] In another aspect, the present application provides methods for preparing the isolated antigen-binding protein, chimeric antigen receptor, or polypeptide described herein. The methods may include culturing the cells described herein under conditions that allow for expression of the isolated antigen-binding protein, chimeric antigen receptor, or polypeptide. For example, the methods may be performed using an appropriate culture medium, at an appropriate temperature and for an appropriate time, as is known to those skilled in the art.

[0148] Pharmaceutical composition, application

[0149] On the other hand, 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.

[0150] 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 counter ion, a metal complex and / or a nonionic surfactant, etc.

[0151] 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, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous reservoir. The pharmaceutical composition can be used to inhibit tumor growth. For example, the pharmaceutical composition of the present application can inhibit or delay the development or progression of a disease, and / or can alleviate and / or stabilize the disease state.

[0152] The pharmaceutical composition described herein can include a preventive and / or therapeutically effective amount of the isolated antigen-binding proteins. The preventive and / or therapeutically effective amount is the dosage required to prevent and / or treat (at least partially treat) a disease or condition and / or any complication thereof in a subject suffering from or having a risk of developing the disease.

[0153] On the other hand, the present application provides a method for detecting or measuring ROR1, which may include using the isolated antigen binding protein, the chimeric antigen receptor, or the polypeptide.

[0154] On the other hand, the present application provides a kit that can be used to detect or determine ROR1 in a sample, and the kit can include the isolated antigen binding protein, the chimeric antigen receptor, or the polypeptide.

[0155] On the other hand, the present application provides a use of 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 in the preparation of a drug for preventing and / or treating a disease and / or condition.

[0156] On the other hand, 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, which are prepared to prevent or treat a disease or condition.

[0157] On the other hand, the present application provides a method for preventing or treating a disease, which comprises administering 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 described herein to a subject in need thereof.

[0158] In the present application, the diseases and / or disorders in the use may be caused or mediated by abnormal expression of ROR1.

[0159] In the present application, the disease and / or condition 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.

[0160] For example, the ROR1-positive tumor can be selected from breast cancer, ovarian cancer, melanoma, pancreatic cancer, lung cancer, leukemia, lymphoma, and / or thyroid cancer.

[0161] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0162] Example

[0163] Example 1 Animal Immunization

[0164] The experimental process is carried out according to the following steps:

[0165] (1) Immunize a 2.5-year-old unimmunized female alpaca five times at two-week intervals with freshly prepared immunogen (see Table 1. Immunization schedule). The immunogen was divided into several portions (each portion was used for one immunization) and stored at -80°C;

[0166] (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 (the second to fifth), 300 μg of immunogen was mixed with an equal volume of Freund's incomplete adjuvant (CFA) to prepare an emulsion (all vaccination experiments were approved by the local ethics committee);

[0167] (3) Before each immunogen administration (1st to 5th time), 5 ml of jugular vein blood sample was collected and serum was recovered as the post-immunization IgG sample for antigen-specific antibody titer;

[0168] (4) 7 days after the last immunization, 50 ml of blood was collected and Ficoll-Paque TM Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by PLUS Media (GE Healthcare, USA), and PBMCs were lysed in Trizol for subsequent total RNA extraction;

[0169] (5) ELISA was used to compare the titers of pre- and post-immunization sera to measure antigen-induced seroconversion. Each diluted serum sample was tested once at each bleeding time point.

[0170] The experimental results are shown in Figure 1. The immune serum showed a significant reaction to the recombinant ROR1 protein, and the ROR1-specific antibody titer in the sera from the 3rd to the 5th time after immunization reached more than 1 / 100,000.

[0171] Table 1. Immunization schedule (*Due to COVID-19 quarantine at the alpaca farm, the 3rd vaccination was delayed by two weeks)

[0172] Example 2 Establishment of immune phage library

[0173] The experimental process is carried out as follows:

[0174] (1) After isolating PBMC from 50 ml of peripheral blood and extracting total RNA, cDNA was prepared using RNA as a template using commercially available reagents. The preparation system is shown in Table 2.

