WO2025026204A1

WO2025026204A1 - Antibodies against ror1 and uses thereof

Info

Publication number: WO2025026204A1
Application number: PCT/CN2024/107761
Authority: WO
Inventors: Zhijin Ye; Hui-han HU; Archie Ngai-Chiu TSE; Jianxin Yang
Original assignee: Tuo Shi Pharmaceuticals Shanghai Co Ltd; CStone Pharmaceuticals Suzhou Co Ltd; Cstone Pharmaceuticals Vistra Cayman Ltd
Current assignee: Tuo Shi Pharmaceuticals Shanghai Co Ltd; CStone Pharmaceuticals Suzhou Co Ltd; Cstone Pharmaceuticals Vistra Cayman Ltd
Priority date: 2023-07-28
Filing date: 2024-07-26
Publication date: 2025-02-06
Anticipated expiration: 2026-01-28

Abstract

The antibodies against ROR1 and uses thereof, specifically antibodies that specifically binds to ROR1 or an antigen binding fragment thereof, nucleic acids encoding the antibodies, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or the vectors. The compositions, kits and conjugates comprise the antibodies, and medical uses of the antibodies.

Description

ANTIBODIES AGAINST ROR1 AND USES THEREOF

The present application claims priority to PCT Application No. PCT/CN2023/109898 filed on July 28, 2023, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to antibodies against ROR1, and uses of such antibodies, in particular their use in cancer detection or diagnosis and in guiding cancer treatment.

BACKGROUND OF THE INVENTION

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, type I membrane protein. ROR1 has 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 frizzled domain and a kringle domain. ROR1 is highly expressed during embryonic and infant development, and the expression level decreases significantly in children and adults. The expression of ROR1 was significantly increased in a variety of blood cancers and solid tumors.

ROR1 expression attenuates during fetal development and, with few exceptions, becomes negligible on most postpartum tissues. In contrast, ROR1 is expressed by multiple human cancers, particularly those that are less differentiated, and is associated with early relapse after therapy or metastasis. Studies using flow cytometry demonstrated cell surface expression of ROR1 in multiple types of cancers including B-CLL, mantle cell lymphoma (MCL) , and a subset of B-cell acute lymphoblastic leukemia (ALL) . Expression of ROR1 enhances tumor cell growth and survival, and promotes epithelial-mesenchymal transition and metastasis of tumors. High ROR1 expression is correlated with shorter overall and metastasis-free survival in triple-negative breast cancer, lung adenocarcinoma, ovarian cancer, as well as other types of cancers. Many data show that ROR1 plays an important role in promoting tumor growth and metastasis, inducing drug resistance, and inhibiting apoptosis.

Due to its expression pattern and function in tumor progression, ROR1 has become a potential biomarker and target for tumor diagnosis and tumor therapy. There exists a need in the art for developing antibodies against ROR1.

SUMMARY OF THE INVENTION

The present disclosure provides a novel antibody targeting ROR1 or antigen binding fragments thereof, which can be used for diagnosis or auxiliary diagnosis, staging and imaging of a ROR1 positive cancer, determination of prognosis of a cancer in a subject, and providing guidance for treatment of a ROR1 positive cancer.

In an aspect, the present disclosure provides an antibody that specifically binds to ROR1, or an antigen binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 12-14 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 15-17 respectively.

In some embodiments of the antibody or the antigen binding fragment thereof disclosed herein, 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 to 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 to SEQ ID NO: 6.

In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 6.

In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

In some embodiments, the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

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 some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the antibody is a rabbit antibody or a humanized antibody.

In some embodiments, the antibody comprises a 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 to SEQ ID NO: 5 and a 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 to SEQ ID NO: 7.

In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof 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 to 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 to SEQ ID NO: 10.

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 to 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 to SEQ ID NO: 11.

In still another aspect, the present disclosure provides a vector comprising the nucleic acid disclosed herein.

In yet another aspect, the present disclosure provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

In another aspect, the present disclosure provides a composition comprising the antibody or the antigen binding fragment thereof disclosed herein.

In still another aspect, the present disclosure provides a kit comprising the antibody or the antigen binding fragment thereof disclosed herein.

In yet another aspect, the present disclosure provides a conjugate, comprising the antibody or the antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

In some embodiments, the chemical moiety is a detectable moiety.

In some embodiments, the detectable moiety is selected from the group consisting of biotins, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent or chemiluminescent molecules.

In another aspect, the present disclosure provides a method for the diagnosis or auxiliary diagnosis of a cancer in a subject comprising:

(i) contacting a biological sample with the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein; and

(ii) detecting the presence of ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of the cancer, wherein the cancer is ROR1 positive.

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, selecting the subject for treatment with an anti-ROR1 therapy.

In some embodiments, the method further comprises administering an anti-ROR1 therapy to the subject.

In some embodiments, the subject is a human subject having or being suspected to have a cancer.

In some embodiments, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

In still another aspect, the present disclosure provides a method for staging a ROR1 positive cancer in a subject comprising:

(ii) determining the level and/or location of ROR1 in the biological sample.

In some embodiments, the biological sample is obtained from the subject.

In yet another aspect, the present disclosure provides a method for determining the prognosis of a cancer in a subject comprising:

(ii) detecting the presence of ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of a poor prognosis.

In some embodiments, the biological sample is obtained from the subject.

In another aspect, the present disclosure provides a method for selecting a subject for treatment with an anti-ROR1 therapy comprising:

(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, selecting the subject for treatment with an anti-ROR1 therapy.

In some embodiments, the biological sample is obtained from the subject.

In yet another aspect, the present disclosure provides a method for treating a cancer in a subject comprising:

(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, administering to the subject an anti-ROR1 therapy;

wherein the cancer is an ROR1-positive.

In some embodiments, the biological sample is obtained from the subject.

In yet another aspect, the present disclosure provides a method for detecting the presence or determining the level of a ROR1 in a sample comprising:

(i) contacting the sample with the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein; and

(ii) detecting the presence of ROR1 or the level of ROR1 bound by the antibody or the conjugate.

In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is obtained from the subject.

In yet another aspect, the present disclosure provides a method for imaging/identifying a cancer cell expressing ROR1 in a sample comprising:

(ii) detecting the presence of a ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of the cancer cell.

In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the biological sample is a tissue sample.

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

In some embodiments, the location of the 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.

