Antibodies against ROR1 and uses thereof

Abstract

The invention relates to an antibody aiming at ROR1 and application thereof, in particular to an antibody specifically bound with ROR1 or an antigen binding fragment thereof, a nucleic acid for coding the antibody, a vector comprising the nucleic acid and a host cell comprising the nucleic acid or the vector. Compositions, kits and conjugates comprising the antibodies, as well as medical uses of the antibodies.

Description

[0001] This application claims priority to PCT application No. PCT / CN2023 / 109898, filed on July 28, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to antibodies against ROR1, and the uses of such antibodies, particularly their use in cancer detection or diagnosis and in guiding cancer treatment. Background Technology

[0003] ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved type I membrane protein. ROR1 possesses a cytoplasmic domain consisting of a tyrosine kinase-like domain, two serine / threonine-rich domains, and a proline-rich domain (PRD); a transmembrane domain; and an extracellular domain consisting of an Ig-like domain, a coiled domain, and a tricyclic domain. ROR1 is highly expressed during embryonic and infant development, and its expression levels are significantly reduced in children and adults. ROR1 expression is significantly increased in various hematologic malignancies and solid tumors.

[0004] ROR1 expression attenuates during fetal development and, with a few exceptions, becomes negligible in most postnatal tissues. In contrast, ROR1 is expressed in a variety of human cancers (particularly those with poor differentiation) and is associated with early recurrence after treatment or metastasis. Flow cytometry studies have demonstrated ROR1 expression on the cell surface in multiple cancer types, including B-cell lymphoma (B-CLL), mantle cell lymphoma (MCL), and a subset of B-cell acute lymphoblastic leukemia (ALL). ROR1 expression enhances tumor cell growth and survival and promotes epithelial-mesenchymal transition and metastasis. In triple-negative breast cancer, lung adenocarcinoma, ovarian cancer, and other cancer types, high ROR1 expression is associated with shorter overall survival and metastasis-free survival. Numerous studies suggest that ROR1 plays a crucial role in promoting tumor growth and metastasis, inducing drug resistance, and inhibiting apoptosis.

[0005] Due to its expression patterns and functions in tumor progression, ROR1 has become a potential biomarker and target for tumor diagnosis and therapy. There is a need in this field to develop antibodies against ROR1. Summary of the Invention

[0006] This disclosure provides novel antibodies or antigen-binding fragments thereof that target ROR1, which can be used for the diagnosis or auxiliary diagnosis, staging and imaging of ROR1-positive cancers, determining the prognosis of cancer in subjects, and providing guidance for the treatment of ROR1-positive cancers.

[0007] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to ROR1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR 1-3 having amino acid sequences as shown in SEQ ID NO: 12-14, and the VL comprises LCDR 1-3 having amino acid sequences as shown in SEQ ID NO: 15-17.

[0008] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 6.

[0009] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO: 4, and VL comprises the amino acid sequence shown in SEQ ID NO: 6.

[0010] In some implementations, the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0011] In some implementations, the antibody has a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0012] In some implementations, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fab'-SH, Fv, scFv, and ds-scFv.

[0013] In some implementations, the antibody is a monoclonal antibody.

[0014] In some implementations, the antibody is a rabbit antibody or a humanized antibody.

[0015] In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 5, and the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7.

[0016] On the other hand, this disclosure provides nucleic acids comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof disclosed herein.

[0017] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 8, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 10.

[0018] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 11.

[0019] In another aspect, this disclosure provides a carrier containing the nucleic acid disclosed herein.

[0020] In another aspect, this disclosure provides a host cell containing the nucleic acids or vectors disclosed herein.

[0021] In another respect, this disclosure provides compositions comprising the antibodies disclosed herein or antigen-binding fragments thereof.

[0022] On the other hand, this disclosure provides kits containing the antibodies or antigen-binding fragments thereof disclosed herein.

[0023] In another aspect, this disclosure provides conjugates comprising the antibody or antigen-binding fragment thereof disclosed herein and the chemical portion conjugated thereto.

[0024] In some implementations, the chemical component is the detectable component.

[0025] In some implementations, the detectable portion is selected from the group consisting of: biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent molecules, phosphorescent molecules, or chemiluminescent molecules.

[0026] On the other hand, this disclosure provides a method for diagnosing or assisting in the diagnosis of cancer in a subject, comprising:

[0027] (i) Contacting a biological sample with the antibody or its antigen-binding fragment disclosed herein, or the conjugate disclosed herein; and

[0028] (ii) Detect the presence of ROR1 in biological samples, where the presence of ROR1 indicates cancer, and cancer is ROR1 positive.

[0029] In some implementations, the method further includes determining the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, a subject is selected for treatment with anti-ROR1 therapy.

[0030] In some implementations, the method further includes administering anti-ROR1 therapy to the subject.

[0031] In some implementations, the subjects are people who have or are suspected of having cancer.

[0032] In some implementations, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

[0033] In another aspect, this disclosure provides a method for staging ROR1-positive cancers in subjects, comprising:

[0034] (i) Contacting a biological sample with the antibody or its antigen-binding fragment disclosed herein, or the conjugate disclosed herein; and

[0035] (ii) Determine the level and / or location of ROR1 in biological samples.

[0036] In some implementations, biological samples are obtained from the subjects.

[0037] In another aspect, this disclosure provides a method for determining the cancer prognosis of a subject, comprising:

[0038] (i) Contacting a biological sample with the antibody or its antigen-binding fragment disclosed herein, or the conjugate disclosed herein; and

[0039] (ii) Detect the presence of ROR1 in biological samples, where the presence of ROR1 indicates a poor prognosis.

[0040] In some implementations, biological samples are obtained from the subjects.

[0041] In some implementations, the method further includes determining the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, a subject is selected for treatment with anti-ROR1 therapy.

[0042] On the other hand, this disclosure provides a method for selecting subjects to be treated with anti-ROR1 therapy, comprising:

[0043] (i) Contacting a biological sample with the antibody or its antigen-binding fragment disclosed herein, or the conjugate disclosed herein; and

[0044] (ii) Determine the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, select subjects to be treated with anti-ROR1 therapy.

[0045] In some implementations, biological samples are obtained from the subjects.

[0046] In some implementations, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

[0047] In another aspect, this disclosure provides a method for treating cancer in a subject, comprising:

[0048] (i) Contacting a biological sample with the antibody or its antigen-binding fragment disclosed herein, or the conjugate disclosed herein; and

[0049] (ii) Determine the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, administer anti-ROR1 therapy to the subject.

[0050] Cancer is ROR1 positive.

[0051] In some implementations, biological samples are obtained from the subjects.

[0052] In some implementations, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

[0053] In another aspect, this disclosure provides a method for detecting the presence of ROR1 in a sample or determining its level, comprising:

[0054] (i) Contacting the sample with the antibody or its antigen-binding fragment disclosed herein, or the conjugate disclosed herein; and

[0055] (ii) Detect the presence of ROR1 bound by antibodies or conjugates or the level of ROR1 bound by antibodies or conjugates.

[0056] In some implementations, the sample is a biological sample. In some implementations, the biological sample is obtained from the subject.

[0057] In another aspect, this disclosure provides a method for imaging or identifying cancer cells expressing ROR1 in a sample, comprising:

[0058] (i) Contacting the sample with the antibody or its antigen-binding fragment disclosed herein, or the conjugate disclosed herein; and

[0059] (ii) Detect the presence of ROR1 in biological samples, where the presence of ROR1 indicates cancer cells.

[0060] In some implementations, the sample is a biological sample. In some implementations, the biological sample is obtained from the subject. In some implementations, the biological sample is a tissue sample.

[0061] In some implementations, the presence or level of ROR1 in a biological sample is determined by a method selected from the group consisting of: flow cytometry, immunohistochemical staining, ELISA assay, Western blotting, immunofluorescence assay, chemiluminescence assay, radioimmunoassay, and multiplex immunoassay.

[0062] In some implementations, the location of ROR1 in biological samples is determined by a method selected from the group consisting of: immunohistochemical staining, immunofluorescence assay, chemiluminescence assay, and multiplex immunoassay.

[0063] On the other hand, this disclosure provides a method for imaging ROR1-positive cancers in a subject, comprising:

[0064] (a) Administering to a subject the antibody or antigen-binding fragment thereof disclosed herein, wherein the antibody is conjugated to a detectable moiety, and

[0065] (b) Detect the presence of the detectable portion.

[0066] In some implementations, the detectable portion is selected from the group consisting of: biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent molecules, phosphorescent molecules, or chemiluminescent molecules.

[0067] In some implementation schemes, cancer is selected from the group consisting of breast cancer (such as triple-negative breast cancer), endometrial cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer, colorectal cancer, uterine cancer, cervical cancer, brain cancer, stomach cancer, bile duct cancer, chondrosarcoma, kidney cancer, thyroid cancer, and skin cancer, or from the group consisting of lymphoma and myeloma, which are ROR1-positive hematologic malignancies.

[0068] In some implementations, lymphoma is selected from the group consisting of: Hodgkin B-cell lymphoma, non-Hodgkin B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. Attached Figure Description

[0069] The features and advantages of the invention will be understood by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments in which the principles of the invention are utilized, and in the accompanying drawings:

[0070] Figure 1 IHC staining of CSTONE-1(CSD)-1-1A in various tumor cell lines is shown.

[0071] Figure 2 IHC staining of CSTONE-1(CSD)-1-1A in various human tissues is shown.

[0072] Figure 3 IHC staining of CSTONE-1(CSD)-2-1A in various tumor cell lines is shown.

[0073] Figure 4 IHC staining of CSTONE-1(CSD)-2-1A in various human tissues is shown.

[0074] Figure 5 IHC staining of CSTONE-1(CSD)-3-1B in various tumor cell lines is shown.

[0075] Figure 6 IHC staining of CSTONE-1(CSD)-3-1B in various human tissues is shown.

[0076] Figure 7 The WB results for CSTONE-1(CSD)-3-1A, CSTONE-1(CSD)-3-1B, CSTONE-1(CSD)-2-1A, CSTONE-1(CSD)-2-1B, CSTONE-1(CSD)-1-1A, and CSTONE-1(CSD)-1-1B are shown.

[0077] Figure 8 The IHC staining of CSTONE-1 (CSD)-1 on multiple human TNBC FFPE samples and the H-score calculated by staining intensity are shown.

[0078] Figure 9 IHC staining of CSTONE-1 (CSD)-1 in various tumor cell lines is shown.

[0079] Figure 10 The distribution of ROR1 protein via IHC and the distribution of ROR1 mRNA via ISH are shown in FFPE samples from the human TNBC PDX model BR1458.