[0175] (2) VHH sequences were amplified from the cDNA library and transformed into TGI cells. The library size was calculated based on the number of colonies in a series of dilutions of the electroporated E. coli cell suspension on a 90 mm plate;

[0176] (3) Perform colony sequencing and bioinformatics analysis.

[0177] The experimental results showed that the first PCR produced two different amplicons with molecular weights of approximately 0.7 kb and 0.9 kb (Figure 2A). The 0.7 kb amplicon was used as a template to amplify VHH, resulting in a DNA fragment of approximately 400 bp (Figure 2B). The library size after amplification was 1.4 × 10 8 ( Figure 3 ), phylogenetic analysis was performed on 90 sequences in the library, and 96 single colonies were randomly picked for colony sequencing. The proportion of VHH correctly inserted into phage was 93.8% (90 / 96), and the proportion of specific VHH sequences was 96.7% (87 / 90) ( Figure 4 ).

[0178] Table 2. Yields of total RNA, cDNA, and PCR products

[0179] Example 3 Screening of antigen-specific antigen-binding proteins

[0180] By monitoring the number of colonies growing on the plates, three rounds of screening were performed to obtain ROR1-specific antigen-binding proteins (Figure 5). The number of colonies in the ROR1-coated group increased after each round of screening, 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.

[0181] Example 4 Antigen Binding Protein Identification and Verification

[0182] (1) Specific antigen-binding proteins after three rounds of screening were verified by ELISA using periplasmic extracts to obtain antigen-positive clones;

[0183] (2) DNA sequences of positive clones are then obtained by colony sequencing to identify specific sequences;

[0184] (3) Recombinant production and purification of specific sequences, and testing of purity to determine whether they meet the requirements of subsequent testing;

[0185] (4) Further detect the affinity and specificity of the antigen binding protein to ROR1 protein.

[0186] The experimental results showed that the OD450 values ​​of 5-26 (VHH sequence shown in SEQ ID NO: 8) and 4-92 (VHH sequence shown in SEQ ID NO: 25) were higher than those of 5-09 (VHH sequence shown in SEQ ID NO: 11) and 5-95 (VHH sequence shown in SEQ ID NO: 12) (Figures 6A and 6B). Among them, 5-26 contains the CDR1 shown in SEQ ID NO: 1, the CDR2 shown in SEQ ID NO: 2, and the CDR3 shown in SEQ ID NO: 3; 4-92 contains the CDR1 shown in SEQ ID NO: 19, the CDR2 shown in SEQ ID NO: 20, and the CDR3 shown in SEQ ID NO: 21. Two non-specific VHHs (VHH1: sequence shown in SEQ ID NO: 9; VHH2: sequence shown in SEQ ID NO: 10) did not bind to the recombinant ROR1 protein, while the anti-ROR1 mAb bound well to the recombinant ROR1 protein (Figure 6C). The experimental results further showed that 5-26, 4-92, 5-09 and 5-95 specifically bound to the recombinant ROR1 protein, but the binding ability of 5-26 and 4-92 was significantly better than that of the other two groups.

[0187] SDS-PAGE results after purification showed that the purity of the antigen-binding proteins (approximately 0.5 mg each) exceeded 90%, meeting the requirements of subsequent functional testing (Figures 7A-7B). The purified antigen-binding proteins 5-26, 4-92, 5-9, and 5-95 specifically bound to the recombinant ROR1 protein (Figures 8A-8B). The negative controls VHH1 and VHH2 did not bind to the ROR1 protein, while the positive control did (Figure 8C). These experimental results indicate that compared to 5-9 and 5-95, 5-26 and 4-92 can bind to the ROR1 protein at higher concentrations and with good specificity at multiple dilutions.

[0188] At the same time, specificity evaluation showed that the purified antigen-binding proteins 5-26, 4-92, 5-9, and 5-95 only bound to recombinant ROR1 protein and did not bind 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) only binds to recombinant ROR1 protein. Anti-his mAb (Sinobiological, #105327-MM02T-H) can bind to all three his-tagged proteins (PD-L1, RBD, ROR1) (Figures 9A-9B). The concentration of the purified antigen-binding proteins used in the experiment was 3μg / ml. The C-terminus of the 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 show that 5-26 and 4-92 have good specificity for ROR1 protein and basically do not bind to PD-L1 and RBD.