In another aspect, the present disclosure provides a method for imaging a ROR1 positive cancer in a subject comprising:

(a) administering the antibody or antigen binding fragment thereof disclosed herein to the subject, wherein the antibody is conjugated to a detectable moiety, and

(b) detecting the presence of the detectable moiety.

In some embodiments, the cancer is ROR1-positve 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, cholangiocarcinoma, chondrosarcoma, kidney cancer, thyroid cancer, and skin cancer, or ROR1-positive hematological malignancies selected from the group consisting of lymphoma and myeloma.

In some embodiments, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Figure 1 shows IHC staining of CSTONE-1 (CSD) -1-1A on multiple tumor cell lines.

Figure 2 shows IHC staining of CSTONE-1 (CSD) -1-1A on multiple human tissues.

Figure 3 shows IHC staining of CSTONE-1 (CSD) -2-1A on multiple tumor cell lines.

Figure 4 shows IHC staining of CSTONE-1 (CSD) -2-1A on multiple human tissues.

Figure 5 shows IHC staining of CSTONE-1 (CSD) -3-1B on multiple tumor cell lines.

Figure 6 shows IHC staining of CSTONE-1 (CSD) -3-1B on multiple human tissues.

Figure 7 shows WB results of 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.

Figure 8 shows IHC staining of CSTONE-1 (CSD) -1 on multiple human TNBC FFPE samples and H-scores calculated by staining intensity.

Figure 9 shows IHC staining of CSTONE-1 (CSD) -1 on multiple tumor cell lines.

Figure 10 shows distributions of ROR1 protein by IHC and ROR1 mRNA by ISH in FFPE sample from human TNBC PDX model BR1458.

Figure 11 shows distributions of ROR1 protein by IHC and ROR1 mRNA by ISH in FFPE sample from human TNBC PDX model BR1282.

Figure 12 shows distributions of ROR1 protein by IHC and ROR1 mRNA by ISH in FFPE sample from human TNBC PDX model BR1474.

Figure 13 shows WB results of CSTONE-1 (CSD) -1 for multiple tumor cell lines.

Figure 14 shows EC50 of CSTONE-1 (CSD) -1 binding to ROR1 as measured by ELISA.

Figure 15 shows H-score of the tested tumor samples, as analyzed by IHC with anti-RORl monoclonal antibody CSTONE-1 (CSD) -1.

Figure 16 shows expression of ROR1 in the tested tumor samples, as analyzed by IHC with anti-RORl monoclonal antibody CSTONE-1 (CSD) -1.

DETAILED DESCRIPTION OF THE INVENTION

The aforementioned features and advantages of the invention as well as additional features and advantages thereof will be more clearly understood hereafter as a result of a detailed description of the following embodiments when taken in conjunction with the drawings.

The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present invention. The embodiments shall not be construed to limit the scope of the present invention. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds., "Amultilingual 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) , as well as the general background art cited above.

Definitions

As used herein, singular forms “a” , “and, ” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an antibody” includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth.

Unless indicated or defined otherwise, the term "comprise" , and variations such as "comprises" and "comprising" , should be understood to imply the inclusion of a stated elements or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

As used herein, the term “antibody” refers to an immunoglobulin molecule which has ability to specifically bind to a specific antigen. Such molecule often comprises two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains of antibodies contain a binding domain that interacts with an antigen. The constant regions of antibodies may 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 pathway of complement activation.

The heavy chain of immunoglobulins can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (fragment crystallizable) . The Fd region comprises the VH and CH1 domains and, in combination with the light chain, forms Fab (antigen-binding fragment) . The Fc fragment is responsible for the immunoglobulin effector functions, which includes, for example, complement fixation and binding to cognate Fc receptors of effector cells. The hinge region, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer that allows the Fab portion to move freely in space relative to the Fc region. The hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses.

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 includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges. The lower hinge region joins the amino terminal end of, and includes residues in the CH2 domain. Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may affect the effector functions of the Fc portion of the antibody.

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

The assignment of amino acids to each VL and VH domain is in accordance with any conventional definition of CDRs. Conventional 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 &Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 878-883, 1989) ; a composite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular’s antibody modelling software; and the CONTACT definition of Martin et al. (world wide web 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. The present disclosure can use CDRs defined according to any of these numbering systems, although preferred embodiments use Kabat defined CDRs.

Based on the amino acid sequence of heavy chain constant regions of the antibody, an immunoglobulin molecule 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, IgA2, etc. The light chain of the antibody can be classified as a lambda (λ) chain or a kappa (κ) chain, based on the amino acid sequence of the light chain.

The term "antibody" as used herein should be understood in its broadest meaning, and includes monoclonal antibodies (including full-length monoclonal antibodies) , polyclonal antibodies, antibody fragments, and multi-specific antibodies containing at least two different antigen binding regions (e.g., bispecific antibodies) . The antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in Fc regions, and mutations in glycosylation sites. Antibodies also include post-translation modified antibodies, fusion proteins containing the antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit desired biological activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, each antibodies constituting the population are the same, except for possible naturally occurring mutations in small amount. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, and should not be interpreted as requiring production of antibodies by any specific method.

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 to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

Examples of antigen binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR) ; (ix) a heavy chain antibody, containing a single heavy chain variable region and two heavy chain constant domains; and (x) a Fab’ -SH fragment, a modified form of the Fab’ fragment in which the sulfur (S) atom of a cysteine residue in the Fab’ fragment is modified to carry a thiol group (-SH) . Furthermore, although the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ) . Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment" of an antibody. Furthermore, the term also includes a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) , which forms an antigen binding region together with a complementary light chain polypeptide, and a modified version of any of the foregoing fragments, which retains antigen binding activity.

These antigen binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

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

Avidity is the measure of the strength of binding between an antibody and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody and the number of pertinent binding sites present on the antibody. Typically, an antibody will bind to an antigen with a dissociation constant (KD) of 10^-5 to 10 ^- ¹² M or less, and preferably 10^-7 to 10 ^-12 M or less and more preferably 10 ^-8 to 10 ^-12 M, and/or with a binding affinity of at least 10⁷ M ^-1, preferably at least 10⁸ M ^-1, more preferably at least 10⁹ M ^-1, such as at least 10¹² M ^-1. Any K_D value greater than 10 ^-4 M is generally considered to indicate non-specific binding. Specifically binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA) , enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.

The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the smallest binding site of an antibody and therefore is the specific target of the antibody or antigen binding fragment thereof.

