CN115819592A - ROR 1-targeted antigen binding proteins - Google Patents

Abstract

The present application relates to an isolated antigen binding protein capable of binding ROR1 (receptor tyrosine kinase orphan receptor 1), said isolated antigen protein comprising CDR1, CDR2 and CDR3. The present application also provides the preparation method and application of the isolated antigen-binding protein.

Description

Antigen binding proteins targeting ROR1

technical field

The present application relates to the field of biomedicine, in particular to an antigen-binding protein targeting ROR1.

Background technique

ROR1 is a transmembrane receptor tyrosine kinase protein. Human ROR1 consists of an extracellular immunoglobulin-like domain (Ig), two cysteine-rich domains (FZD), a proximal membrane kringle domain, single transmembrane structure plus an intracellular tyrosine kinase domain (TKD), two serine/threonine rich domains (S/TRD) and a proline rich domain (PRD) composition. Regulates cell division, proliferation, migration, and cell chemotaxis by mediating signaling through multiple signaling pathways. Highly expressed during early embryonic development. With the process of fetal development, the expression of ROR1 gradually decreased.

ROR1 is low or not expressed in human normal tissues, but highly expressed in various hematological and solid tumors. Hematological malignancies that highly express ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid blood cancers. Among solid tumors, ROR1-expressing tumors 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 therapy.

Contents of the invention

The application provides an isolated antigen binding protein targeting ROR1. In this application, the isolated antigen-binding protein can specifically bind ROR1 antigen and has better binding activity. The isolated antigen binding proteins described herein are capable of binding to ROR1 expressed on the surface of cells (eg, A549 cells, MDA MB231 cells, MEC-ROR1 cells, JeKo-1 cells). The isolated antigen-binding protein described in this application can kill tumors.

In one aspect, the application provides an isolated antigen-binding protein capable of binding to ROR1 (receptor tyrosine kinase orphan receptor 1), the isolated antigenic protein comprising CDR1, CDR2 and CDR3, the amino acid of the CDR1 The sequence is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3.

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

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

In certain embodiments, the amino acid sequence of the VHH is shown in SEQ ID NO:8.

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

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

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

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

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

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

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

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.

In another aspect, the present application also provides a drug molecule comprising the isolated antigen-binding protein.

In another aspect, the present application also provides a nucleic acid molecule encoding the isolated antigen-binding protein.

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

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

On the other hand, the present application also provides a pharmaceutical composition comprising the isolated antigen-binding protein, the drug molecule, the nucleic acid molecule, the carrier, and/or the cell, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present application also provides a kit comprising the isolated antigen-binding protein, and the kit is capable of detecting the presence and/or content of ROR1 in a sample.

In another aspect, the present application also provides the use of the isolated antigen-binding protein, the drug molecule, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition in the preparation of a medicament, The medicament is used for preventing and/or treating ROR1-related diseases and/or conditions.

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

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

In certain embodiments, the tumor comprises a ROR1 positive tumor.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows the ROR1-specific antibody titers in alpaca serum before and after immunization collected from the 1st to the 5th day after immunization described in this application.

What Figure 2 shows is the size of the amplified sequence from the cDNA library described in this application. Figure 2A shows that the first PCR produces two groups of different amplicons with molecular weights of about 0.7kb and 0.9kb. The amplicon is amplified as a template to obtain a DNA fragment with a fragment size of about 400 bp.

Figure 3 shows the plate used to estimate the size of the library by counting the number of colonies in the plate by serial dilution as described in the present application.

Figure 4 shows the phylogenetic analysis of 90 sequences in the library described in this application.

Figure 5 shows the plate used for the three rounds of continuous screening of ROR1-specific antigen-binding proteins described in this application.

Figure 6 shows the results of the ELISA described in this application, Figure 6A shows the OD450 value of the sequence after screening, and Figure 6B shows the OD450 value of VHH1, VHH2, and anti-ROR1 mAb.

Figure 7 shows the results of SDS-PAGE of the purified antigen-binding protein described in this application.