[0189] Anti-ROR 1 mAb: ROR1 mouse monoclonal antibody, from Proteintech (catalog No.: 66923-1-Ig), molecular weight 130 kDa, can be used in FC, IF, IHC, WB, ELISA and other applications.

[0190] Example 5 Detection of the ability of antigen binding proteins to bind to cell lines expressing ROR1

[0191] ROR1 was expressed in A549, MB231, and JeKo-1 cells at 1.5-2.0 × 10 cells per well. 5The cells were incubated with the antigen-binding protein at a concentration of 1 μg / ml at 4°C for 30 minutes and stained with anti-HA APC. Flow cytometric analysis showed that in A549 cells, the binding rate of 5-26 reached 97.0%, 5-9 18.5%, and 5-95 11.8% ( FIG10A ), and the binding rate of 4-92 reached 34.6%, 5-9 18.5%, and 5-95 11.8% ( FIG10B ). In MB231 cells, the binding rate of 5-26 reached 96.8%, 5-9 61.7%, and 5-95 53.3% ( FIG11A ), and the binding rate of 4-92 reached 74.3%, 5-9 61.7%, and 5-95 53.3% ( FIG11B ). In JeKo-1 cells, 5-26 bound to 99.7% of cells, 5-9 to 1.90%, and 5-95 to 1.18% (Figure 12A). 4-92 bound to 2.96% of cells, 5-9 to 1.90%, and 5-95 to 1.18% (Figure 12B). These results suggest that 5-26 and 4-92 have stronger binding abilities to ROR1-expressing cell lines than 5-9 and 5-95.

[0192] Example 6 Detection of the ability of humanized antigen binding proteins to bind to cell lines expressing ROR1

[0193] The sequence of 5-26 was humanized to obtain the amino acid sequence of hu5-26. The amino acid sequence of CDR1 of hu5-26 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR3 is shown in SEQ ID NO: 13, the amino acid sequence of FR1 is shown in SEQ ID NO: 14, the amino acid sequence of FR2 is shown in SEQ ID NO: 15, the amino acid sequence of FR3 is shown in SEQ ID NO: 16, the amino acid sequence of FR4 is shown in SEQ ID NO: 17, and the amino acid sequence of VHH is shown in SEQ ID NO: 18. Hu5-26 was connected to IgG Fc to obtain a hu5-26 single-domain antibody. The ability of hu5-26 to bind to different cell lines (MEC) expressing ROR1 was detected using the method in Example 5. The experimental results are shown in Figures 13A-13D. The experimental results showed that in MEC cells, the ratio of hu5-26 binding cells reached 99.6% (Figure 13A); in JeKo-1 cells, the ratio of hu5-26 binding cells reached 92.5% (Figure 13B); in A549 cells, the ratio of hu5-26 binding cells reached 80.1% (Figure 13C); in MB231 cells, the ratio of hu5-26 binding cells reached 90.0% (Figure 13D).

[0194] In the present application, hu5-26 has a stronger ROR1 binding ability than other humanized antibodies and has a stronger affinity for almost all ROR1-positive cells.

[0195] Example 7 Preparation and Expression of ROR1 Nano-chimeric Antigen Receptor

[0196] Cell culture

[0197] HEK293T, MEC1 and MDA-MB-231 cell lines were obtained from ATCC, USA. MEC1-ROR1 cell line is a ROR1 stable expression strain generated in-house. HEK293T cell line was maintained in full culture medium (DMEM containing 10% heat-inactivated FBS, 100U / mL penicillin / streptomycin and 2mM L-glutamine). MDA-MB-231, MEC1 and MEC1-ROR1 cell lines were maintained in full culture medium (IMDM containing 10% heat-inactivated FBS, 100U / mL penicillin / streptomycin and 2mM L-glutamine). PBMCs were isolated from whole blood of healthy donors using Ficoll-Paque and plated at 2x10 7 1 mL of sample was aliquoted at a concentration of 10 cells / mL and stored frozen in a liquid nitrogen tank. The culture medium was heat-inactivated FBS supplemented with 10% DMSO (vol / vol).