As used herein, the term “sequence identity” refers to the extent to which two sequences (amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X%identical to SEQ ID NO: Y” refers to X%identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X%of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, 1988) , FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997) , BLASTP, BLASTN, or GCG (Devereux et al., 1984) .

Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly 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.

Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

The term “pharmaceutically acceptable” means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof and/or that such carrier or excipient is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to humans.

As used herein, the terms "treatment" , "treating" , “treat” , and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptom of the disease. "Treatment" , as used herein, may include treatment of a disease or disorder (e.g. cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease) ; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the antibodies or compositions or conjugates disclosed herein to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with diseases (e.g., cancers) .

As used herein, the term “diagnosis” means detecting a disease or determining the stage or degree of a disease. Usually, a diagnosis of a disease is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or disorder. Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease, e.g., there may be differential diagnoses that can be inferred from a diagnostic factor or symptom. Likewise, there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease. The term “diagnosis” also encompasses determining the therapeutic effect of a drug therapy or predicting the pattern of response to a drug therapy. The diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical arts for a particular disease.

The term “prognosis” as used herein refers to a prediction of the probable course and outcome of a clinical condition or disease. A prognosis is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease. The phrase “determining the prognosis” as used herein refers to the process by which the skilled artisan 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. Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.

The term “detection” includes any means of detecting, including direct and indirect detection.

The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

The term "effective amount" as used herein means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

The term “subject” , as used herein, refers to any mammalian subject for whom diagnosis, treatment or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc.

The term “biological sample” is meant a collection of similar cells obtained from a subject or patient. A biological sample can be a tissue or a cell sample. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The biological sample can also be obtained from in vitro tissue or cell culture. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. Examples of biological samples herein include, but are not limited to, tumor biopsies, circulating tumor cells, serum or plasma, circulating plasma proteins, ascitic fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, as well as preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

The term “biomarker” as used herein refers to an indicator, e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample, for example, ROR1. The biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and/or clinical features. In some embodiments, a 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 numbers) , polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications) , carbohydrates, and/or glycolipid-based molecular markers.

The terms “level, ” “level of expression, ” or “expression level” in general are used interchangeably and generally refer to the amount of a polynucleotide, mRNA, or an amino acid product or protein in a biological sample. “Expression” generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, according to the invention “expression” of a gene (e.g., the ROR1 gene) may refer to transcription into a polynucleotide, translation into a protein, or even posttranslational modification of the protein. Fragments of the transcribed polynucleotide, the translated protein, or the post-translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the protein, e.g., by proteolysis. In some embodiments, “expression level” refers to amount of a protein (e.g., ROR1) in a biological sample as 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, for example Fluorescence-Activated Cell Sorting (FACS^TM) , or Enzyme-Linked Immunosorbant Assay (ELISA) .

Anti-ROR1 antibodies

The present disclosure provides an antibody that specifically binds to ROR1, or an antigen binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 12-14 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 15-17 respectively.

In some embodiments, CDR sequences are defined according to Kabat numbering system.

When CDR sequences are defined according to Kabat numbering system, the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth 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 comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 15 (QASQSIYNNKNLA) , SEQ ID NO: 16 (AASNLAS) and SEQ ID NO: 17 (LGEFSCSSGDCFA) respectively.

In some embodiments, 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 to 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 to SEQ ID NO: 6.

In some embodiments, the VH comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 4 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to ROR1. In some embodiments, the VL comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 6 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to ROR1.

The functional variant comprises or consists of 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 to the amino acid sequence of the parent polypeptide.

In the context of the functional variant, the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of the inserted, deleted and/or substituted amino acid can be 1-20, preferably 1-10, more preferably 1-7, still more preferably 1-5, and most preferably 1-2. In a preferred embodiment, the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the insertion, deletion and/or substitution can be performed at framework (FR) regions, e.g., at FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) . Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly 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, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

In a preferred embodiment, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 6.

Based on the amino acid sequence of heavy chain constant regions of the antibody, a immunoglobulin molecule 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, IgA2, etc. The light chain of the antibody can be classified as a lambda (λ) chain or a kappa (κ) chain, based on the amino acid sequence of the light chain. The antibodies disclosed herein can be of any classes or subtypes above.

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

The antibody disclosed herein can be an intact antibody or the antigen binding fragment thereof. The antigen binding fragment can be any fragments of the antibody that retain the ability to specifically bind to ROR1. Examples of antigen binding fragments include but are not limited to a Fab fragment; a F (ab') 2 fragment; a Fab' fragment; Fab’ -SH, a Fd fragment; a Fd' fragment; a Fv fragment; a scFv fragment; a dAb fragment; an isolated complementarity determining region (CDR) ; a nanobody; a linear antibody comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) , and a modified version of any of the foregoing fragments, which retains antigen binding activity.

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 another preferred embodiment, the antigen binding fragment is scFv.

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 non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 5 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to ROR1. In some embodiments, the light chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 7 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to ROR1.

In some embodiments, the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of the inserted, deleted and/or substituted amino acid can be 1-50, preferably 1-20, more preferably 1-10, still more preferably 1-5. In a preferred embodiment, the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the insertion, deletion and/or substitution can be performed at framework (FR) regions, e.g., at FR1, FR2, FR3 and/or FR4; and/or constant regions, e.g., CL, CH1, CH2 and/or CH3.

In some embodiments, the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) . Examples of conservative substitutions are as described above.

In a preferred embodiment, the antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 5 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 7.

Nucleic acids

The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof disclosed herein.

The term "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

For example, the invention provides nucleic acid molecules encoding the heavy chain variable region sequence disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding any one of the heavy chain variable region sequence 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 to SEQ ID NO: 8.

For example, the invention provides nucleic acid molecules encoding the light chain variable region sequence disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding any one of the light chain variable region sequence 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 to SEQ ID NO: 10.

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

For example, the invention provides nucleic acid molecules encoding a heavy chain variable region sequence that comprises the CDR sequences of any one of the heavy chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that encode a heavy chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the heavy chain variable region sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding a light chain variable region sequence that comprises the CDR sequences of any one of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that encode a light chain variable region sequence that comprises CDR sequences that are at least 90%, at least 95%, at least 98%or at least 99%identical to the CDR sequences of any one of the light chain variable region sequences disclosed herein.