Figure 8 shows the results of specific binding of the purified antigen-binding protein described in the present application to the recombinant ROR1 protein, Figure 8A shows the antigen-binding protein after screening, and Figure 8B shows the binding effect of the negative control and positive control.

Figure 9 shows the results of specificity verification of the antigen-binding proteins described in this application.

Figure 10 shows the flow cytometric results of the binding of the antigen binding protein described in this application to A549 cells expressing ROR1.

Figure 11 shows the flow cytometric results of the binding of the antigen binding protein described in this application to MB231 cells expressing ROR1.

Figure 12 shows the flow cytometry results of the binding of the antigen binding protein described in this application to JeKo-1 cells expressing ROR1.

Detailed ways

The implementation of the invention of the present application will be described by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone moiety that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding protein to the antigen. Examples of 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 substances, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity. An "isolated antigen binding protein" of the present application may comprise an antigen-binding moiety and, optionally, a scaffold or framework moiety that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding moiety to antigen.

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

In this application, the term "polypeptide" generally refers to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are an analog or mimetic of the corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers. The term may also include amino acid polymers that are modified, for example, by the addition of sugar residues to form glycoproteins or by phosphorylation. Polypeptides may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of a native protein, or having one or more amino acid deletions, additions, and/or deletions from the native sequence or substituted molecules. The term "polypeptide" may include an antigen-binding fragment, an antibody, or a sequence having deletions, additions and/or substitutions of one or more amino acids of an antigen-binding fragment.

In this application, the term "nucleic acid" generally refers to a polymer of nucleotides (such as 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, cDNA or mRNA. For example, a nucleic acid molecule can be synthetic (eg, chemically synthesized) or recombinant. Nucleic acids can include nucleic acids that contain analogs or derivatives of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. A nucleic acid sequence may also include conservatively modified variants thereof (eg degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as explicitly indicated sequences. The term is not limited by the length of the polymer. Nucleic acids can be single- or double-stranded, and generally contain 5'-3' phosphodiester linkages, and nucleotide analogs can have other linkages.

In the present application, the term "constant region" generally refers to the sum of domains of an antibody other than the variable region. The constant regions are not directly involved in antigen binding, but display distinct effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are classified 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 that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively.

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

In this application, the term "variable region" or "variable domain" generally refers to a part of an antibody light chain and/or heavy chain, generally including about the amino terminal 120-130 amino acids of the heavy chain and the light chain The approximately 100-110 amino-terminal amino acids in . Variable regions typically differ widely in amino acid sequence, even among antibodies of the same species. The variable regions of antibodies can determine the binding and specificity of each particular antibody for its particular antigen. Sequence variability is concentrated in those regions called complementarity determining regions (CDRs), while the more conserved regions of the variable domain are called framework regions (FR). The CDRs of the light and heavy chains may contain within them amino acids that are largely responsible for the direct interaction of the antibody with the antigen.

In this application, the term "drug molecule" may generally include any drug form known in the art. For example, the drug molecule can be a protein. For example, the drug molecule can be a polypeptide. For example, the drug molecule may be a conjugate of different forms of drugs. For example, small molecule drugs may be included in the drug molecules.

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

In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and/or between cells (eg, host cells). The vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having various functions as described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell. Typically, the vector produces the desired expression product by culturing appropriate cells containing the vector. In the present application, the vector may be a plasmid.

In this application, 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. Such formulations may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, supplemental immunopotentiating agents such as adjuvants and cytokines and optionally other therapeutic agents, such as chemotherapeutics.

In this application, the term "complementarity determining region" or the term "CDR" generally refers to a complementarity determining region within the variable sequence of an antibody. In each variable region of the heavy and/or light chain there are 3 CDRs, named CDR1, CDR2 and CDR3 for each variable region. As used herein, a CDR combination may refer to a set of 3 CDRs that occur in a single variable domain capable of binding 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 unambiguous residue numbering applicable to any variable region of an antibody system, also provides precise residue boundaries defining the 3 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 subportions within Kabat CDRs adopt nearly identical Peptide backbone conformations, despite large diversity at the amino acid sequence level. These subdivisions 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 can be referred to as Chothia CDRs, which have overlapping borders with Kabat CDRs. Other boundaries defining CDRs overlapping with Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). In other CDR boundary definitions that may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, although predictions or experimental findings that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding, they can shorten or lengthen. The CDRs described in this article can be defined using the IMGT division method.