[0080] Figure 11The distribution of ROR1 protein via IHC and the distribution of ROR1 mRNA via ISH are shown in FFPE samples from the human TNBC PDX model BR1282.

[0081] Figure 12 The distribution of ROR1 protein via IHC and the distribution of ROR1 mRNA via ISH are shown in FFPE samples from the human TNBC PDX model BR1474.

[0082] Figure 13 The results of Western blot analysis of CSTONE-1 (CSD)-1 against various tumor cell lines are shown.

[0083] Figure 14 EC50 of the combination of CSTONE-1 (CSD)-1 and ROR1 as measured by ELISA is shown.

[0084] Figure 15 The H-scores of the test tumor samples analyzed by IHC using the anti-ROR1 monoclonal antibody CSTONE-1 (CSD)-1 are shown.

[0085] Figure 16 The expression of ROR1 in the test tumor sample was shown by IHC analysis using the anti-ROR1 monoclonal antibody CSTONE-1 (CSD)-1. Detailed Implementation

[0086] The above-described features and advantages of the invention, as well as its additional features and advantages, will become more clearly understood below when considered in conjunction with the accompanying drawings, due to the detailed description of the following embodiments.

[0087] The embodiments described herein with reference to the accompanying drawings are illustrative and explanatory, and are intended to provide a general understanding of the invention. These embodiments should not be construed as limiting the scope of the invention. Throughout the specification, identical or similar elements and elements having identical or similar functions are indicated by similar reference numerals.

[0088] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be clear to those skilled in the art. For example, refer to standard manuals such as Leuenberger, H.G., Nagel, B., and Klbl, H. (eds.), "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al. (eds.), "Current protocols in molecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al., "Immunology" (6th ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York. (2005), and the general background techniques cited above.

[0089] definition

[0090] As used herein, unless the context clearly indicates otherwise, the singular forms of “a,” “an,” “the,” and “the” also include plural referents. Thus, for example, references to “an antibody” include multiple antibodies, and in some embodiments, references to “an antibody” include multiple antibodies, and so on.

[0091] Unless otherwise indicated or defined, the terms “comprising,” “including,” and variations thereof shall be understood to imply the inclusion of the said element or step or group of elements or steps, but not to exclude any other element or step or group of elements or steps.

[0092] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Such molecules typically consist of two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (or domain) (abbreviated VH herein) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (or domain) (abbreviated VL herein) and a light chain constant region. The light chain constant region contains one domain: CL. The variable regions of both the heavy and light chains of an antibody contain binding domains that interact with the antigen. The constant regions of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, the first component in the classical complement initiation pathway.

[0093] The heavy chain of immunoglobulins can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region contains the VH and CH1 domains and combines with the light chain to form the Fab (antigen-binding fragment). The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to homologous Fc receptors on effector cells. The hinge region, found in the immunoglobulin classes IgG, IgA, and IgD, acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region. The structure of the hinge domain is highly diverse, differing in both sequence and length among immunoglobulin classes and subclasses.

[0094] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided structurally and functionally into three regions: the upper hinge, the core hinge, and the lower hinge (Shin et al., Immunological Reviews 130:87, 1992). The upper hinge comprises amino acids from the carboxyl terminus of CH1 to the first motility-restricting residue in the hinge (typically the first cysteine ​​residue that forms an interchain disulfide bond between the two heavy chains). The length of the upper hinge region is related to the segmental flexibility of the antibody. The core hinge region contains the interchain disulfide bonds. The lower hinge region connects to the amino terminus of the CH2 domain and includes residues within the CH2 domain. The conformational changes allowed by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence can affect the effector function of the antibody Fc moiety.

[0095] The light chain variable region (VL) or heavy chain variable region (VH) consists of three complementarity-determining regions (CDRs) and a frame region that is interrupted by them. The frame region is used to align the CDRs for specific binding to the antigenic epitope. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following frame (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; CDRs 1, 2, and 3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0096] The amino acid assignments for each VL and VH domain are based on any conventional definition of the CDR. Common definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a complex of the Chothia-Kabat CDR, wherein CDR-H1 is a complex of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition by Martin et al. (bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. This disclosure may use CDRs defined according to any of these numbering systems, although the preferred embodiment uses the Kabat definition of the CDR.

[0097] Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the light chain, antibody light chains can be classified as lambda (λ) chains or kappa (κ) chains.

[0098] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two distinct antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and any other modified immunoglobulin molecules containing antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

[0099] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies. That is, each antibody that makes up this group is identical, except that it may be due to a small number of naturally occurring mutations. Monoclonal antibodies are highly specific and directed against a single antigen. The term "monoclonal antibody" in this document is not limited to antibodies produced by hybridoma technology and should not be construed as requiring any particular method to produce the antibody.

[0100] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind antigens. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0101] Examples of antigen-binding fragments covered in the term "antigen-binding moiety" of antibodies include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments consisting of two Fab fragments linked by disulfide bonds at the hinge region; (iii) Fab' fragments, which are essentially Fab fragments with a partially hinge region; (iv) Fd fragments consisting of VH and CH1 domains; (v) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) Fv fragments consisting of the VL and VH domains of a single antibody arm; (vii) dAb fragments consisting of a VH domain; (viii) separated complementarity-determining regions (CDRs); (ix) heavy chain antibodies containing a single heavy chain variable region and two heavy chain constant domains; and (x) The Fab'-SH fragment is a modified form of the Fab' fragment in which the sulfur (S) atom of the cysteine ​​residue is modified to carry a thiol group (-SH). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked via synthetic linkers using recombinant methods, allowing them to be made into a single protein chain where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibody. Additionally, the term also includes "linear antibodies" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1) forming an antigen-binding region together with a complementary light chain polypeptide, and modified versions of either of the aforementioned fragments that retain antigen-binding activity.

[0102] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and their utility can be screened in the same manner as for intact antibodies.

[0103] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as the non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity, represented by the equilibrium constant (KD) for antigen-antibody dissociation, is a measure of the strength of binding between the antigenic determinant and the antigen-binding site on the antibody: the smaller the KD value, the stronger the binding between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), i.e., 1 / KD.

[0104] Affinity is a measure of the strength of binding between an antibody and its associated antigen. Affinity is related to both the affinity between the antigenic determinant and its antigen-binding site on the antibody, and the number of associated binding sites present on the antibody. Typically, antibodies will bind at a rate of 10... -5 Up to 10-12 M or smaller, preferably 10⁻⁷ to 10⁻¹², and more preferably 10 -8 Up to 10 -12 The dissociation constant of M (K) D ) bind antigen, and / or at least 10 7 M -1 Preferably at least 10 8 M -1 More preferably at least 10 9 M -1 Such as at least 10 12 M -1 The binding affinity of [agent] to the antigen is [increased]. Any [amount] greater than 10 [units]... -4 M of K D Values ​​are generally considered to represent nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined by any known suitable method, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays, as well as various variants thereof known in the art.

[0105] The term "epitope" refers to a site on an antigen that an antibody binds to. Epitopes can be formed from one or more proteins through ternary folding of consecutive or discontinuous amino acids. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least three, and more often at least five, or eight to ten amino acids in a unique spatial conformation. Epitopes define the minimal binding site of an antibody and are therefore specific targets for antibodies or their antigen-binding fragments.

[0106] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions in an alignment. For example, "amino acid sequence identical to SEQ ID NO: YX%" means that the amino acid sequence is X% identical to SEQ ID NO: Y, and more specifically, that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in SEQ ID NO: Y. Such calculations are typically performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and BLAST with vacancies (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0107] Furthermore, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which one amino acid residue is replaced by another amino acid residue having a similar chemical structure and having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.

[0108] Such conservative substitutions are preferably the following, wherein an amino acid within groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0109] The particularly preferred conservative substitutions are as follows: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted with Ile or Val; Lys is substituted with Arg, Gln, or Glu; Met is substituted with Leu, Tyr, or Ile; Phe is substituted with Met, Leu, or Tyr; Ser is substituted with Thr; Thr is substituted with Ser; Trp is substituted with Tyr; Tyr is substituted with Trp; and / or Phe is substituted with Val, Ile, or Leu.

[0110] As used in this article, the term "carrier" is intended to refer to a nucleic acid molecule that can transport another nucleic acid to which it is attached.

[0111] As used in this article, the term "host cell" refers to the cell in which the expression vector has been introduced.

[0112] The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with the other components of the composition and is substantially harmless to the recipient and / or that such carrier or excipient is approved or may be approved for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0113] As used herein, the term “treatment” refers to the administration of a drug or procedure for the purpose of achieving an effect. The effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and may be therapeutic in relation to the partial or complete cure of a disease and / or its symptoms. As used herein, “treatment” can include treating a disease or condition (e.g., cancer) in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease or its symptoms in subjects who may be susceptible to the disease but have not yet been diagnosed with it (e.g., including diseases that may be associated with or caused by a primary disease); (b) suppressing the disease, i.e., halting its development; and (c) alleviating the disease, i.e., causing its regression. Treatment can refer to any sign of success in the treatment, improvement, or prevention of cancer, including any objective or subjective parameter such as elimination; relief; reduction of symptoms or making the patient more tolerant of the disease condition; slowing the rate of degeneration or decline; or making the endpoint of degeneration less severe. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician’s examination. Therefore, the term "treatment" includes the administration of antibodies or compositions or conjugates disclosed herein to prevent or delay, alleviate or stop or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer).

[0114] As used herein, the term "diagnosis" means the detection of a disease or the determination of its stage or extent. Typically, the diagnosis of a disease is based on the evaluation of one or more factors and / or symptoms that indicate the disease. That is, a diagnosis can be made based on the presence, absence, or quantity of factors that indicate the presence or absence of a disease or symptom. Each factor or symptom considered to indicate a specific disease does not need to be specifically associated with that specific disease; for example, a differential diagnosis may exist that can be inferred from a diagnostic factor or symptom. Similarly, there may be situations where factors or symptoms indicating a specific disease are present in individuals who do not have that specific disease. The term "diagnosis" also encompasses determining the therapeutic effect of a drug therapy or predicting a pattern of response to a drug therapy. Diagnostic methods can be used independently or in combination with other diagnostic and / or staging methods known in the medical field for a specific disease.

[0115] As used herein, the term "prognosis" refers to the prediction of the likely course and outcome of a clinical condition or disease. Prognosis is typically made by evaluating disease factors or symptoms that indicate a favorable or unfavorable course or outcome. As used herein, the phrase "determine prognosis" refers to the process by which a person skilled in the art can predict the course or outcome of a condition in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy. Rather, a person skilled in the art will understand that the term "prognosis" refers to an increased probability that a particular course or outcome will occur; that is, the course or outcome is more likely to occur in patients presenting with a given condition compared to those individuals who did not present with a given condition.