For example, the invention provides nucleic acid molecules encoding the heavy chain sequence disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding the heavy chain sequence 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 to SEQ ID NO: 9.

For example, the invention provides nucleic acid molecules encoding the light chain sequence disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%or at least 99%identical to nucleic acids encoding the light chain sequence 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 to SEQ ID NO: 11.

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.

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

In some embodiments, the deoxyribonucleic acid (DNA) may be introduced into the cells of a human body in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the invention is comprised in a vector or a delivering agent. In some embodiments, the deoxyribonucleic acid (DNA) of the invention is integrated into the genome of a cell.

In some embodiments, the ribonucleic acid (RNA) may be introduced into the cells of a human body in vivo. In some embodiments, the ribonucleic acid (RNA) of the invention is comprised in a vector or a delivering agent.

Vectors

The present disclosure provides a vector comprising the nucleic acid disclosed herein.

In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising a heavy or light chain variable region of the antibody. For example, the invention provides expression vectors comprising any of the nucleic acid molecules mentioned above.

Any vector may be suitable for the present 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 adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector (AAV) , a lentiviral vector, or any combination thereof. Suitable exemplary vectors include e.g., 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, MSCV IRES Luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

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

Recombinant expression vectors may be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley &Sons, NY, 1994. Constructs of expression vectors, which are circular or linear, may be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems may be derived, e.g., from ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.

For example, the vector may be an adenoviral vector comprising a nucleotide sequence encoding the antibody disclosed herein. The vector may be administered into the body of a subject, and then enter into a cell of the subject in vivo, thereby the nucleotide sequence encoding the antibody disclosed herein is integrated into the genome of the cell, and subsequently the cell expresses the antibody disclosed herein.

Host Cells

The present disclosure provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

Any cell may be used as a host cell for the nucleic acids or the vectors of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cells such as a mammalian cell. Suitable prokaryotic cells include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans, and Shigella; Bacilli such as B. subtilis and B. licheniformis; Pseudomonas such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T and HEK293FS.

The host cell of the invention is prepared by introducing the vector disclosed herein or the nucleic acid disclosed herein in vitro or ex vivo. The host cell of the invention may be administered into the body of a subject, and the host cell expresses the antibody disclosed herein in vivo.

The invention provides host cells into which any of the vectors mentioned above have been introduced. The invention further provides a method of preparing the antibody of the invention, wherein the method comprises a) culturing the host cell of the fourth aspect of the invention under a condition suitable for the production of the antibody; and b) obtaining the antibody from the culture.

Compositions

The present disclosure provides a composition comprising the antibody or the antigen binding fragment thereof disclosed herein.

Kits

As set forth herein, the present disclosure provides diagnostic methods for determining the expression level of ROR1. In one particular aspect, the present disclosure provides a kit comprising the antibody or the antigen binding fragment thereof disclosed herein for performing these methods as well as instructions for carrying out the methods of the present disclosure such as collecting tissue and/or performing the screen, and/or analyzing the results.

The kit of the invention may comprise one or more containers filled with the antibodies or the antigen binding fragment disclosed herein.

The kit comprises, or alternatively consists essentially of, or yet further consists of, a ROR1 antibody composition (e.g., monoclonal antibodies) disclosed herein, and instructions for use. The kits are useful for detecting the presence of ROR1 in a biological sample, e.g., any body fluid including, but not limited to, e.g., sputum, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, acitic fluid or blood and including biopsy samples of body tissue. The test samples may also be a tumor cell, a normal cell adjacent to a tumor, a normal cell corresponding to the tumor tissue type, a blood cell, a peripheral blood lymphocyte, or combinations thereof. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are known in the art and can be readily adapted in order to obtain a sample which is compatible with the system utilized.

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

The kit can also comprise, e.g., a buffering agent, a preservative or a protein-stabilizing agent. The kit can further comprise components necessary for detecting the detectable label, e.g., an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present disclosure may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.

As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components may be provided in solution or as a liquid dispersion or the like.

In one embodiment, the kit is provided for detecting ROR1 in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Kits for detecting a polypeptide will typically comprise an anti-ROR1 antibody. In a further embodiment, the antibody is labeled with a detectable moiety (for example, with a fluorescent, radioactive, or an enzymatic label) .

In one embodiment, a kit includes instructional materials disclosing means of use of an anti-ROR1 antibody. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files) . The kits may also include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like) . The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. The method of detecting ROR1 in a biological sample generally includes the steps of contacting the biological sample with an anti-ROR1 antibody. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

Conjugates

The present disclosure provides a conjugate comprising the antibody or the antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

In the context of the present 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 label, protein, nucleic acid, lipid, nanoparticle, carbohydrate or recombinant virus. An antibody conjugate is often referred to as an "immunoconjugate" . When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent) , the conjugate is often referred to as an "antibody-drug conjugate" or "ADC" .

The term "conjugated" or "linked" may refer to making two polypeptides into one contiguous polypeptide molecule. In one embodiment, an antibody is joined to a chemical moiety. In another embodiment, an antibody joined to a chemical moiety is further joined to a lipid or other molecule to a protein or peptide to increase its half-life in the body. The linkage can be either by chemical or recombinant means. In one embodiment, the linkage is chemical, wherein a reaction between the antibody moiety and the chemical moiety has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the chemical moiety.

A chemical moiety can be linked to the antibody of the invention using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching a chemical moiety to the antibody varies according to the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH) , free amine (-NH₂) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules. The linker can be any molecule used to join the antibody to the chemical moiety. The linker is capable of forming covalent bonds to both the antibody and to the chemical moiety. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the chemical moiety are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the chemical moiety from the antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site.

Cleavage of the linker to release the chemical moiety from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules) , drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein) . In general, the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody) , a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a strep tavidin core region or a polyhistidine tag) .

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types) . Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate) . Such linkers are commercially available.

In some embodiments of the conjugate disclosed herein, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immune stimulatory molecule.

In some embodiments, the chemical moiety is a therapeutic agent. In some embodiments, the therapeutic agent includes but is not limited to immunomodulators, radioactive compounds, enzymes (for example perforin) , chemotherapeutic agents (for example cis-platin) , or a toxin. In some embodiments, the therapeutic agent can be such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g., auristatins) , or minor groove binding agents such as calicheamicin.