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 comprise the antigen binding protein described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the carrier described herein and/or The cells described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations. Acceptable ingredients of the compositions are nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention may include, but are not limited to, liquid, frozen and lyophilized compositions.

In this application, the term "ROR1" generally refers to the receptor tyrosine kinase orphan receptor 1. In the present application, the term covers the full length of ROR1 as well as functionally active fragments or variants thereof. For example, the term may include the Ig-like domain and/or the Frizzled 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 can be described under GeneBank accession number Q01973.

The proteins described in this application may also include functional variants, derivatives, analogs, homologues and fragments thereof.

The term "functional variant" refers to a polypeptide having substantially the same amino acid sequence or encoded by a substantially identical nucleotide sequence as the 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 is one 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 A sequence of endogenous functions. Variant sequences may be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the The original functional activity is sufficient.

In the present application, the term "derivative" generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and and/or added, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

In this application, the term "analogue" generally refers to polypeptides or polynucleotides, including any mimetic of polypeptides or polynucleotides, that is, having at least one endogenous function of the polypeptide or polynucleotide simulated by the mimetic of chemical compounds.

Typically, amino acid substitutions, e.g., at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made so long as the modified sequence remains substantially as desired. activity or ability. Amino acid substitutions may involve the use of non-naturally occurring analogs.

The proteins or polypeptides used in this 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 on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar headgroups with similar hydrophilicity values include aspartic acid. Paragine, Glutamine, Serine, Threonine and Tyrosine.

Detailed description of the invention

isolated antigen binding protein

In one aspect, the application provides an isolated antigen-binding protein, which may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the CDR may be HCDR1, HCDR2 and/or HCDR3. The heavy chain variable region VH may comprise the amino acid sequence shown in SEQ ID NO:8. In the present application, the HCDR sequence of the isolated antigen-binding protein may comprise the HCDR sequence obtained by using any method, as long as the VH sequence is identical to the amino acid sequence shown in SEQ ID NO: 8, the HCDR sequence obtained by using any method All are within the scope of protection of this application. In some implementations, IMGT may be used to divide the CDRs.

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.

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.

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.

In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2 sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.

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.

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 SEQ ID NO : Amino acid sequence shown in 4.

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 SEQ ID NO:5 amino acid sequence.

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 SEQ ID NO:6 amino acid sequence.

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 SEQ ID NO : Amino acid sequence shown in 7.

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

In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and/or H-FR4, and the C-terminus of the H-FR1 is connected to the The N-terminal of the HCDR1 is directly or indirectly connected, 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-terminal of the H-FR4 directly or indirectly connected to the C-terminus of the HCDR3. In the present application, direct or indirect connection may be connected by intermolecular force, or may be connected by a linker.

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.

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, eg, a human IgG Fc region; eg, an IgG1 Fc region; eg an IgG4 Fc region. For example, the Fc fragment may include variants thereof, which may include substitutions, deletions and/or additions of one or more amino acids to the amino acid sequence of the Fc fragment. For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; or may encompass homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%) identical to the amino acid sequence of the Fc fragment %, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology of amino acid sequences.

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

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

In the present application, the isolated antigen-binding protein, wherein the antibody can be selected from the group consisting of monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody and fully human antibody.

single domain antibody

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

In the present application, the single domain antibody may comprise at least one CDR in the variable region VHH of the heavy chain of the antibody, 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.

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.

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.

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.

In the present application, the single domain antibody may comprise CDR1, CDR2 and CDR3, 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.

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.

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.

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.

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.

In the present application, the single domain antibody may comprise FR1, FR2, FR3 and FR4, the FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, and 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.