[0116] The term "detection" includes any means of detection, including both direct and indirect detection.

[0117] The term "therapeutic effect" refers to the reduction, elimination, or prevention of a subject's disease, symptoms of disease, or side effects of disease.

[0118] As used in this article, the term "effective amount" refers to the amount that is sufficient to treat a disease when administered to a subject to treat that disease.

[0119] As used herein, the term “subject” refers to any mammalian subject for whom a diagnosis, treatment, or therapy is desired. “Mammalian” for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0120] The term "biological sample" refers to a collection of similar cells obtained from a subject or patient. A biological sample can be a tissue or cell sample. The source of a tissue or cell sample can be solid tissue from fresh, frozen, and / or preserved organ or tissue samples or biopsies or aspirates; blood or any blood composition; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Biological samples can also be obtained from in vitro tissue or cell cultures. Tissue samples may contain compounds that do not naturally mix with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of biological samples in this document include, but are not limited to, tumor biopsies, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like characteristics, and preserved tumor samples, such as formalin-fixed, paraffin-embedded, or frozen tumor samples.

[0121] As used herein, the term "biomarker" refers to an indicator (e.g., ROR1) that can be detected in a sample, such as predictive, diagnostic, and / or prognostic indicators. Biomarkers can serve as indicators of a specific subtype of a disease or condition (e.g., cancer) characterized by certain molecular, pathological, histological, and / or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide copy number alterations (e.g., DNA copy number), peptides, peptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate and / or glycolipid-based molecular markers.

[0122] The terms “level,” “level of expression,” or “expression level” are generally used interchangeably and typically refer to the amount of polynucleotides, mRNA, or amino acid products or proteins in a biological sample. “Expression” generally refers to the process by which information encoded by a gene is transformed into a structure present and manipulated in the cell. Therefore, according to the invention, “expression” of a gene (e.g., the ROR1 gene) can refer to transcription into a polynucleotide, translation into a protein, or even post-translational modifications of a protein. Fragments of transcribed polynucleotides, translated proteins, or post-translational modified proteins should also be considered expressed, whether they originate from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of proteins, such as proteolysis. In some embodiments, “expression level” refers to the amount of protein (e.g., ROR1) in a biological sample determined using methods known in the art or described herein, including but not limited to immunohistochemistry (IHC), immunoblotting (e.g., Western blotting), immunofluorescence (IF), flow cytometry, such as fluorescence activated cell sorting (FACS™), or enzyme-linked immunosorbent assay (ELISA).

[0123] Anti-ROR1 antibody

[0124] This disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to ROR1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR 1-3 having amino acid sequences as shown in SEQ ID NO: 12-14, and the VL comprises LCDR 1-3 having amino acid sequences as shown in SEQ ID NO: 15-17.

[0125] In some implementations, the CDR sequence is defined according to the Kabat numbering system.

[0126] When the CDR sequence is defined according to the Kabat numbering system, the VH of the antibody disclosed herein contains HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 12 (SYAVT), SEQ ID NO: 13 (IIYTDASAYYATWAKG), and SEQ ID NO: 14 (GRV), respectively, and the VL of the antibody disclosed herein contains LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 15 (QASQSIYNNKNLA), SEQ ID NO: 16 (AASNLAS), and SEQ ID NO: 17 (LGEFSCSSGDCFA), respectively.

[0127] In some embodiments, VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 6.

[0128] In some embodiments, VH comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 4, formed by inserting, deleting, and / or substituting one or more amino acids in SEQ ID NO: 4, provided that the functional variant retains the ability to bind to ROR1. In some embodiments, VL comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 6, formed by inserting, deleting, and / or substituting one or more amino acids in SEQ ID NO: 6, provided that the functional variant retains the ability to bind to ROR1.

[0129] The functional variant comprises or consists of the following: an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

[0130] In the case of functional variants, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parental amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1-20, preferably 1-10, more preferably 1-7, even more preferably 1-5, and most preferably 1-2. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0131] In some implementations, insertions, deletions, and / or substitutions may be performed at frame (FR) areas, such as FR1, FR2, FR3, and / or FR4.

[0132] In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions in which one amino acid in groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0133] The particularly preferred conservative substitutions are as follows: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted with Ile or Val; Lys is substituted with Arg, Gln, or Glu; Met is substituted with Leu, Tyr, or Ile; Phe is substituted with Met, Leu, or Tyr; Ser is substituted with Thr; Thr is substituted with Ser; Trp is substituted with Tyr; Tyr is substituted with Trp; and / or Phe is substituted with Val, Ile, or Leu.

[0134] In a preferred embodiment, VH comprises the amino acid sequence shown in SEQ ID NO: 4, and VL comprises the amino acid sequence shown in SEQ ID NO: 6.

[0135] Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the light chain, antibody light chains can be classified as lambda (λ) chains or kappa (κ) chains. The antibodies disclosed herein can possess any of the above classes or subtypes.

[0136] In some embodiments, the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody has a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, the antibody is an IgG1 antibody.

[0137] The antibodies disclosed herein may be complete antibodies or their antigen-binding fragments. The antigen-binding fragment may be any fragment of an antibody that retains the ability to specifically bind to ROR1. Examples of antigen-binding fragments include, but are not limited to: Fab fragments; F(ab')2 fragments; Fab' fragments; Fab'-SH, Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity-determining regions (CDRs); nanobodies; linear antibodies comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1); and modified versions of one of the aforementioned fragments that retain antigen-binding activity.

[0138] In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fab'-SH, Fv, scFv, and ds-scFv. In a preferred embodiment, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In yet another preferred embodiment, the antigen-binding fragment is scFv.

[0139] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a rabbit antibody or a humanized antibody. A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all at least one and generally both variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. The humanized antibody may optionally comprise at least a portion of the antibody constant region derived from the human antibody. An antibody, for example, in a “humanized form” of a non-human antibody, refers to an antibody that has undergone humanization.

[0140] In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 5, and the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7.

[0141] In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 5, formed by inserting, deleting, and / or substituting one or more amino acids in SEQ ID NO: 5, provided that the functional variant retains its ability to bind to ROR1. In some embodiments, the light chain comprises a functional variant of the amino acid sequence shown in SEQ ID NO: 7, formed by inserting, deleting, and / or substituting one or more amino acids in SEQ ID NO: 7, provided that the functional variant retains its ability to bind to ROR1.

[0142] The functional variant comprises or consists of the following: an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the parent polypeptide.

[0143] In some embodiments, the number of inserted, deleted, and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the parental amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1-50, preferably 1-20, more preferably 1-10, and even more preferably 1-5. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0144] In some implementations, insertions, deletions, and / or substitutions may be performed at frame (FR) regions (e.g., at FR1, FR2, FR3, and / or FR4) and / or at constant regions (e.g., CL, CH1, CH2, and / or CH3).

[0145] In some implementations, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0146] In a preferred embodiment, the antibody comprises a heavy chain including the amino acid sequence shown in SEQ ID NO: 5 and a light chain including the amino acid sequence shown in SEQ ID NO: 7.

[0147] Nucleic acid

[0148] This disclosure provides nucleic acids comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof disclosed herein.

[0149] The term "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides, deoxyribonucleotides, or modified forms of any type of nucleotide. These modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linking modifications such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phosphoroanilothioate, phoshoraniladate, and aminophosphates.

[0150] For example, the present invention provides nucleic acid molecules encoding the heavy chain variable region sequences disclosed herein. The present invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 98%, or at least 99% identity with a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 8.

[0151] For example, the present invention provides nucleic acid molecules encoding the light chain variable region sequences disclosed herein. The present invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 98%, or at least 99% identity with a nucleic acid encoding any of the light chain variable region sequences disclosed herein. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 10.

[0152] In a preferred embodiment, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 8 and the nucleotide sequence shown in SEQ ID NO: 10.

[0153] For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a CDR sequence of any of the heavy chain variable region sequences disclosed herein.

[0154] For example, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a light chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

[0155] For example, the present invention provides nucleic acid molecules encoding the heavy chain sequences disclosed herein. The present invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 98%, or at least 99% identity with the nucleic acid encoding the heavy chain sequences disclosed herein. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9.

[0156] For example, the present invention provides nucleic acid molecules encoding the light chain sequences disclosed herein. The present invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 98%, or at least 99% identity with the nucleic acid encoding the light chain sequences disclosed herein. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 11.

[0157] In a preferred embodiment, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO: 9 and a nucleotide sequence as shown in SEQ ID NO: 11.

[0158] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an antibody disclosed herein.

[0159] In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell.

[0160] In some embodiments, ribonucleic acid (RNA) can be introduced into human cells in vivo. In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.

[0161] carrier

[0162] This disclosure provides a carrier containing the nucleic acid disclosed herein.

[0163] In some embodiments, the vector is an expression vector capable of expressing a polypeptide containing a heavy chain variable region or a light chain variable region of an antibody. For example, the present invention provides an expression vector comprising any of the above-described nucleic acid molecules.

[0164] Any vector may be suitable for use in this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an EB virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCVIRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFPT2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0165] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. For example, vectors can be selected from the pUC series (Fermentas LifeSciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors that can be used in this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0166] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmids, λ, SV40, bovine papillomavirus, etc.

[0167] For example, the vector could be an adenovirus vector containing a nucleotide sequence encoding the antibody disclosed herein. The vector can be administered to a subject and then enter the subject's cells in vivo, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the cell's genome, and subsequently, the cells express the antibody disclosed herein.

[0168] host cells

[0169] This disclosure provides a host cell containing the nucleic acids or vectors disclosed herein.

[0170] Any cell can be used as the host cell for the nucleic acids or vectors disclosed herein. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescans and Shigella; Bacilli, such as B. subtilis and B. licheniformis; and Pseudomonas, such as Pseudomonas aeruginosa. (aeruginosa); and Streptomyces. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0171] The host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present invention can be administered to a subject, and the host cells express the antibodies disclosed herein in vivo.

[0172] This invention provides a host cell in which any of the above-described vectors has been introduced. The invention further provides a method for preparing the antibody of the invention, wherein the method comprises a) culturing the host cell of the fourth aspect of the invention under conditions suitable for antibody production; and b) obtaining the antibody from the culture.

[0173] Composition

[0174] This disclosure provides compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein.