Other suitable therapeutic agents include such as, small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e. proteins or fragments thereof with the ability to kill mammalian cells, for example, ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase; radio-nuclides, i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of a or β particles, or γ rays, for example, iodine-131 , rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225 and astatine-213; chelating agents may be used to facilitate the association of these radionuclides to the molecules, or multimers thereof.

In some embodiments, the chemical moiety is a detectable moiety. In some embodiments, the detectable moiety can be selected from the group consisting of biotin, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent, or chemiluminescent molecules. A detectable moiety for diagnostic purposes includes for instance, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.

The antibody can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, Western Blot, 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 tomography (CAT) scans, magnetic resonance imaging (MRI) , nuclear magnetic resonance imaging NMRI) , magnetic resonance tomography (MTR) , ultrasound, fiberoptic examination, and laparoscopic examination) . Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI) . For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) .

The antibody or antigen binding fragment can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. The antibody or antigen binding fragment may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

The antibody may be fused to a self-labelling protein tag (e.g. HaloTag) . For example, the protein tag could be cloned at the end of a constant region. HaloTag is a self-labelling protein tag derived from a bacterial enzyme (a haloalkane dehalogenase) , designed to covalently bind to a synthetic ligand. In some instances, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3 (6) : 373-82) .

The antibody may be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) , and manganese.

Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels. The antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) . In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

The antibody can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect expression of a target antigen by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

In some embodiments, the immune stimulatory molecule is an immune effector molecule which stimulates immune response. For example, the immune stimulatory molecule can be cytokines such as IL-2 and IFN-γ, chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, complement activators; viral/bacterial protein domains, or viral/bacterial peptides.

Methods for diagnosis, auxiliary diagnosis, detection, staging, determination of prognosis, imaging, subject selection, and cancer treatment

The present disclosure provides a method for detecting ROR1 protein in vitro. In some cases, ROR1 expression is detected in a biological sample. The sample can be any sample, including, but not limited to, blood samples, tissue from biopsies, autopsies and pathology specimens. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.

The present disclosure also provides a method of determining if a subject has a ROR1 positive cancer by contacting a sample from the subject with anti-ROR1 antibodies disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample identifies the subject as having the cancer. In some embodiments, the control sample is a sample from a subject without the cancer. In a particular embodiment, the sample is a blood or tissue sample.

In another embodiment, the present disclosure provides a method for diagnosis or auxiliary diagnosis of a cancer in a subject, comprising contacting a biological sample with anti-ROR1 antibodies or the antigen binding fragment thereof disclosed herein; and detecting the presence of a ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of the 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, selecting the subject for treatment with an anti-ROR1 therapy. In some embodiments, the method further comprises administering an anti-ROR1 therapy to the subject. In some embodiments, the subject is a human subject having or being suspected to have a cancer. In some embodiments, the anti-ROR1 therapy is an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

The present disclosure also provides a method for staging a ROR1 positive cancer in a subject comprising: (i) contacting a biological sample with the antibody or the antigen binding fragment thereof disclosed herein or the 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.

The present disclosure further provides a method for determining the prognosis of a cancer in a subject comprising: (i) contacting a biological sample with the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein; (ii) detecting the presence of ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of 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, selecting the subject 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.

The present disclosure also provides a method for selecting a subject for treatment with an anti-ROR1 therapy comprising: (i) contacting a biological sample with the antibody or the antigen binding fragment thereof disclosed herein or the 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, selecting the subject 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.

The present disclosure further provides a method for treating a cancer in a subject comprising: (i) contacting a biological sample with the antibody or the antigen binding fragment thereof disclosed herein or the 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, administering to the subject an anti-ROR1 therapy; wherein the cancer is an 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.

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

The present disclosure also provides a method for imaging/identifying a cancer cell expressing ROR1 in a sample comprising: (i) contacting the sample with the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein; and (ii) detecting the presence of a ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of the cancer cell. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is obtained from the subject.

In some embodiments of the methods for diagnosis, auxiliary diagnosis, detection, staging, determination of prognosis, subject selection, and cancer treatment disclosed herein, the biological sample can be any sample, including, but not limited to, blood samples, tissue from biopsies, autopsies and pathology specimens. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate. In a preferred embodiment, the biological sample is a tissue sample.

In some embodiments, the presence, level or location of the ROR1 in the biological sample is determined by ELISA, Western Blot, 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 tomography (CAT) scans, magnetic resonance imaging (MRI) , nuclear magnetic resonance imaging NMRI) , magnetic resonance tomography (MTR) , ultrasound, fiberoptic examination, and laparoscopic examination) . Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI) .

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

In another aspect, a method is provided of detecting ROR1 in a sample, comprising contacting a sample from a human with any of the aforementioned antibodies under conditions that allow binding of the antibody to ROR1, and detecting the bound antibody. In one embodiment, a first antibody to ROR1 is immobilized on a solid support, as a capture reagent, and a second antibody to ROR1 is used as a detection reagent. In a related aspect, the amount of ROR1 in the sample is quantitated by measuring the amount of the bound antibody. The detection methods can be used in a variety of diagnostic, prognostic and monitoring methods, including methods of diagnosing an ROR1-related disorder and methods of monitoring therapy with an anti-ROR1 antibody. In such methods, a level of ROR1 at or above a certain threshold is correlated with the presence of ROR1-positive cancer, while a level below said threshold indicates that the patient is unlikely to have ROR1-positive cancer.

Methods of setting an appropriate threshold for diagnosis of the disease states described herein are well known in the art. By way of example, levels of ROR1 in a sample from a sufficient representative number of normal subjects (e.g., healthy population without the condition to be detected) are analyzed relative to the ROR1 level from a sufficient representative number of diseased subjects (e.g., population confirmed to have the disease or condition) using the same protocols. A threshold cutoff can be determined that differentiates most of the normal population from most of the diseased population. Alternatively, useful end point values for negative, uncertain and positive results can be determined from the data. For example, a normal range (indicative of a negative result) can be determined, which includes ROR1 of most of the normal population, but which exclude almost all of the diseased population. Correspondingly, a range indicative of a positive result can be determined, which includes ROR1 of most of the diseased population, but which exclude almost all of the normal population. Appropriate endpoint values for the threshold may be determined to optimize the desired specificity or sensitivity, and may also take account of overall medical and epidemiological factors. Factors to be considered include the clinical objective of the IVD test and whether it is necessary to have a high positive predictive value, or a high negative predictive value, as well as prevalence of the disease in the tested population.

In preferred embodiments, the location of the 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.