[0198] Clone construction

[0199] All relevant gene structures were constructed and synthesized by GeneWiz. The ROR1 nano-chimeric antigen receptor adopts the structure of the second-generation CAR (Figure 14). The gene structure encoding hu5-26ROR1VHH CAR (hereinafter referred to as ROR1VHH CAR) (ROR1VHH CDR1 is shown in SEQ ID NO: 1, CDR2 is shown in SEQ ID NO: 2, CDR3 is shown in SEQ ID NO: 13, VHH is shown in SEQ ID NO: 18, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 26, the amino acid sequence of the hinge region is shown in SEQ ID NO: 27, the amino acid sequence of the transmembrane domain is shown in SEQ ID NO: 28, the amino acid sequence of the costimulatory domain is shown in SEQ ID NO: 29, and the amino acid sequence of the intracellular signaling domain is shown in SEQ ID NO: 30) was inserted into the pALD expression plasmid through the BamHI and SalI cloning sites. After double enzyme digestion verification and sequencing verification, the expression plasmid was amplified in large quantities for lentiviral packaging.

[0200] Lentivirus preparation

[0201] All lentiviruses were prepared from HEK293T cells. Freshly resuspended HEK293T cells were washed in PBS and seeded into 10-cm culture dishes at a cell density of 90%-95% of the dish area. A mixture of lentiviral packaging plasmid and transfection plasmid was mixed with Lipofectamine-3000 and transiently transfected into the seeded HEK293T cells. After 48 hours of culture, the supernatant was collected, filtered through a 0.45 μm filter, and aliquoted into 1 ml tubes for storage at -80°C.

[0202] T cell activation and transduction

[0203] In order to transduce the lentivirus encoding the corresponding structure into PBMC, the frozen PBMC was revived and added to a 24-well plate, and CD3 and CD28-coupled magnetic beads and IL2 (500IU / ml) were added for 24-72 hours of activation. After the activated PBMC cells were distributed into multiple wells of a 24-well plate, the corresponding lentivirus and polybrene (8g / ml) were added. After centrifugation at 2000g for 2 hours in a desktop centrifuge, they were transferred to an incubator at 37°C and 5% CO2 for culture. Among them, the lentivirus was replaced with complete culture medium in the control group, and the other steps were the same. The transfected cells were cultured for 48 hours in X-VIVO-15 cell culture medium containing IL2 (500IU / ml) for subsequent experiments and analysis.

[0204] Flow cytometry

[0205] Flow cytometry was performed using a flow cytometer (Beckman Coulter) in semi-automatic or plate mode, and data were analyzed using FlowJo software. Cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2mM EDTA, pH 7.0) and resuspended to 1-5×10 6 Cells were stained with a fluorophore-containing antibody at a concentration of 10 cells / mL and placed on ice prior to staining. After incubation at 4°C, protected from light, for 40 minutes, 7-AAD was added and staining continued for 5 minutes. After staining, unbound antibodies were washed away with a large amount of FACS buffer. After centrifugation at 300 x g for 3 minutes, the supernatant was removed. After washing and resuspending in FACS buffer, cells were analyzed by flow cytometry.

[0206] Enzyme-linked immunosorbent assay

[0207] The supernatant after co-culture was carefully aspirated after brief centrifugation and used for enzyme-linked immunosorbent assay after appropriate dilution. TMDeluxe Set Human IFN-γ Kit (Biolegend; 430104) and ELISA MAX TM The secretion level of cytokines in the culture medium was measured using the Standard Set Human IL-2 (Biolegend; 431801) kit.

[0208] The construct containing the hu5-26ROR1CAR was successfully cloned and packaged into lentiviral vectors. As shown in Figure 15, 72 hours after lentiviral infection, high levels of CAR expression were detected in activated human PBMCs, and the CAR construct was able to bind to the PE-labeled ROR1 protein.

[0209] Example 8 In vitro functional verification of ROR1 nano-chimeric antigen receptor

[0210] Tumor cell lines

[0211] MEC1 (B-cell chronic lymphocytic leukemia) tumor cells were engineered to stably express the ROR1 antigen. The MDA-MB-231 cell line already expresses the ROR1 antigen. These three tumor cell lines were transfected to express GFP and used in subsequent experiments.