[0175] Reagent test kit

[0176] As described herein, this disclosure provides diagnostic methods for determining the expression level of ROR1. In one particular aspect, this disclosure provides kits comprising the antibodies disclosed herein or antigen-binding fragments thereof for performing these methods, and instructions for performing the methods of this disclosure (such as collecting tissues and / or performing screening and / or analyzing results).

[0177] The kit of the present invention may comprise one or more containers filled with the antibody or antigen-binding fragments disclosed herein.

[0178] The kit contains, or alternatively constitutes, essentially constitutes, or further constitutes, the ROR1 antibody composition disclosed herein (e.g., a monoclonal antibody) and instructions for use. The kit can be used to detect the presence of ROR1 in biological samples, such as any bodily fluid, including but not limited to sputum, serum, plasma, lymph, cystic fluid, urine, feces, cerebrospinal fluid, ascites, or blood, and includes biopsy samples of body tissue. Test samples can also be tumor cells, normal cells adjacent to a tumor, normal cells corresponding to a tumor tissue type, blood cells, peripheral blood lymphocytes, or combinations thereof. The test samples used in the above methods will vary based on the assay format, the nature of the detection method, and the tissue, cells, or extracts used as the test sample. Methods for preparing protein extracts or membrane extracts of cells are known in the art and can be readily adapted to obtain samples compatible with the system used.

[0179] In some aspects, the kit may comprise: one or more anti-ROR1 antibodies capable of binding to ROR1 in a biological sample (e.g., an antibody or antigen-binding fragment thereof having the same antigen-binding specificity as the anti-ROR1 antibody CSTONE-1); means for determining the expression level of ROR1 in the sample; and means for comparing the expression level of ROR1 in the sample with a standard. One or more anti-ROR1 antibodies may be labeled. Kit components (e.g., reagents) may be packaged in suitable containers. The kit may further comprise instructions for using the kit to detect ROR1. In some aspects, the kit comprises a first antibody that binds to ROR1, for example, which is attached to a solid support; and optionally; 2) a different second antibody that binds to ROR1 or the first antibody and is conjugated to a detectable label.

[0180] The kit may also contain, for example, buffers, preservatives, or protein stabilizers. The kit may further contain components necessary for detecting the detectable label, such as enzymes or substrates. The kit may also contain a control sample or a series of control samples that can be measured and compared with the test sample. Each component of the kit may be packaged in a separate container, and all the different containers may be in a single package along with instructions for interpreting the results of assays performed using the kit. The kits disclosed herein may have written product documentation on or included with the kit container. The written product describes how to use the reagents contained in the kit.

[0181] If appropriate, these recommended kit components can be packaged in a manner commonly used by those skilled in the art. For example, these recommended kit components can be provided in the form of solutions or liquid dispersions.

[0182] In one embodiment, a kit is provided for detecting ROR1 in biological samples, such as blood or tissue samples. For example, to confirm a cancer diagnosis in a subject, a biopsy may be performed to obtain a tissue sample for histological examination. Kits for detecting peptides typically contain anti-ROR1 antibodies. In a further embodiment, the antibody is labeled with a detectable portion (e.g., labeled with fluorescence, radioactivity, or enzyme).

[0183] In one embodiment, the kit includes instructional material disclosing the means of using the anti-ROR1 antibody. The instructional material may be written in electronic form (such as a computer floppy disk or optical disc) or visual (such as a video file). The kit may also include additional components to facilitate the application of the kit design. Thus, for example, the kit may additionally contain means for detecting labels (such as enzyme substrates for enzymatic labeling, filter sets for detecting fluorescent labels, suitable secondary labels such as secondary antibodies, etc.). The kit may additionally include buffers and other reagents conventionally used to practice the specific method. Such kits and suitable contents are well known to those skilled in the art.

[0184] In one implementation, the diagnostic kit includes an immunoassay. Methods for detecting ROR1 in biological samples typically involve contacting the biological sample with an anti-ROR1 antibody. This allows the antibody to specifically bind under immunoreactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0185] Conjugate

[0186] This disclosure provides conjugates comprising the antibody or antigen-binding fragment thereof disclosed herein and the chemical portion conjugated thereto.

[0187] In the context of this disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, toxin, therapeutic agent, detectable marker, protein, nucleic acid, lipid, nanoparticle, carbohydrate, or recombinant virus. Antibody conjugates are commonly referred to as "immunoconjugates." When a conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is commonly referred to as an "antibody-drug conjugate" or "ADC."

[0188] The terms "conjugated" or "linked" can refer to the formation of a single continuous polypeptide molecule from two polypeptides. In one embodiment, an antibody is linked to a chemical moiety. In another embodiment, the antibody linked to the chemical moiety is further linked to a lipid or other molecule, a protein, or a peptide to increase its half-life in vivo. Linkage can be performed chemically or recombinantly. In one embodiment, the linking is chemical, wherein a reaction between the antibody moiety and the chemical moiety has generated a covalent bond between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the chemical moiety.

[0189] The chemical moiety can be attached to the antibody of the present invention using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. The procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Peptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to result in the binding of the chemical moiety. Alternatively, the antibody can be derivatized to expose or attach additional reactive functional groups. Derivatization can involve attachment of any of a number of known linker molecules. The linker can be any molecule used to attach the antibody to the chemical moiety. The linker is capable of forming a covalent bond with both the antibody and the chemical moiety. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the chemical moiety are peptides, the linker can be attached via their side groups (such as by disulfide linkage with cysteine) to the constituent amino acid or to the α-carbon amino group and carboxyl group attached to the terminal amino acid.

[0190] In some cases, when an immunoconjugate reaches its target site, it is desirable to release a chemical moiety from the antibody. Therefore, in these cases, the immunoconjugate will contain a cleavable linker near the target site.

[0191] The cleavage of the linker to release the chemical moiety from the antibody can be facilitated by enzymatic activity or conditions experienced by the immunoconjugate within or near the target cell.

[0192] Given the numerous methods already reported for attaching various radiodiagnostic compounds, radiotherapy compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, those skilled in the art will be able to determine suitable methods for attaching a given agent to an antibody or other peptide.

[0193] The antibodies disclosed herein can be derivatized to or linked to another molecule (such as another peptide or protein). Typically, the antibody or a portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, the antibody can be functionally linked to one or more other molecular entities (through chemical coupling, genetic fusion, non-covalent association, or other means), such as another antibody (e.g., a bispecific antibody or a biantibody), a detection agent, a pharmaceutical formulation, and / or a protein or peptide that can mediate association between the antibody or an antibody portion and another molecule (such as a streptavidin core region or a multihistidine tag).

[0194] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same or different types). Suitable crosslinking agents include those that are heterobifunctional (having a suitable spacer such as m-maleimide benzoyl-N-hydroxysuccinimide) or homobifunctional (e.g., disuccinimide octanoate). Such linkers are commercially available.

[0195] In some embodiments of the conjugates disclosed herein, the chemical portion is selected from the group consisting of: therapeutic agents, detectable portions, and immunostimulatory molecules.

[0196] In some embodiments, the chemical component is a therapeutic agent. In some embodiments, the therapeutic agent includes, but is not limited to, immunomodulators, radioactive compounds, enzymes (e.g., perforin), chemotherapeutic agents (e.g., cisplatin), or toxins. In some embodiments, the therapeutic agent may be such as maytansine, gerdemycin, microtubule inhibitors such as microtubule binding agents (e.g., olprestatins), or minor groove binding agents such as chaziomycin.

[0197] Other suitable therapeutic agents include small molecule cytotoxic agents, which are compounds with a molecular weight of less than 700 Daltons that have the ability to kill mammalian cells. These compounds may also contain toxic metals capable of exerting cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, which are compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, chalcone, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, and trimetreate glucuronide. glucuronate), vincristine and doxorubicin; peptide cytotoxins, which are proteins or fragments thereof capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase and RNase; radionuclides, which are unstable isotopes of elements that decay with the simultaneous emission of one or more alpha or beta particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225 and astartaryin-213; chelating agents can be used to promote the association of these radionuclides with molecules or polymers thereof.

[0198] In some embodiments, the chemical component is the detectable component. In some embodiments, the detectable component may be selected from the group consisting of: biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent molecules, phosphorescent molecules, or chemiluminescent molecules. Detectable components for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

[0199] Antibodies can be conjugated to detectable biomarkers; for example, biomarkers detectable by ELISA, Western blotting, spectrophotometry, flow cytometry, immunohistochemical staining, immunofluorescence assays, chemiluminescence assays, radioimmunoassays, multiplex immunoassays, and microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optic examination, and laparoscopy). Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable biomarkers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent biomarkers are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP).

[0200] Antibody or antigen-binding fragments can also be conjugated to detectable enzymes, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When an antibody or antigen-binding fragment is conjugated to a detectable enzyme, it can be detected by adding additional reagents to produce a distinguishable reaction product. For example, in the presence of horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is visually detectable. Antibody or antigen-binding fragments can also be conjugated to biotin and detected by indirectly measuring avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to enzymes or fluorescent labels.

[0201] Antibodies can be fused to self-labeled protein tags (e.g., HaloTag). For example, protein tags can be cloned at the end of a constant region. HaloTag is a self-labeled protein tag derived from a bacterial enzyme (haloalkane dehalogenase) and designed to bind covalently to a synthetic ligand. In some cases, the synthetic ligand contains a chloroalkane linker attached to a fluorophore (such as a near-infrared fluorophore) (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

[0202] Antibodies can be labeled with magnetic reagents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0203] Paramagnetic particles, such as superparamagnetic iron oxide, can also be used as labels. Antibodies can also be labeled with predetermined peptide epitopes recognized by secondary reporter antibodies (such as leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, and epitope tags). In some embodiments, the labels are attached via spacer arms of varying lengths to reduce potential steric hindrance.

[0204] Antibodies can also be labeled with radioactively labeled amino acids. Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect the expression of target antigens by X-rays, emission spectroscopy, or other diagnostic techniques. Examples of labeling for peptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I.

[0205] In some implementations, immunostimulatory molecules are immune effector molecules that stimulate an immune response. For example, immunostimulatory molecules can be cytokines such as IL-2 and IFN-γ, chemokines such as IL-8, platelet-4, melanoma growth-stimulating protein, complement activators; viral / bacterial protein domains or viral / bacterial peptides.

[0206] Methods for diagnosis, diagnostic aids, detection, staging, prognostic determination, imaging, subject selection, and cancer treatment.