In some embodiments, the anti-ROR1 antibody is directly labeled with a detectable label. In another embodiment, the anti-ROR1 antibody (the first antibody) is unlabeled and a second antibody or other molecule that can bind the first is labeled. As is well known to one of skill in the art, a secondary antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.

Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-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, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, ROR1 can be assayed in a biological sample by a competition immunoassay utilizing ROR1 protein standards labeled with a detectable substance and an unlabeled anti-ROR1 antibody. In this assay, the biological sample, the labeled ROR1 protein standards and the anti-ROR1 antibody are combined and the amount of labeled ROR1 protein standard bound to the unlabeled antibody is determined. The amount of ROR1 in the biological sample is inversely proportional to the amount of labeled ROR1 protein standard bound to the anti-ROR1 antibody.

The immunoassays and methods disclosed herein can be used for a number of purposes. In one embodiment, the anti-ROR1 antibody may 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 another embodiment, the antibody can be used to detect the location of ROR1 in a biological sample.

In some embodiments of the method for imaging disclosed herein, the detectable moiety is selected from the group consisting of biotins, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent or chemiluminescent molecules. In preferred embodiments, the detectable moiety is fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.

In some embodiments of the methods disclosed herein, the cancer is ROR1-positve solid tumors or ROR1-positive hematological malignancies.

In some embodiments, the ROR1-positve 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, cholangiocarcinoma, chondrosarcoma, kidney cancer, thyroid cancer, and skin cancer.

In some embodiments, the ROR1-positve hematological malignancies are selected from the group consisting of lymphoma and myeloma. In some embodiments, the lymphoma is selected from the group consisting of Hodgkin B-cell lymphoma (such as diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma) , non-Hodgkin B-cell lymphoma (Burkitt lymphoma, lymphoblastic lymphoma, anaplastic large cell lymphoma) , mantle cell lymphoma, chronic lymphocytic leukemia, and diffuse large B cell lymphoma. In some embodiments, myeloma may include, but not limited to solitary myeloma, multiple myeloma, diffuse myeloma, extramedullary myeloma, and leukemia myeloma.

Medical uses

The present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein in the manufacture of a kit or a composition for the diagnosis or auxiliary diagnosis of a cancer in a subject. In some embodiments, the subject is a human subject having or being suspected to have a cancer.

The present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein for use in the diagnosis or auxiliary diagnosis of a cancer in a subject. In some embodiments, the subject is a human subject having or being suspected to have a cancer.

The present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein in the manufacture of a kit or a composition for staging a ROR1 positive cancer in a subject.

The present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein for use in staging a ROR1 positive cancer in a subject.

The present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein in the manufacture of a kit or a composition for determining the prognosis of a cancer in a subject.

The present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein for use in determining the prognosis of a cancer in a subject.

The present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein in the manufacture of a kit or a composition for imaging a ROR1 positive cancer in a subject.

The present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein for use in imaging a ROR1 positive cancer in a subject.

The present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein in the manufacture of a kit or a composition for selecting a subject for treatment with an anti-ROR1 therapy.

The present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein for use in selecting a subject for treatment with an anti-ROR1 therapy.

The present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein in the manufacture of a kit or a composition for treating a cancer in a subject; wherein a biological sample obtained from the subject is contacted with the antibody or the antigen binding fragment thereof disclosed herein or the conjugate 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, an anti-ROR1 therapy is administered to the subject; and wherein the cancer is an ROR1-positive.

The present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein or the conjugate disclosed herein for use in treating a cancer in a subject; wherein a biological sample obtained from the subject is contacted with the antibody or the antigen binding fragment thereof disclosed herein or the conjugate 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, an anti-ROR1 therapy is administered to the subject; and wherein the cancer is an ROR1-positive.

In some embodiments of the uses disclosed herein, the biological sample can be any sample, including, but not limited to, blood samples, tissue from biopsies, autopsies and pathology specimens. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate. In a preferred embodiment, the biological sample is a tissue sample. In some embodiments, the presence, level or location of the ROR1 in the biological sample is determined by ELISA, Western Blot, 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 tomography (CAT) scans, magnetic resonance imaging (MRI) , nuclear magnetic resonance imaging NMRI) , magnetic resonance tomography (MTR) , ultrasound, fiberoptic examination, and laparoscopic examination) . Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI) . In some embodiments, the detectable moiety is selected from the group consisting of biotins, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent or chemiluminescent molecules.

The present disclosure further provides use of the antibody or the antigen binding fragment thereof disclosed herein, the nucleic acid disclosed herein, the vector disclosed herein, the host cell disclosed herein, the composition disclosed herein, the kit disclosed herein, or the conjugate disclosed herein in the manufacture of a medicament or a product for treating a cancer in a subject.

The present disclosure also provides the antibody or the antigen binding fragment thereof disclosed herein, the nucleic acid disclosed herein, the vector disclosed herein, the host cell disclosed herein, the composition disclosed herein, the kit disclosed herein, or the conjugate disclosed herein for use in treating a cancer in a subject.

In some embodiments of the uses disclosed herein, the cancer is an ROR1 positive cancer. In some embodiments, the cancer is ROR1-positve solid tumors or ROR1-positive hematological malignancies.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1. Immunization of animals

1.1 Preparation of Immunogens

The following recombinant proteins and peptides were synthesized and used for the immunization of animals:

1) The recombinant protein P4577 which is the ECD domain of human ROR-1 (amino acids 30-403) expressed by HEK293 cells having the following amino acid sequences:

2) A mixture of CSTONE-1a peptide and CSTONE-1b peptide having the following amino acid sequences:

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

CSTONE-1b peptide (amino acids 779-800 of human ROR-1)

1.2. Animal Immunization

Six healthy New Zealand white rabbits (QINGDAO KANGDA BIOLOGICAL TECHNOLOGY) were used for animal immunization, with a total of five injections according to conventional injection method. The immunization interval was 3+2+2+2 weeks. Three rabbits (K10047R, K10048R and K10049R) were immunized with the recombinant protein P4577. The other three rabbits (K10044D2, K10045D2, K10046D2) were immunized with the mixture of CSTONE-1a and CSTONE-1b peptides. Before immunization, 0.1-0.2mg of immunogen was mixed with Freund adjuvant at a ratio of 1: 1, and fully emulsified to ensure the effect of immunization. The immunogen had been emulsified with Freund' s complete adjuvant in the first time and with Freund' s incomplete adjuvant in the latter times. During the immunization procedure, rabbit serum was collected for titer test to evaluate the immune effect.