In the present application, the single domain antibody may comprise CDR1, CDR2, CDR3, FR1, FR2, FR3 and/or FR4, and the C-terminus of the FR1 is directly or indirectly connected to the N-terminus of the CDR1, and the FR2 Located between the CDR1 and the CDR2, the FR3 is located between the CDR2 and the CDR3, and the N-terminal of the FR4 is directly or indirectly connected to the C-terminal of the CDR3. In the present application, direct or indirect connection may be connected by intermolecular force, or may be connected by a linker.

In this 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.

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

Polypeptides, drug molecules, nucleic acids, vectors, cells, preparation methods

On the other hand, the present application provides a polypeptide, which may comprise the isolated antigen-binding protein.

On the other hand, the application provides a drug molecule, which may comprise the isolated antigen-binding protein

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

In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may contain one or more such nucleic acid molecules. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may contain a variety of expression-controlling elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. In addition, the vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.

In another aspect, the present application provides a cell, which may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein. In certain embodiments, each or each cell may comprise one or more of the nucleic acid molecules or vectors described herein. In certain embodiments, each or each cell may comprise a plurality (eg, 2 or more) or a plurality (eg, 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into said cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS/0 cells, HEK293 cells, or other eukaryotic cells, such as cells from plants, fungi or yeast cells etc. The vectors described in this application can be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.

In another aspect, the present application provides methods for preparing the isolated antigen-binding protein or the polypeptide described herein. The method may comprise culturing the cells described herein under conditions such that the isolated antigen binding protein or the polypeptide is expressed. For example, by using appropriate medium, appropriate temperature and incubation time, etc., these methods are understood by those of ordinary skill in the art.

pharmaceutical composition, application

In another aspect, the present application provides a pharmaceutical composition, which comprises the isolated antigen-binding protein, the polypeptide, the drug molecule, the nucleic acid molecule, the carrier, the cells, and/or a pharmaceutically acceptable carrier.

For example, the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or nonionic Surfactant etc.

In the present application, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at tumor site, inhalation, rectal administration, vaginal administration, transdermal administration Administration or via a subcutaneous depot. 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.

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

In another aspect, the present application provides a method for detecting or measuring ROR1, which may include using the isolated antigen-binding protein or the polypeptide.

In another aspect, the present application provides a kit for detecting or measuring ROR1 in a sample, and the kit may include the isolated antigen-binding protein or the polypeptide.

In another aspect, the present application provides a kind of said isolated antigen-binding protein, said polypeptide, said drug molecule, said isolated nucleic acid molecule, said carrier, said cell and/or Use of the pharmaceutical composition in the preparation of medicines for preventing and/or treating diseases and/or diseases.

In another aspect, the present application provides the isolated antigen-binding protein, the polypeptide, the drug molecule, the isolated nucleic acid molecule, the carrier, the cell and/or the A pharmaceutical composition prepared for the prevention or treatment of a disease or condition.

In another aspect, the present application provides a method for preventing or treating a disease, which comprises administering the isolated antigen-binding protein described in the present application, the polypeptide, the drug molecule, the The isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.

In the present application, the diseases and/or conditions in the uses may be caused or mediated by abnormal expression of ROR1.

In the present application, the diseases and/or conditions may comprise tumors. For example, the tumor may comprise a solid tumor and/or a hematological tumor. For example, the tumor can be a ROR1 positive tumor.

The application also provides the following embodiments:

CLAIMS 1. An isolated antigen binding protein comprising at least one CDR in an antibody heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:8.

2. The isolated antigen binding protein according to embodiment 1, comprising HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, said HCDR2 comprising SEQ ID NO:

2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

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

The amino acid sequence shown in 4.

4. The isolated antigen binding protein of any one of embodiments 1-3, comprising an H-FR2 located between said HCDR1 and said HCDR2, and said H-FR2 comprising Amino acid sequence shown in SEQ ID NO:5.

5. The isolated antigen binding protein of any one of embodiments 1-4, comprising an H-FR3 located between said HCDR2 and said HCDR3, and said H-FR3 comprising Amino acid sequence shown in SEQ ID NO:6.