[0207] This disclosure provides a method for in vitro detection of ROR1 protein. In some cases, ROR1 expression is detected in biological samples. Samples can be any type of sample, including but not limited to blood samples, tissues from biopsies, autopsies, and pathological specimens. Biological samples further include bodily fluids such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0208] This disclosure also provides a method for determining whether a subject has ROR1-positive cancer, which involves contacting a sample from the subject with an anti-ROR1 antibody disclosed herein; and detecting the binding of the antibody to the sample to determine whether the subject has ROR1-positive cancer. An increase in antibody binding to a sample, compared to binding to a control sample, identifies the subject as having cancer. In some embodiments, the control sample is a sample from a subject without cancer. In specific embodiments, the sample is a blood or tissue sample.

[0209] In another embodiment, this disclosure provides a method for diagnosing or assisting in the diagnosis of cancer in a subject, comprising contacting a biological sample with an anti-ROR1 antibody or an antigen-binding fragment thereof disclosed herein; and detecting the presence of ROR1 in the biological sample, wherein the presence of ROR1 indicates cancer, and wherein the cancer is ROR1 positive. In some embodiments, the biological sample is obtained from the subject. In some embodiments, the method further comprises determining the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, the subject is selected for treatment with an anti-ROR1 therapy. In some embodiments, the method further comprises administering the anti-ROR1 therapy to the subject. In some embodiments, the subject is a person who has or is suspected of having cancer. In some embodiments, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

[0210] This disclosure also provides a method for staging ROR1-positive cancers in a subject, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof disclosed herein or a conjugate disclosed herein; and (ii) determining the level and / or location of ROR1 in the biological sample. In some embodiments, the biological sample is obtained from the subject.

[0211] This disclosure also provides a method for determining cancer prognosis in a subject, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof disclosed herein, or a conjugate disclosed herein; and (ii) detecting the presence of ROR1 in the biological sample, wherein the presence of ROR1 indicates a poor prognosis. In some embodiments, the method further comprises determining the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, the subject is selected for treatment with an anti-ROR1 therapy. In some embodiments, the biological sample is obtained from the subject. In some embodiments, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

[0212] This disclosure also provides a method for selecting a subject to be treated with an anti-ROR1 therapy, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof disclosed herein or a conjugate disclosed herein; and (ii) determining the expression level of ROR1 in the sample, wherein a subject is selected to be treated with the anti-ROR1 therapy if the expression level of ROR1 in the sample is at or above a predetermined threshold level. In some embodiments, the biological sample is obtained from the subject. In some embodiments, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

[0213] This disclosure also provides a method for treating cancer in a subject, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof disclosed herein or a conjugate disclosed herein; and (ii) determining the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, an anti-ROR1 therapy is administered to the subject; wherein the cancer is ROR1 positive. In some embodiments, the biological sample is obtained from the subject. In some embodiments, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

[0214] This disclosure also provides methods for detecting the presence of ROR1 in a sample or determining its level, comprising: (i) contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein or a conjugate disclosed herein; and (ii) detecting the presence of ROR1 bound by the antibody or conjugate or the level of ROR1 bound by the antibody or conjugate. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is obtained from a subject.

[0215] This disclosure also provides methods for imaging or identifying cancer cells expressing ROR1 in a sample, comprising: (i) contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein or a conjugate disclosed herein; and (ii) detecting the presence of ROR1 in the biological sample, wherein the presence of ROR1 indicates cancer cells. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is obtained from a subject.

[0216] In some embodiments of the methods disclosed herein for diagnosis, auxiliary diagnosis, detection, staging, prognosis determination, subject selection, and cancer treatment, the biological sample can be any sample, including but not limited to blood samples, tissues from biopsies, autopsies, and pathological specimens. Biological samples further include bodily fluids such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates. In a preferred embodiment, the biological sample is a tissue sample.

[0217] In some implementations, the presence, level, or location of ROR1 in biological samples is determined by ELISA, Western blotting, spectrophotometry, flow cytometry, immunohistochemical staining, immunofluorescence assay, chemiluminescence assay, radioimmunoassay, multiplex immunoassay, and microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optic examination, and laparoscopy). Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme-linked compounds, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI).

[0218] In a preferred embodiment, the presence or level of ROR1 in a biological sample is determined by a method selected from the group consisting of: flow cytometry, immunohistochemical staining, ELISA assay, Western blotting, immunofluorescence assay, chemiluminescence assay, radioimmunoassay, and multiplex immunoassay.

[0219] On the other hand, a method for detecting ROR1 in a sample is provided, comprising contacting a sample from a human with any of the aforementioned antibodies under conditions allowing antibodies to bind to ROR1, and detecting the bound antibodies. In one embodiment, a first antibody against ROR1 is immobilized on a solid support as a capture reagent, and a second antibody against ROR1 is used as a detection reagent. Relatedly, the amount of ROR1 in the sample is quantified by measuring the amount of bound antibody. The detection method can be used in a variety of diagnostic, prognostic, and monitoring methods, including methods for diagnosing ROR1-related conditions and methods for monitoring therapy administered with anti-ROR1 antibodies. In such methods, ROR1 levels at or above a certain threshold are associated with the presence of ROR1-positive cancer, while levels below said threshold indicate that a patient is unlikely to have ROR1-positive cancer.

[0220] The methods for setting appropriate thresholds for diagnosing disease states as described herein are well known in the art. For example, using the same protocol, ROR1 levels in samples from a sufficiently representative number of normal subjects (e.g., a healthy population without the condition to be tested) can be analyzed relative to ROR1 levels from a sufficiently representative number of diseased subjects (e.g., a group confirmed to have the disease or symptom). A threshold cutoff that distinguishes the majority of the normal population from the majority of the diseased population can be determined. Alternatively, useful endpoint values ​​for negative, indeterminate, and positive results can be determined from the data. For example, a normal range (indicating a negative result) can be determined, which includes the ROR1 of the majority of the normal population but excludes almost all diseased individuals. Accordingly, a range indicating a positive result can be determined, which includes the ROR1 of the majority of the diseased population but excludes almost all normal individuals. Appropriate endpoint values ​​for the thresholds can be determined to optimize the desired specificity or sensitivity, and overall medical and epidemiological factors can also be considered. Factors to consider include the clinical purpose of the IVD test and whether a high positive predictive value or a high negative predictive value is required, as well as the prevalence of the disease in the tested population.

[0221] In a preferred embodiment, the location of ROR1 in the biological sample is determined by a method selected from the group consisting of: immunohistochemical staining, immunofluorescence assay, chemiluminescence assay, and multiplex immunoassay.

[0222] In some embodiments, the anti-ROR1 antibody is directly labeled with a detectable marker. In another embodiment, the anti-ROR1 antibody (the first antibody) is unlabeled, and the second antibody or other molecule that can bind to the first antibody is labeled. As is well known to those skilled in the art, a specific type and class of second antibody is selected that can specifically bind to the first antibody. For example, if the first antibody is human IgG, the second antibody could be anti-human IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available.

[0223] Suitable labeling for antibodies or secondary antibodies includes various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium; and a non-limiting exemplary radioactive labeling includes… 125 I, 131 I, 35 S or 3H.

[0224] In an alternative embodiment, ROR1 in a biological sample can be determined by a competitive immunoassay using a ROR1 protein standard labeled with a detectable substance and an unlabeled anti-ROR1 antibody. In this assay, the biological sample, the labeled ROR1 protein standard, and the anti-ROR1 antibody are combined, and the amount of labeled ROR1 protein standard binding to the unlabeled antibody is measured. The amount of ROR1 in the biological sample is inversely proportional to the amount of labeled ROR1 protein standard binding to the anti-ROR1 antibody.

[0225] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, an anti-ROR1 antibody can be used to detect the presence or production of ROR1 in a biological sample. In another embodiment, the antibody can be used to detect the amount or level of ROR1 in a biological sample. In yet another embodiment, the antibody can be used to detect the location of ROR1 in a biological sample.

[0226] In some embodiments of the imaging methods disclosed herein, the detectable portion is selected from the group consisting of: biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent molecules, phosphorescent molecules, or chemiluminescent molecules. In preferred embodiments, the detectable portion is a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, and a contrast agent.

[0227] In some embodiments of the methods disclosed herein, the cancer is a ROR1-positive solid tumor or a ROR1-positive hematologic malignancy.

[0228] In some implementations, ROR1-positive solid tumors are selected from the group consisting of: breast cancer (such as triple-negative breast cancer), endometrial cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer, colorectal cancer, uterine cancer, cervical cancer, brain cancer, gastric cancer, bile duct cancer, chondrosarcoma, kidney cancer, thyroid cancer, and skin cancer.

[0229] In some implementations, ROR1-positive hematologic malignancies are selected from the group consisting of lymphoma and myeloma. In some implementations, lymphomas are selected from the group consisting of Hodgkin's B-cell lymphomas (such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma), non-Hodgkin's B-cell lymphomas (Burkit lymphoma, lymphoblastic lymphoma, anaplastic large cell lymphoma), mantle cell lymphoma, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. In some implementations, myeloma may include, but is not limited to, solitary myeloma, multiple myeloma, diffuse myeloma, extramedullary myeloma, and leukemic myeloma.

[0230] Medical use

[0231] This disclosure provides for the use of the antibodies or antigen-binding fragments thereof disclosed herein, or the conjugates disclosed herein, in the preparation of kits or compositions for the diagnosis or auxiliary diagnosis of cancer in a subject. In some embodiments, the subject is a person who has or is suspected of having cancer.

[0232] This disclosure provides antibodies or antigen-binding fragments thereof disclosed herein, or conjugates disclosed herein, for the diagnosis or auxiliary diagnosis of cancer in a subject. In some embodiments, the subject is a person who has or is suspected of having cancer.

[0233] This disclosure provides for the use of the antibodies or antigen-binding fragments thereof disclosed herein, or the conjugates disclosed herein, in the preparation of kits or compositions for staging ROR1-positive cancers in subjects.

[0234] This disclosure provides antibodies or antigen-binding fragments thereof disclosed herein, or conjugates disclosed herein, for staging ROR1-positive cancers in subjects.

[0235] This disclosure provides for the use of the antibodies or antigen-binding fragments thereof disclosed herein, or the conjugates disclosed herein, in the preparation of kits or compositions for determining the prognosis of a subject for cancer.

[0236] This disclosure provides antibodies or antigen-binding fragments thereof disclosed herein, or conjugates disclosed herein, for the purpose of determining cancer prognosis in subjects.

[0237] This disclosure provides the use of the antibodies or antigen-binding fragments thereof disclosed herein, or the conjugates disclosed herein, in the preparation of kits or compositions for imaging ROR1-positive cancers in subjects.

[0238] This disclosure provides antibodies or antigen-binding fragments thereof disclosed herein, or conjugates disclosed herein, for imaging ROR1-positive cancers in subjects.