1.3. Serum titer

After immunization by the last injection, appropriate amount of rabbit blood was collected and rabbit serum was prepared for test of titer. Serum titer was tested by conventional ELISA with immunogenic peptides or recombinant proteins as antigens. The results are shown in the table 1.

Table 1

It can be seen that the serum titer of three rabbit K10048R, K10045D2 and K10044D2 were positive when the dilution times reached 1: 64000.

Example 2. Antibody screening

2.1. fusion and subcloning

After the immunization, rabbits (K10048R, K10045D2, K10044D2) with expected immune effect were euthanatized, and the spleens of the rabbits were immediately isolated. Active rabbit spleen cells were separated as soon as possible and fused with rabbit myeloma cells (240E1 cell, Abcam) to develop hybridoma cells. Cell fusion was performed by conventional PEG method. After the cell fusion, the cell mixture was supplemented with FBS and other nutrients, and was placed in the 96-well cell culture plate with adjusted density, and cultured in the cell incubator for two to three weeks. After observing the growth of hybridoma cells and forming cell clones (colonies) , the hybridoma culture supernatant was collected for ELISA detection, and positive hybridoma clones were screened out.

The positive hybridoma clones were developed by limited dilution method, and the positive monoclonal hybridoma cells were obtained.

2.2. Screening of hybridomas

129 positive clones among 960 clones were initially screened by ELISA. 25 positive clones among these 129 clones were screened by IHC (Immunohistochemistry) using an engineered chronic myeloid leukemia cell line K562 with stable transfection of ROR1 or ROR2 to overexpress ROR1 and ROR2 respectively (iCareAb) . Three candidate clones among these 25 clones were further screened by IHC using human TMA samples. Finally the three candidate clones were selected for recombinant antibody development.

The three positive hybridoma cells were cultured for 2-3 days to ensure the healthy state of hybridoma cells. Appropriate amount of hybridoma cells were collected and lysed to prepare antibody mRNA template, which was used in RT-PCR to amplify the heavy chain and light chain genes (cDNA) of antibody, and then the expression vector (Abcam) was constructed after sequencing confirmation. The mammalian cells HEK293 were transfected with the antibody expression plasmid to express the recombinant antibody.

The antibodies expressed by the three positive hybridoma cells were named as CSTONE-1(CSD) -3, CSTONE-1 (CSD) -2, CSTONE-1 (CSD) -1. The CSTONE-1 (CSD) -1 antibody has the following amino acid sequences and nucleotide encoding sequences.

Amino acid sequence of heavy chain variable region

Amino acid sequence of HCDR1

Amino acid sequence of HCDR2

Amino acid sequence of HCDR3

GRV (SEQ ID NO: 14)

Amino acid sequence of heavy chain

Amino acid sequence of light chain variable region

Amino acid sequence of LCDR1

Amino acid sequence of LCDR2

Amino acid sequence of LCDR3

Amino acid sequence of light chain

Nucleotide sequence of heavy chain variable region

Nucleotide sequence of heavy chain

Nucleotide sequence of light chain variable region

Nucleotide sequence of light chain

2.3 Tests for the binding of recombinant antibodies

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 blot (WB) were performed. The antibody clones shown as “1A” and “1B” were two parallel clones derived from the same clone. For example, CSTONE-1 (CSD) -1-1A and CSTONE-1 (CSD) -1-1B were both derived from CSTONE-1 (CSD) -1.

IHC experiment was performed with various cell specimens and tissue specimens using commonly used procedures in the art. Briefly, cell line specimens 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) cells and tissue specimens from human esophagus, human colon, human stomach, human spleen, human bone marrow, human breast cancer, human colon cancer, suspected TNBC tissues were prepared to Formalin-Fixed and Parrffin-Embedded (FFPE) slides. For antigen retrieval, the FFPE slides were placed into a dyeing tank containing the antigen retrieval solution Tris EDTA buffer, pH 9.0 (ab93684, 1: 100 dilution) , and the dyeing tank was placed into the pressure cooker at 110℃ for 15-30 min. Then, the FFPE slides were soaked in 3%hydrogen peroxide for 10 min and washed. Block in 10%normal serum with 1%BSA in TBS for 30min at room temperature. The primary antibodies (CSTONE-1(CSD) -3, CSTONE-1 (CSD) -2, and CSTONE-1 (CSD) -1) were diluted with the diluent at dilution ratio of 1: 1000-2000 according to the requirements or instructions, added to the slides, and incubated in a wet box at 4℃ overnight. Goat Anti-Rabbit IgG H&L (HRP polymer) (ab 214880) as secondary antibody was added to the slides and incubated at room temperature for 30 min. Freshly prepared DAB was added for chromogen development (about 0.5-2 min) . Hematoxylin dye solution was added for counterstain (1-10 min) , and the slides were rinsed in TBST for about 10s. The slides were dehydrated with gradient alcohol and xylene or tissue transparent agent, dried, and then sealed with permanent tablet. The IHC results of the three recombinant antibodies were shown in Figures 1-6 and summarized in Table 2 below.

The results in Table 2 and Figures 1-6 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 other four types of cells. CSTONE-1 (CSD) -2-1A and CSTONE-1 (CSD) -2-1B showed nearly almost no staining on all human tissues.

Table 2

WB experiment was performed with ROR1-positive K-562 ROR1 cells using commonly used procedures in the art. Briefly, lysate from K-562 ROR1 cells was collected for western bolt. The antibodies (CSTONE-1 (CSD) -3, CSTONE-1 (CSD) -2, and CSTONE-1 (CSD) -1) diluted at 1: 10 or 1: 40 were used as primary antibodies, and goat Anti-Rabbit IgG H&L (HRP polymer) (ab 214880) was used as secondary antibody. The WB results of the three recombinant antibodies are shown in Figure 7.

Figure 7 showed that a 130 kDa band (corresponding to ROR1) was observed for CSTONE-1 (CSD) -1-1A and CSTONE-1 (CSD) -1-1B, a non-specific 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.

The results from IHC and WB suggest 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 deserves for further development.