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

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

8. The isolated antigen binding protein of any one of embodiments 1-7, further comprising an antibody heavy chain constant region.

9. The isolated antigen binding protein of embodiment 8, wherein the antibody heavy chain constant region comprises an Fc fragment.

10. The isolated antigen binding protein of embodiment 9 comprising an antibody or antigen binding fragment thereof.

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

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

13. The isolated antigen binding protein of any one of embodiments 1-12, which is a single domain antibody.

14. The isolated antigen binding protein of embodiment 13, wherein said single domain antibody comprises CDR1, CDR2 and CDR3, said CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, said CDR2 comprising SEQ ID NO : the amino acid sequence shown in 2, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3.

15. The isolated antigen binding protein of any one of embodiments 1-14, comprising a VHH comprising the amino acid sequence set forth in SEQ ID NO:8.

16. The isolated antigen binding protein of any one of embodiments 1-15, which 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.

17. The isolated antigen binding protein of embodiment 16, wherein said ROR1 comprises the Ig-like domain and the Frizzled domain of ROR1.

18. The isolated antigen binding protein of any one of embodiments 16-17, wherein said ROR1 comprises the amino acid sequence Q30-D305 of ROR1.

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

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

21. A nucleic acid molecule encoding the isolated antigen binding protein of any one of embodiments 1-18, or the polypeptide of embodiment 19.

22. A vector comprising the nucleic acid molecule of embodiment 21.

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

24. A method of preparing the isolated antigen binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19, said method comprising the step of making the isolated antigen binding protein of any one of embodiments 1-18 The cell according to embodiment 23 is cultured under conditions in which the antigen binding protein of the present invention or the polypeptide according to embodiment 19 is expressed.

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

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

27. A kit comprising the isolated antigen binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19 for detecting or determining the presence and/or presence of ROR1 in a sample content.

28. The isolated antigen binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the drug molecule of embodiment 20, the nucleic acid molecule of embodiment 21, the polypeptide of embodiment 22 The use of the carrier described in the embodiment 23, the cell described in the embodiment 23, and the pharmaceutical composition described in the embodiment 25 in the preparation of medicines for preventing and/or treating diseases and/or diseases related to ROR1 expression.

29. The use according to embodiment 28, wherein the disease and/or condition comprises a tumor.

30. The use according to embodiment 29, wherein the tumor comprises a solid tumor and/or a hematological tumor.

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

32. A method for preventing and/or treating a disease and/or disorder associated with ROR1 expression, comprising administering the isolated antigen-binding protein of any one of embodiments 1-18 to a subject in need, performing The polypeptide of embodiment 19, the drug molecule of embodiment 20, the nucleic acid molecule of embodiment 21, the carrier of embodiment 22, the cell of embodiment 23 and/or the drug of embodiment 25 combination.

33. The method of embodiment 32, wherein the disease and/or condition comprises a tumor.

34. The method of embodiment 33, wherein the tumor comprises a solid tumor and/or a hematological tumor.

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

36. The isolated antigen binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the drug molecule of embodiment 20, the nucleic acid molecule of embodiment 21, the polypeptide of embodiment 22 The above-mentioned carrier, the cell according to embodiment 23 and/or the pharmaceutical composition according to embodiment 25, which are used for preventing and/or treating diseases and/or disorders related to ROR1 expression.

37. The isolated antigen binding protein, said polypeptide, said drug molecule, said nucleic acid molecule, said carrier, said cell and/or said pharmaceutical composition according to embodiment 36 , wherein the disease and/or condition comprises a tumor.