[0239] This disclosure provides for the use of the antibodies or antigen-binding fragments thereof disclosed herein, or the conjugates disclosed herein, in the preparation of kits or compositions for selecting subjects to be treated with anti-ROR1 therapy.

[0240] This disclosure provides antibodies or antigen-binding fragments thereof disclosed herein, or conjugates disclosed herein, for use in selecting subjects to be treated with anti-ROR1 therapy.

[0241] This disclosure provides the use of the antibodies disclosed herein or their antigen-binding fragments or conjugates disclosed herein in the preparation of a kit or composition for treating a subject with cancer; wherein a biological sample obtained from a subject is contacted with the antibodies disclosed herein or their antigen-binding fragments or conjugates disclosed herein, and the expression level of ROR1 in the sample is determined, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, anti-ROR1 therapy is administered to the subject; and wherein the cancer is ROR1 positive.

[0242] This disclosure provides the antibody or antigen-binding fragment thereof disclosed herein or the conjugate thereof disclosed herein for treating a subject with cancer; wherein a biological sample obtained from the subject is contacted with the antibody or antigen-binding fragment thereof disclosed herein or the conjugate thereof disclosed herein, and the expression level of ROR1 in the sample is determined, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, anti-ROR1 therapy is administered to the subject; and wherein the cancer is ROR1 positive.

[0243] In some embodiments of the uses disclosed herein, the biological sample can be any sample, including but not limited to blood samples, tissues from biopsies, autopsies, and pathological specimens. Biological samples further include bodily fluids such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates. In a preferred embodiment, the biological sample is a tissue sample. In some embodiments, the presence, level, or location of ROR1 in the biological sample is determined by ELISA, Western blotting, spectrophotometry, flow cytometry, immunohistochemical staining, immunofluorescence assay, chemiluminescence assay, radioimmunoassay, multiplex immunoassay, and microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopy). Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme-linked molecules, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). In some implementations, the detectable portion is selected from the group consisting of: biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent molecules, phosphorescent molecules, or chemiluminescent molecules.

[0244] This disclosure further provides the use of the antibodies or antigen-binding fragments thereof disclosed herein, the nucleic acids disclosed herein, the vectors disclosed herein, the host cells disclosed herein, the compositions disclosed herein, the kits disclosed herein, or the conjugates disclosed herein in the preparation of a medicament or product for treating a subject with cancer.

[0245] This disclosure also provides the antibodies or antigen-binding fragments thereof disclosed herein, the nucleic acids disclosed herein, the vectors disclosed herein, the host cells disclosed herein, the compositions disclosed herein, the kits disclosed herein, or the conjugates disclosed herein for the treatment of cancer in a subject.

[0246] In some embodiments of the uses disclosed herein, the cancer is ROR1-positive cancer. In some embodiments, the cancer is a ROR1-positive solid tumor or a ROR1-positive hematologic malignancy.

[0247] In some implementations, ROR1-positive solid tumors are selected from the group consisting of: breast cancer (such as triple-negative breast cancer), endometrial cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer, colorectal cancer, uterine cancer, cervical cancer, brain cancer, gastric cancer, bile duct cancer, chondrosarcoma, kidney cancer, thyroid cancer, and skin cancer.

[0248] In some implementations, ROR1-positive hematologic malignancies are selected from the group consisting of lymphoma and myeloma. In some implementations, lymphomas are selected from the group consisting of Hodgkin's B-cell lymphomas (such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma), non-Hodgkin's B-cell lymphomas (Burkit lymphoma, lymphoblastic lymphoma, anaplastic large cell lymphoma), mantle cell lymphoma, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. In some implementations, myeloma may include, but is not limited to, solitary myeloma, multiple myeloma, diffuse myeloma, extramedullary myeloma, and leukemic myeloma.

[0249] Example

[0250] The following examples are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the invention in any way. These examples, together with the methods described herein, represent preferred embodiments and are exemplary, and are not intended to limit the scope of the invention. Variations and other uses thereof will be apparent to those skilled in the art, encompassing the spirit of the invention as defined by the scope of the claims.

[0251] Example 1. Immunization of animals

[0252] 1.1 Preparation of Immunogen

[0253] The following recombinant proteins and peptides were synthesized and used for animal immunization:

[0254] 1) Recombinant protein P4577, which is the ECD domain (amino acids 30-403) of human ROR-1 expressed in HEK293 cells, has the following amino acid sequence:

[0255] QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLS DKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKME (SEQ ID NO: 1)

[0256] 2) A mixture of CSTONE-1a and CSTONE-1b peptides having the following amino acid sequence:

[0257] CSTONE-1a peptide (amino acids 779-807 of human ROR-1)

[0258] SNLSNPRYPNYMFPSQGITPQGQIAGFIGC (SEQ ID NO: 2)

[0259] CSTONE-1b peptide (amino acid 779-800 of human ROR-1)

[0260] SNLSNPRYPNYMFPSQGITPQGC (SEQ ID NO: 3)

[0261] 1.2. Animal immunization

[0262] Six healthy New Zealand white rabbits (QINGDAO KANGDA BIOLOGICAL TECHNOLOGY) were immunized with a total of five injections according to the standard injection method. The immunization interval was 3+2+2+2 weeks. Three rabbits (K10047R, K10048R, and K10049R) were immunized with recombinant protein P4577. The other three rabbits (K10044D2, K10045D2, and K10046D2) were immunized with a mixture of CSTONE-1a and CSTONE-1b peptides. Before immunization, 0.1–0.2 mg of immunogen was mixed with Freund's adjuvant at a 1:1 ratio and thoroughly emulsified to ensure the immunization effect. The immunogen was emulsified with Freund's complete adjuvant for the first time, and with Freund's incomplete adjuvant for the subsequent second emulsification. Rabbit serum was collected during the immunization program for titer testing to evaluate the immune effect.

[0263] 1.3. Serum titer

[0264] Following the final immunization, a suitable amount of rabbit blood was collected and rabbit serum was prepared for titer testing. Serum titers were tested using a routine ELISA assay with immunogenic peptides or recombinant proteins as antigens. The results are shown in Table 1.

[0265] Table 1

[0266] serial number serum 1:250 1:1000 1:4000 1:16000 1:64000 1:256000 Wrapped Notes 1 K10047R 0.098 0.084 0.077 0.076 0.077 0.071 P4577 Before immunization 2 K10048R 0.105 0.078 0.082 0.089 0.078 0.07 P4577 Before immunization 3 K10049R 0.113 0.087 0.082 0.08 0.083 0.072 P4577 Before immunization 4 K10047R 0.863 0.319 0.15 0.098 0.085 0.081 P4577 After the third immunization 5 K10048R 2.695 2.392 1.679 0.835 0.346 0.164 P4577 After the third immunization 6 K10049R 1.798 0.984 0.413 0.173 0.106 0.083 P4577 After the third immunization 7 K10047R 1.092 0.553 0.214 0.116 0.09 0.079 P4577 After the 4th immunization 8 K10048R 2.737 2.311 1.622 0.829 0.297 0.169 P4577 After the 4th immunization 9 K10049R 2.077 1.229 0.5 0.216 0.109 0.08 P4577 After the 4th immunization 10 K10044D2 0.095 0.074 0.069 0.07 0.072 0.069 CSTONE-1a peptide Before immunization 11 K10045D2 0.095 0.074 0.07 0.069 0.074 0.073 CSTONE-1a peptide Before immunization 12 K10046D2 0.092 0.074 0.068 0.067 0.072 0.069 CSTONE-1a peptide Before immunization 13 K10044D2 1.052 0.646 0.305 0.146 0.1 0.093 CSTONE-1a peptide After the third immunization 14 K10045D2 1.913 0.66 0.396 0.212 0.123 0.104 CSTONE-1a peptide After the third immunization 15 K10046D2 1.249 0.499 0.222 0.11 0.089 0.082 CSTONE-1a peptide After the third immunization 16 K10044D2 1.109 0.712 0.354 0.16 0.099 0.087 CSTONE-1a peptide After the 4th immunization 17 K10045D2 1.815 1.108 0.521 0.215 0.158 0.097 CSTONE-1a peptide After the 4th immunization 18 K10046D2 0.974 0.666 0.299 0.141 0.094 0.088 CSTONE-1a peptide After the 4th immunization 19 K10044D2 0.097 0.076 0.069 0.069 0.07 0.066 CSTONE-1b peptide Before immunization 20 K10045D2 0.095 0.077 0.075 0.069 0.072 0.069 CSTONE-1b peptide Before immunization 21 K10046D2 0.118 0.092 0.086 0.085 0.082 0.08 CSTONE-1b peptide Before immunization 22 K10044D2 2.542 1.832 1.014 0.397 0.175 0.103 CSTONE-1b peptide After the third immunization 23 K10045D2 2.684 1.911 1.204 0.542 0.226 0.117 CSTONE-1b peptide After the third immunization 24 K10046D2 2.248 1.416 0.684 0.249 0.136 0.089 CSTONE-1b peptide After the third immunization 25 K10044D2 2.348 1.711 0.866 0.351 0.156 0.095 CSTONE-1b peptide After the 4th immunization 26 K10045D2 2.344 1.895 0.998 0.408 0.179 0.107 CSTONE-1b peptide After the 4th immunization 27 K10046D2 2.199 1.502 0.667 0.261 0.135 0.092 CSTONE-1b peptide After the 4th immunization

[0267] It can be seen that when the dilution ratio reaches 1:64000, the serum titers of the three rabbits K10048R, K10045D2 and K10044D2 are all positive.

[0268] Example 2. Antibody Screening

[0269] 2.1. Fusion and Subcloning

[0270] Following immunization, rabbits exhibiting the expected immune response (K10048R, K10045D2, K10044D2) were euthanized, and their spleens were immediately isolated. Live rabbit spleen cells were isolated as quickly as possible and fused with rabbit myeloma cells (240E1 cells, Abcam) to form hybridoma cells. Cell fusion was performed using the standard PEG method. After cell fusion, the cell mixture was supplemented with FBS and other nutrients and placed at an adjusted density in 96-well cell culture plates, where it was cultured for two to three weeks. After observing hybridoma cell growth and colony formation, the hybridoma culture supernatant was collected for ELISA detection, and positive hybridoma clones were screened.

[0271] Positive hybridoma clones were developed using the limiting dilution method, and positive monoclonal hybridoma cells were obtained.

[0272] 2.2. Screening of hybridomas

[0273] 129 positive clones were initially screened from 960 clones using ELISA. Using engineered chronic myeloid leukemia cell lines K562 (iCareAb) stably transfected with ROR1 or ROR2 to overexpress ROR1 and ROR2 respectively, 25 positive clones were screened from these 129 clones by IHC (immunohistochemistry). Three candidate clones were further screened from these 25 clones using human TMA samples via IHC. Finally, three candidate clones were selected for recombinant antibody development.