[0212] CAR-T short-term tumor killing experiment (flow cytometry detection method)

[0213] The above structure (ROR1VHH CAR) was transduced into PBMC cells and cultured for 72 hours before the expression of CAR was detected by flow cytometry. The effector cells and target cells were accurately counted and then the effector-target ratio was 1:1 and 1 = 5x10 4 The cells were resuspended in 200 μl of cell culture medium at a density of 100 μl and plated in 96-well plates. After 24 hours, the plates were removed, centrifuged, and the supernatant was used to measure the secretion of interferon-γ and IL-2 using an enzyme-linked immunosorbent assay.

[0214] At the same time, the co-cultured cells were washed with PBS and suspended, and then stained with 7-AAD in the dark for 5 minutes. The proportion of target cells in the surviving cells was detected by flow cytometry.

[0215] CAR-T long-term tumor killing experiment (Incucyte real-time imaging detection)

[0216] The above structure (ROR1VHH CAR) was transduced into PBMC cells and cultured for 72 hours before the expression of CAR was detected by flow cytometry. The effector cells and target cells were accurately counted and then the effector-target ratio was 1:1 and 1=1x10 4Cells were resuspended in 200 μl of cell culture medium at a density of 100 μl and plated in a flat-bottom 96-well plate. The 96-well plate was placed in the IncuCyte for real-time imaging, allowing the 48-hour growth curve of GFP-labeled target cells to be monitored.

[0217] In the flow cytometry short-term killing test, because the target cells are GFP-labeled, the target cells that are still alive in the entire co-culture system can be counted. As shown in Figure 16, after 24 hours, the ROR1VHH CAR group showed a strong killing ability against ROR1-expressing target cells, and the target cell ratio dropped from 50% at 0 hours to 1.925% (MEC-ROR1) and 2.955% (MB231). For target cells that do not express ROR1, there is no significant difference from the control group, showing good specificity.

[0218] At the same time, in the process of killing ROR1-positive target cells, the hu5-26ROR1VHH CAR group can detect higher levels of γ-interferon and IL2 secretion (Figure 17). As shown in Figures 18A-18C, in the IncuCyte real-time imaging experiment, the ROR1VHH CAR group can effectively inhibit the in vitro proliferation of ROR1-positive tumor cells and can basically eliminate target cells within 48 hours (MB231 kills 80%; MEC-ROR kills 90%. Similarly, this tumor killing ability also shows good specificity and has good safety in practical applications.

[0219] 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 changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

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

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

25.

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

4. The isolated antigen-binding protein according to any one of claims 1 to 3, 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. The isolated antigen binding protein according to any one of claims 1 to 4, comprising at least one VHH fragment.

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

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

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

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

10. The isolated antigen-binding protein according to any one of claims 1 to 9, having at least one of the following properties: (1) can specifically bind to ROR1; and (2) Able to bind to ROR1 expressed on the cell surface.

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

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

13. The chimeric antigen receptor of claim 12, wherein the transmembrane domain comprises a transmembrane domain 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 according to claim 13, wherein the transmembrane domain is derived from the transmembrane domain of CD28, and the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

28.

15. The chimeric antigen receptor of any one of claims 12-14, wherein the co-stimulatory domain comprises a co-stimulatory domain selected from one or more of the following proteins: 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 of 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 shown in SEQ ID NO:

29.

17. The chimeric antigen receptor of any one of claims 12-16, wherein the intracellular signaling domain comprises an intracellular signaling region 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 PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising 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 shown in SEQ ID NO:

30.

19. The chimeric antigen receptor according to any one of claims 11-18, 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 shown in SEQ ID NO:

27.

22. The chimeric antigen receptor according to 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 11-22.

24. The immune cell according to claim 23, which is selected from: T cells, NK cells, iNKT cells, CIK cells or γδT cells.

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

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

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. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-10, the immune cell of any one of claims 23-24, 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. A kit 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, wherein the kit is capable of detecting the presence and / or amount of ROR1 in a sample.

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

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

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

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