38. The isolated antigen binding protein of embodiment 37, wherein said tumor is a solid tumor and/or a hematological tumor

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

Not intending to be limited by any theory, the following examples are only for explaining various technical solutions of the invention of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Embodiment 1 animal immunization

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

(1) Immunize a 2.5-year-old unimmunized female alpaca five times every two weeks with the freshly prepared immunogen (see Table 1. Immunization schedule), and divide the immunogen into several parts (each part is used for one immunization), and stored at -80°C;

(2) For the first immunization, mix 300 μg of immunogen with an equal volume of Freund’s complete adjuvant (CFA) to make an emulsion, and for subsequent immunizations (second to fifth) mix 300 μg of immunogen with an equal volume of CFA Incomplete Freund's adjuvant (CFA) mixed adjuvant (IFA) made into emulsion (all vaccination experiments were approved by the local ethics committee);

(3) Before each immunogen administration (1st to 5th time), 5ml of jugular vein blood sample was taken, and serum was recovered as IgG-containing sample after immunization for antigen-specific antibody titer;

(4) 7 days after the last immunization, collect 50ml of blood and use Ficoll-Paque ^TM PLUS Media (GE Healthcare, USA) to separate peripheral blood mononuclear cells (PBMC) from whole blood, and lyse PBMC in Trizol for subsequent Total RNA extraction;

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

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 titers in the 3rd to 5th sera after immunization all reached above 1/100,000.

Table 1. Immunization schedule

(*Due to COVID-19 quarantine at alpaca farm, 3rd immunization delayed by two weeks)

Example 2 Establishment of immune phage library

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

(1) After isolating PBMC from 50ml of peripheral blood and extracting total RNA, use RNA as a template to prepare cDNA through commercially available reagents. The preparation system is shown in Table 2;

(2) Amplify the VHH sequence from the cDNA library, and after being transformed into TGI cells, calculate the library size according to the number of colonies in a series of dilutions of the electroporated E. coli cell suspension on the 90mm plate;

(3) Perform colony sequencing and bioinformatics analysis.

The experimental results showed that the first PCR produced two different amplicons with a molecular weight of about 0.7kb and 0.9kb (Figure 2A). Using the 0.7kb amplicon as a template to amplify VHH, the resulting fragment size was about 400bp The size of the amplified library was 1.4×10 ⁸ (Figure 3). Phylogenetic analysis was performed on 90 sequences in the library, and 96 single colonies were randomly selected for colony sequencing. The VHH was correctly inserted into the phage The ratio of VHH sequence was 93.8% (90/96), and the ratio of specific VHH sequence was 96.7% (87/90) ( FIG. 4 ).

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

Example 3 Antigen-specific antigen-binding protein screening

By monitoring the number of colonies grown on the plate, three consecutive rounds of screening were performed to obtain the specific antigen-binding protein of ROR1 ( FIG. 5 ). After each round of selection, the number of colonies in the ROR1-coated group increased, indicating that ROR1-specific antigen-binding proteins were enriched. R1, R2, and R3 represent the screening results of the first, second, and third rounds, respectively, and NC represents the negative control.

Example 4 Identification and Verification of Antigen Binding Proteins

(1) Verify the specific antigen-binding protein after 3 rounds of screening, use periplasmic extracts for ELISA identification, and obtain antigen-positive clones;

(2) Then obtain the DNA sequence of the positive clone by colony sequencing, and identify the specific sequence;

(3) Perform recombinant production and purification of the specific sequence, and test the purity to determine whether it meets the requirements for subsequent testing;

(4) Further testing the affinity and specificity of the antigen-binding protein to the ROR1 protein.

Experimental results show that the OD450 value of 5-26 (VHH sequence shown in SEQ ID NO: 8) is higher than 5-09 (VHH sequence shown in SEQ ID NO: 11) and 5-95 (VHH sequence shown in SEQ ID NO :12) (Figure 6A). Wherein, 5-26 comprises CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3. The two non-specific VHHs (VHH1: the sequence shown in SEQ ID NO:9; VHH2: the sequence shown in SEQ ID NO:10) did not bind to the recombinant ROR1 protein, and the anti-ROR1 mAb bound well to the recombinant ROR1 protein (Fig. 6B). The experimental results further indicated that 5-26, 5-09 and 5-95 specifically bind to the recombinant ROR1 protein, but the binding ability of 5-26 is significantly better than that of the other two groups.