[0274] The three types of positive hybridoma cells were cultured for 2-3 days to ensure their health. A suitable amount of hybridoma cells were collected and lysed to prepare an antibody mRNA template. This template was used for RT-PCR to amplify the heavy and light chain genes (cDNA) of the antibody, and then, after sequencing confirmation, an expression vector (Abcam) was constructed. Mammalian HEK293 cells were transfected with the antibody expression plasmid to express the recombinant antibody.

[0275] The antibodies expressed by these three positive hybridoma cells were named CSTONE-1(CSD)-3, CSTONE-1(CSD)-2, and CSTONE-1(CSD)-1. The CSTONE-1(CSD)-1 antibody has the following amino acid sequence and nucleotide coding sequence.

[0276] The amino acid sequence of the heavy chain variable region

[0277] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAVTWVRQAPGKGLEWIGIIYTDASAYYATWAKGRFTISKTSTTVDLEITSPTTEDTATYLCSTGRVWGPGTLVTVSS (SEQ ID NO: 4)

[0278] amino acid sequence of HCDR1

[0279] SYAVT (SEQ ID NO: 12)

[0280] amino acid sequence of HCDR2

[0281] IIYTDASAYYATWAKG (SEQ ID NO: 13)

[0282] amino acid sequence of HCDR3

[0283] GRV (SEQ ID NO: 14)

[0284] amino acid sequence of heavy chain

[0285] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAVTWVRQAPGKGLEWIGIIYTDASAYYATWAKGRFTISKTSTTVDLEITSPTTEDTATYLCS TGRVWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPS TCSKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEK TISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 5)

[0286] Amino acid sequence of the light chain variable region

[0287] MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSVAVGGTVTINCQASQSIYNNKNLAWFQQKPGQPPKRLISAASNLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCLGEFSCSSGDCFAFGGGTEVVVK (SEQ IDNO: 6)

[0288] The amino acid sequence of LCDR1

[0289] QASQSIYNNKNLA (SEQ ID NO: 15)

[0290] The amino acid sequence of LCDR2

[0291] AASNLAS (SEQ ID NO: 16)

[0292] The amino acid sequence of LCDR3

[0293] LGEFSCSSGDCFA (SEQ ID NO: 17)

[0294] Amino acid sequence of the light chain

[0295] MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSVAVGGTVTINCQASQSIYNNKNLAWFQQKPGQPPKRLISAASNLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCLGEFSCSSGDCFAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 7)

[0296] Nucleotide sequence of the heavy chain variable region

[0297] ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGTTATGCAGTGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTTATACTGATGCTAGCGCATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGGAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTATGCAGTACGGGTAGAGTCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 8)

[0298] Nucleotide sequence of the heavy chain

[0299]

[0300] Nucleotide sequence of the light chain variable region

[0301] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGTAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTATTTATAATAACAAAAATTTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTCTGCTGCATCCAATCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGAATTTAGTTGTAGTAGTGGTGATTGTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA (SEQ ID NO: 10)

[0302] Nucleotide sequence of the light chain

[0303] ATGGACACGAGGGCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGTAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTATTTATAATAACAAAAATTTAGCCTGGTTT CAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTCTGCTGCATCCAATCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGAATTTAGTTGTAGTAGT GGTGATTGTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACC ACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG (SEQ ID NO: 11)

[0304] 2.3 Assay for binding of recombinant antibodies

[0305] To determine the binding activity of the recombinant antibodies CSTONE-1(CSD)-3, CSTONE-1(CSD)-2, and CSTONE-1(CSD)-1, IHC and Western blotting (WB) were performed. The antibody clones shown as "1A" and "1B" are two parallel clones derived from the same clone. For example, both CSTONE-1(CSD)-1-1A and CSTONE-1(CSD)-1-1B are derived from CSTONE-1(CSD)-1.

[0306] IHC experiments were performed on a variety of cell and tissue samples using procedures commonly used in the art. Briefly, cell lines from K-562 (iCareAb), K-562 ROR1 (iCareAb), K-562 ROR2 (iCareAb), MCF-7 (ATCC, Cat. No: HTB-22), and MDA-MB-231 (SIBS, Cat. No: HCHu227), and tissue samples from human esophagus, human colon, human stomach, human spleen, human bone marrow, human breast cancer, human colon cancer, and suspected TNBC tissue were prepared onto formalin-fixed and paraffin-embedded (FFPE) slides. For antigen retrieval, the FFPE slides were placed in staining jars containing Tris EDTA buffer, pH 9.0 (ab93684, 1:100 dilution), and the jars were placed in an autoclave at 110°C for 15–30 min. Next, FFPE slides were immersed in 3% hydrogen peroxide for 10 min and washed. Blocked at room temperature in 10% normal serum and 1% BSA in TBS solution for 30 min. Primary antibodies (CSTONE-1(CSD)-3, CSTONE-1(CSD)-2, and CSTONE-1(CSD)-1) were diluted with diluent at a ratio of 1:1000–1:2000 as required or according to instructions, added to the slides, and incubated overnight in a humidified chamber at 4°C. Goat anti-rabbit IgG H&L (HRP polymer) (ab 214880) was added to the slides as a secondary antibody and incubated at room temperature for 30 min. Freshly prepared DAB was added for chromogenic development (approximately 0.5–2 min). Hematoxylin dye solution was added for counterstaining (1–10 min), and the slides were rinsed in TBST for approximately 10 s. The slides were dehydrated with gradient alcohols and xylene or tissue clearing agents, dried, and then sealed with permanent tablets. IHC results of three recombinant antibodies are shown in Figure 1-6 The results are summarized in Table 2 below.

[0307] Table 2 and Figure 1-6The results showed that CSTONE-1(CSD)-3-1B exhibited specific staining on ROR1-positive K-562 ROR1 and MDA-MB-231 cells, and non-specific staining on ROR1-negative K-562, K-562 ROR2, and MCF-7 cells. CSTONE-1(CSD)-2-1A, CSTONE-1(CSD)-2-1B, CSTONE-1(CSD)-1-1A, and CSTONE-1(CSD)-1-1B exhibited specific staining on K-562 ROR1 cells, and negative or weak background staining on the other four cell types. CSTONE-1(CSD)-2-1A and CSTONE-1(CSD)-2-1B showed almost no staining on any human tissue.

[0308] Table 2

[0309] clone result CSTONE-1(CSD)-3-1A For cell sedimentation, K-562 ROR1, MCF-7, and MDA-MB-231 cells showed membrane and cytoplasmic staining, while the other two cell types showed weak staining. All normal and tumor tissues showed positive staining, such as immune cells, smooth muscle, and stromal tissue. Many breast cancers and other types of tumors show positive staining on stromal tissue; this is nonspecific staining. CSTONE-1(CSD)-3-1B The staining pattern is similar to CSD-3-1A, but the staining intensity is slightly stronger. CSTONE-1(CSD)-2-1A Of all the cells and tissues tested, only K-562 ROR1 cells showed positive staining. CSTONE-1(CSD)-2-1B Same staining as CSD-2-1A. CSTONE-1(CSD)-1-1A K-562 ROR1 cells showed sporadic membrane and cytoplasmic staining, while the other four cell types showed background staining. All human tissues showed weak staining or no staining. CSTONE-1(CSD)-1-1B The same staining as clone CSD-1-1A.

[0310] Western blot (WB) experiments were performed using ROR1-positive K-562 ROR1 cells using procedures commonly used in the art. Briefly, lysates from K-562 ROR1 cells were collected for Western blotting. Antibodies diluted 1:10 or 1:40 (CSTONE-1(CSD)-3, CSTONE-1(CSD)-2, and CSTONE-1(CSD)-1) were used as primary antibodies, and goat anti-rabbit IgG H&L (HRP polymer) (ab 214880) was used as secondary antibodies. WB results for the three recombinant antibodies are shown below. Figure 7 As shown.

[0311] Figure 7 The results showed that a 130 kDa band (corresponding to ROR1) was observed for CSTONE-1(CSD)-1-1A and CSTONE-1(CSD)-1-1B, a nonspecific band was observed for CSTONE-1(CSD)-3-1A and CSTONE-1(CSD)-3-1B, and no obvious band was observed for CSTONE-1(CSD)-2-1A and CSTONE-1(CSD)-2-1B.

[0312] The results of IHC and WB indicate that CSTONE-1(CSD)-1 has better binding activity and specificity against ROR1-positive cells and tissues than CSTONE-1(CSD)-3 and CSTONE-1(CSD)-2, and is worthy of further development.

[0313] Example 3. Recombinant antibody CSTONE-1(CSD)-1 for IHC

[0314] To confirm the sensitivity and specificity of CSTONE-1 (CSD)-1 in detecting ROR1-positive tumors, membrane ROR1 IHC staining was performed using FFPE slides from various cell lines (K-562 (iCareAb), K-562 ROR1 (iCareAb), K-562 ROR2 (iCareAb), MCF-7 (ATCC, Cat. No: HTB-22), and MDA-MB-231 (SIBS, Cat. No: HCHu227) cells) and human tumor TNBC (Bioaitech, China). The entire staining process was performed on a Leica Bond III, and the detailed staining protocol is as follows:

[0315] Cut 4 μm sections and stain with Leica Bond III (Leica Biosystems). Pretreat slides with antigen retrieval ER2 (pH 9.0) (40 min), block with 3% hydrogen peroxide (5 min), then treat with primary antibody (10 ug / ml) of CSTONE-1 (CSD)-1 mAb for 30 min, and detect with BOND Polymer Detection (DS9800) for 10 min.

[0316] The results are as follows Figure 8 and Figure 9 As shown. To quantify the ROR1 membrane expression level in human TNBC FFPE slides, the histochemical score (H-score, a numerical value expressed as a weighted sum of staining percentages, interpreting both staining intensity and the percentage of cells at that intensity) was calculated using the following formula: H-score = (3 x percentage of cells with strong staining) + (2 x percentage of cells with moderate staining) + (1 x percentage of cells with mild staining).

[0317] Figure 8 The IHC results showed that CSTONE-1 (CSD)-1 had clear membrane staining in multiple human TNBC FFPE samples and obtained different H-scores for six different human TNBC FFPE slides (145, 80, 50, 30, 15 and 0, respectively), indicating different ROR1 expression levels in human TNBC samples. Figure 9 The IHC results showed that positive IHC signals were detected in ROR1-positive K562-ROR1 and MDA-MB-231 cells, while no IHC signals were detected in ROR1-negative K562, K562-ROR2, and MCF-7 cells. These results indicate that CSTONE-1 (CSD)-1 has high sensitivity and specificity for ROR1-positive cell lines.