Purified SDS-PAGE results showed that the purity of the antigen-binding proteins (about 0.5 mg each) was over 90%, meeting the requirements for subsequent functional tests (Figure 7). The purified antigen-binding proteins 5-26, 5-9, 5-95 specifically binds to recombinant ROR1 protein ( FIG. 8A ), negative controls VHH1 and VHH2 do not bind to ROR1 protein, and positive control binds to ROR1 protein ( FIG. 8B ). The results of this experiment showed that, compared with 5-9 and 5-95, 5-26 can bind to ROR1 protein at a higher concentration at multiple dilutions with good specificity.

At the same time, the specificity evaluation showed that the purified antigen-binding proteins 5-26, 5-9, and 5-95 only combined with the recombinant ROR1 protein, but not with the other two his-tagged proteins (PD-L1 and RBD), Indicates that the selected antigen-binding protein does not recognize other tags. Anti-ROR1 mAb (positive control) binds only recombinant ROR1 protein. Anti-his mAb (Sinobiological, #105327-MM02T-H) can bind to all three his-tagged proteins (PD-L1, RBD, ROR1) (Figure 9). The concentration of the purified antigen-binding protein used in the experiment was 3 μg/ml, and the C-terminus of the protein contained a his tag and an HA tag. The secondary antibody used for recognition was HRP anti-HA tag antibody (Abcam, #ab1190). The results of this experiment show that 5-26 has good specificity to ROR1 protein and basically does not bind to PD-L1 and RBD.

anti-ROR 1mAb: ROR1 mouse monoclonal antibody, from Proteintech (catalogNo.: 66923-1-Ig), molecular weight 130kDa, can be used in FC, IF, IHC, WB, ELISA and other applications.

Example 5 Antigen-binding protein binding to cell lines expressing ROR1

Express ROR1 in A549, MB231, and JeKo-1 cells, 1.5-2.0×10 ⁵ cells per well, incubate the antigen-binding protein with the cells at a concentration of 1 μg/ml at 4°C for 30 minutes, use anti-HA APC Staining was carried out, and the results of flow cytometry analysis showed that in A549 cells, the ratio of 5-26 binding cells reached 97.0%, 5-9 was 18.5%, and 5-95 was 11.8% (Figure 10). In MB231 cells, 5- The ratio of 26-binding cells reached 96.8%, 5-9 was 61.7%, and 5-95 was 53.3% (Figure 11). In JeKo-1 cells, the ratio of 5-26-binding cells reached 99.7%, and 5-9 was 1.90 %, 5-95 is 1.18% (Figure 12). The results of this experiment indicated that compared with 5-9 and 5-95, 5-26 had a stronger ability to bind to cell lines expressing ROR1.

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims (20)

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

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.

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

4. The isolated antigen binding protein of any one of claims 1-3, wherein the antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

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

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

7. The isolated antigen binding protein of any one of claims 1-6, 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 of any one of claims 1-9, having at least one of the following properties:

(1) Is capable of specifically binding to ROR1; and

(2) Capable of binding to ROR1 expressed on the surface of cells.

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

12. A nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-10.

13. A vector comprising the nucleic acid molecule of claim 12.

14. A cell comprising the nucleic acid molecule of claim 12, and/or the vector of claim 13.

15. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-10, the pharmaceutical molecule of claim 11, the nucleic acid molecule of claim 12, the vector of claim 13, and/or the cell of claim 14, and optionally a pharmaceutically acceptable carrier.

16. A kit comprising the isolated antigen binding protein of any one of claims 1-10, said kit capable of detecting the presence and/or amount of ROR1 in a sample.

17. Use of the isolated antigen binding protein of any one of claims 1-10, the pharmaceutical molecule of claim 11, the nucleic acid molecule of claim 12, the vector of claim 13, the cell of claim 14, the pharmaceutical composition of claim 15, for the preparation of a medicament for the prevention and/or treatment of a ROR 1-associated disease and/or disorder.

18. The use of claim 17, wherein the disease and/or condition comprises a tumor.

19. The use of claim 18, wherein the tumor is a solid tumor and/or a hematological tumor.

20. The use of any one of claims 18-19, wherein the tumor is a ROR1 positive tumor.

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