[0318] Example 4. Recombinant antibody CSTONE-1(CSD)-1 co-localized via IHC and RNAscope® ISH

[0319] To determine the distribution of ROR1 protein in tumor cells, IHC and ISH (in situ hybridization) were performed using various TNBC tumor FFPE slides (such as BR1458, BR1282, and BR1474) from human TNBC patient-derived tumor xenograft (PDX) models (CrownBio Beijing). IHC experiments were performed using TNBC PDX tumor FFPE slides as described in Example 3. ISH experiments were performed using TNBC PDX tumor FFPE slides following the ACD RNAscope® SOP. The probe used was RNAscope® Probe-Hs-ROR1-alltv (ACD, 411091).

[0320] ROR1 expression in a sample was determined using tumor proportion score (TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. Tumor cell membrane ROR1 expression was quantified based on the H-score. The H-score was calculated using the following formula: H-score = (3 x percentage of cells with strong staining) + (2 x percentage of cells with moderate staining) + (1 x percentage of cells with light staining).

[0321] Results from FFPE slides of human TNBC PDX models BR1458, BR1282, and BR1474 are shown in Figure 10-12 And in Table 3.

[0322] Table 3

[0323] These results demonstrate that the ROR1 protein detected in tumor samples by IHC staining with CSTONE-1 (CSD)-1 (in this example, a diffuse membranous stain) is reflected by the detection of ROR1 mRNA in the cytoplasm of tumor cells (identified as punctate mRNAs at the points where the probe binds to the mRNA transcript). ISH mRNA distribution confirms the IHC signal, demonstrating the good specificity of CSTONE-1 (CSD)-1 binding to ROR1 in FFPE tissue samples.

[0324] Example 5. Binding of recombinant antibody CSTONE-1 (CSD)-1 to human ROR1

[0325] To determine the binding affinity and specificity of CSTONE-1 (CSD)-1 to human ROR1 protein, ELISA and Western blotting were performed. For Western blotting, lysates from K562, K562-ROR1, K562-ROR2, MCF-7, and MDA-MB-231 cells were used. The experiment consisted of three groups: a, b, and c. In group a, CSTONE-1 (CSD)-1 was used as the primary antibody. In group b, anti-GAPDH and anti-fouling protein antibodies were used as primary antibodies. In group c, CSTONE-1 (CSD)-1 was used as the primary antibody. Western blotting results are shown below. Figure 13 As shown.

[0326] ELISA was performed using recombinant protein P4577 (the ECD domain of human ROR as described in the examples) as the antigen. Briefly, the antigen was coated overnight at 4°C. Samples were added to serial dilutions starting at 4 μg / ml (purified antibody) and incubated at room temperature for 1.5 hours. AffiniPure goat anti-rabbit IgG peroxidase and F(ab')2 fragment-specific secondary antibody were added at room temperature for 1 hour. Substrate solution was added and development was performed at room temperature for 5 minutes. Absorbance was measured at 405 nm. ELISA results are as follows. Figure 14 As shown.

[0327] Figure 13 The results showed that a 130 kDa band representing the ROR1 protein was observed in ROR1-positive K562-ROR1 and MDA-MB-231 cells, but not in ROR1-negative K562, K562-ROR2 and MCF-7 cells, demonstrating the high specificity of CSTONE-1(CSD)-1 binding to ROR1. Figure 14 The EC50 of CSTONE-1(CSD)-1 antibody binding to ROR1 was 26.44 ng / ml (approximately 2.3 x 10⁻¹⁰ M), demonstrating the high binding affinity of CSTONE-1(CSD)-1 to the ROR1 epitope.

[0328] Example 6. Identification of solid tumors expressing ROR1 using the anti-ROR1 monoclonal antibody CSTONE-1 (CSD)-1

[0329] This embodiment uses solid tumor samples expressing ROR1 to determine whether the anti-ROR1 monoclonal antibody CSTONE-1 (CSD)-1 can detect endogenous ROR1 in tissues such as diseased tissues.

[0330] The purified anti-ROR1 monoclonal antibody CSTONE-1 (CSD)-1 was used for IHC assays of formalin-fixed-paraffin-embedded (FFPE) samples from non-small cell lung cancer (NSCLC), colorectal cancer (CRC), triple-negative breast cancer (TNBC), pancreatic cancer, endometrial cancer, ovarian cancer, gastric cancer, and esophageal squamous cell carcinoma (ESCC) tissues.

[0331] IHC samples were prepared as described in Example 3 above. ROR1 on the cell surface of tumor tissues was scored by a qualified pathologist. Specifically, ROR1 was found to be highly expressed in subgroups of patients with NSCLC, CRC, TNBC, pancreatic cancer, endometrial cancer, ovarian cancer, gastric cancer, and ESCC tissues. Figure 15 and Figure 16 In this study, 45%, 60%, 50%, 30%, 40%, 50%, 60%, and 40% of NSCLC, CRC, TNBC, pancreatic cancer, endometrial cancer, ovarian cancer, gastric cancer, and ESCC had tumor cell membrane ROR1 expression H-scores ≥50 (Table 4). Figure 16 Clear cell surface staining was observed in tissues of non-small cell lung cancer (NSCLC), colorectal cancer (CRC), triple-negative breast cancer (TNBC), pancreatic cancer (PC), endometrial cancer (EC), ovarian cancer, and gastric and esophageal squamous cell carcinoma (ESCC). These results demonstrate that CSTONE-1 (CSD)-1 effectively detects endogenous ROR1 in tissues.

[0332] Table 4

[0333] Tumor types H-score ≥ 50 H-score ≥10 H-score ≥1 NSCLC 45% (9 / 20) 55% (11 / 20) 75%(15 / 20) CRC 60% (6 / 10) 60% (6 / 10) 60% (6 / 10) TNBC 50% (5 / 10) 60% (6 / 10) 60% (6 / 10) pancreatic cancer 30% (6 / 20) 60% (12 / 20) 70% (14 / 20) Endometrial cancer 40% (4 / 10) 40% (4 / 10) 40% (4 / 10) Ovarian cancer 50% (5 / 10) 70% (7 / 10) 90% (9 / 10) Stomach cancer 60% (6 / 10) 60% (6 / 10) 70% (7 / 10) ESCC 40% (4 / 10) 50% (5 / 10) 90% (9 / 10)

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to ROR1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR 1-3 having amino acid sequences as shown in SEQ ID NO: 12-14, and the VL comprises LCDR 1-3 having amino acid sequences as shown in SEQ ID NO: 15-17.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 4, and the VL comprises the amino acid sequence shown in SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody has a subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fab'-SH, Fv, scFv and ds-scFv.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody is a monoclonal antibody.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody is a rabbit antibody or a humanized antibody.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 5, and the light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

7.

10. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-9.

11. The nucleic acid of claim 10, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 8, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

10.

12. The nucleic acid according to claim 10 or 11, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

11.

13. A vector comprising the nucleic acid according to any one of claims 10-12.

14. A host cell comprising the nucleic acid according to any one of claims 10-12 or the vector according to claim 13.

15. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-9.

16. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-9.

17. A conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-9, and a chemical moiety conjugated thereto.

18. The conjugate according to claim 17, wherein the chemical portion is a detectable portion.

19. The conjugate according to claim 18, wherein the detectable portion is selected from the group consisting of: biotin, streptavidin, enzymes or their catalytically active fragments, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent molecules, phosphorescent molecules or chemiluminescent molecules.

20. Methods for diagnosing or assisting in the diagnosis of cancer, including: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a conjugate according to any one of claims 17-19; and (ii) Detecting the presence of ROR1 in the biological sample, wherein the presence of ROR1 indicates the cancer. The cancer in question is ROR1 positive.

21. The method of claim 20, wherein the subject is a person who has or is suspected of having cancer.

22. A method for staging ROR1-positive cancers in subjects, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a conjugate according to any one of claims 17-19; and (ii) Determine the level and / or location of ROR1 in the biological sample.

23. A method for determining the prognosis of a subject's cancer, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a conjugate according to any one of claims 17-19; and (ii) Detect the presence of ROR1 in the biological sample, wherein the presence of ROR1 indicates a poor prognosis.

24. A method for selecting subjects to be treated with anti-ROR1 therapy, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a conjugate according to any one of claims 17-19; and (ii) Determine the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, the subject is selected for treatment with anti-ROR1 therapy.

25. A method for treating a subject's cancer, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a conjugate according to any one of claims 17-19; and (ii) Determine the expression level of ROR1 in the sample, wherein if the expression level of ROR1 in the sample is at or above a predetermined threshold level, administer anti-ROR1 therapy to the subject; The cancer in question is ROR1 positive.

26. A method for detecting the presence of ROR1 in a sample or determining its level, comprising: (i) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a conjugate according to any one of claims 17-19; and (ii) Detect the presence or level of ROR1 bound by the antibody or the conjugate. Optionally, the sample is a biological sample.

27. A method for imaging or identifying cancer cells expressing ROR1 in a sample, comprising: (i) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a conjugate according to any one of claims 17-19; and (ii) Detecting the presence of ROR1 in the biological sample, wherein the presence of ROR1 indicates the cancer cells. Optionally, the sample is a biological sample.

28. The method according to any one of claims 20-27, wherein the biological sample is obtained from the subject.

29. The method according to any one of claims 20-28, wherein the biological sample is a tissue sample.

30. The method according to any one of claims 20-29, wherein the presence or level of ROR1 in the biological sample is determined by a method selected from the group consisting of: flow cytometry, immunohistochemical staining, ELISA assay, Western blotting, immunofluorescence assay, chemiluminescence assay, radioimmunoassay, and multiplex immunoassay.

31. The method of any one of claims 22, wherein the location of ROR1 in the biological sample is determined by a method selected from the group consisting of: immunohistochemical staining, immunofluorescence assay, chemiluminescence assay, and multiplex immunoassay.

32. The method according to any one of claims 20-25, 27-31, wherein the cancer is a ROR1-positive solid tumor selected from the group consisting of breast cancer (such as triple-negative breast cancer), endometrial cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer, colorectal cancer, uterine cancer, cervical cancer, brain cancer, gastric cancer, bile duct cancer, chondrosarcoma, kidney cancer, thyroid cancer, and skin cancer, or a ROR1-positive hematologic malignancy selected from the group consisting of lymphoma and myeloma.

33. The method of claim 32, wherein the lymphoma is selected from the group consisting of: Hodgkin B-cell lymphoma, non-Hodgkin B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma.

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