Example 3. The recombinant antibody CSTONE-1 (CSD) -1 for IHC

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 from human tumor TNBC (Bioaitech, China) . The whole staining process was performed on Leica Bond III with detailed staining protocol below:

4-μm sections were cut and stained with the Leica Bond III (Leica Biosystems) . Slides were pretreated with antigent retrieval ER2 (PH 9.0) (40 minutes) , blocked with 3%hydrogen peroxide (5 minutes) followed by the primary antibody of CSTONE-1 (CSD) -1 mAb (10ug/ml) for 30 minutes and detected using BOND Polymer Detection (DS9800) for 10 minutes.

The results were shown in figures 8 and 9. To quantify the ROR1 membrane expression level in human TNBC FFPE slides, a histochemical score (H-score, which is a numerical value represented by a weighted summation of percent staining which accounts for both the staining intensity and the percentage of cells at that intensity) was calculated using the following formula: H-score= (3 × %cells with strong intensity staining) + (2×%cells with moderate intensity staining) + (1 × %cells with mild intensity staining) .

The IHC results in figure 8 showed that CSTONE-1 (CSD) -1 has clear membrane staining in multiple human TNBC FFPE samples, and different H-scores (145, 80, 50, 30, 15 and 0, respectively) were obtained for six different human TNBC FFPE slides, suggesting different ROR1 expression level in human TNBC samples. The IHC results in figure 9 showed that positive IHC signal was detected in ROR1-positive K562-ROR1 and MDA-MB-231 cells, and no IHC signal was detected in ROR1-negative K562, K562-ROR2, and MCF-7 cells. These results suggest that CSTONE-1 (CSD) -1 has high sensitivity and specificity for ROR1-positive cell lines.

Example 4. The recombinant antibody CSTONE-1 (CSD) -1 for co-location by IHC andISH

To determine the distribution of ROR1 protein in tumor cells, IHC and ISH (in situ hybrid) were performed using various TNBC tumor FFPE slides from human TNBC Patient-Derived tumor Xenograft (PDX) models (CrownBio Beijing) , such as BR1458, BR1282 and BR1474. 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 ACDSOP. The probe wasProbe-Hs-ROR1-alltv (ACD, 411091) .

ROR1 expression in the specimen was determined by 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 a H-score. H-scores were calculated using the following formula: H-score= (3 × %cells with strong intensity staining) + (2×%cells with moderate intensity staining+ (1 × %cells with mild intensity staining) .

The results for FFPE slides from human TNBC PDX models BR1458, BR1282 and BR1474 were shown in Figures 10-12 and Table 3.

Table 3

These results showed that ROR1 protein detected in tumor samples by IHC staining with CSTONE-1 (CSD) -1 (diffusely membranous in this example) is mirrored by detection of ROR1 mRNA within tumor cell cytoplasm (mRNA identified as punctate dots at points of probe binding to mRNA transcripts) . ISH mRNA distribution corroborated IHC signal, demonstrating the good specificity of CSTONE-1 (CSD) -1 for binding to ROR1 in FFPE tissue samples.

Example 5. The binding of the recombinant antibody CSTONE-1 (CSD) -1 to human ROR1

To determine binding affinity and specificity of CSTONE-1 (CSD) -1 to human ROR1 protein, ELISA and WB were performed. For WB, lysate from K562, K562-ROR1 K562-ROR2, MCF-7 and MDA-MB-231 cells were used. The experiments contain three group, a, b and c. In group a, CSTONE-1 (CSD) -1 was used as primary antibody. In group b, anti-GAPDH antibody and anti-vinculin antibody were used as primary antibodies. In group c, CSTONE-1(CSD) -1 was used as primary antibody. The WB results were shown in Figure 13.

ELISA was performed using the recombinant protein P4577 (ECD domain of human ROR as described in Example) as the antigen. Briefly, antigen was coated overnight at 4℃. Samples were added in serial dilutions starting at 4 μg/ml (purified antibody) and incubated at room temperature for 1.5 hours. Peroxidase AffiniPure Goat Anti-Rabbit IgG, F (ab') 2 fragment specific secondary antibody was added at room temperature for 1 hour. Substrate solution was added and developed for 5 minutes at room temperature. Absorbance is measured at 405 nm. The ELISA results were shown in Figure 14.

Figure 13 showed that a 130 kDa band which refers to ROR1 protein was observed in ROR1 positive K562-ROR1 and MDA-MB-231 cells, and was absent in ROR1 negative K562, K562-ROR2 and MCF-7 cells, demonstrating high specificity of CSTONE-1 (CSD) -1 binding to ROR1. Figure 14 showed that EC50 of CSTONE-1 (CSD) -1 antibody binding to ROR1 is 26.44 ng/ml (～2.3ⅹ10-10 M) , demonstrating high binding affinity of CSTONE-1 (CSD) -1 binding the ROR1 epitope.

Example 6. Identification of solid tumors expressing ROR1 through anti-ROR1 monoclonal antibody CSTONE-1 (CSD) -1

The present example uses RORl-expressing solid tumor samples to determine whether anti-RORl monoclonal antibody CSTONE-1 (CSD) -1 could detect endogenous RORl in tissues such as diseased tissues.

Purified anti-RORl monoclonal antibody CSTONE-1 (CSD) -1 was used in IHC assay on formalin-fixed-paraffin-embedded (FFPE) samples of 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 carcinoma (ESCC) tissues.

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

Table 4

Claims

An antibody that specifically binds to ROR1, or an antigen binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 12-14 respectively, and the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 15-17 respectively.
The antibody or the 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 to 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 to SEQ ID NO: 6.
The antibody or the antigen binding fragment thereof according to claim 2, wherein the VH comprises an amino acid sequence as set forth in SEQ ID NO: 4 and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 6.
The antibody or the antigen binding fragment thereof according to any one of claims 1-3, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.
The antibody or the antigen binding fragment thereof according to any one of claims 1-3, wherein the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
The antibody or the 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.
The antibody or the antigen binding fragment thereof according to any one of claims 1-6, wherein the antibody is a monoclonal antibody.
The antibody or the antigen binding fragment thereof according to any one of claims 1-7, wherein the antibody is a rabbit antibody or a humanized antibody.
The antibody or the antigen binding fragment thereof according to claim 1, wherein the antibody comprises a 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 to SEQ ID NO: 5 and a 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 to SEQ ID NO: 7.
A nucleic acid encoding the antibody or the antigen binding fragment thereof according to any one of claims 1-9.
The nucleic acid according to 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 to 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 to SEQ ID NO: 10.
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 to 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 to SEQ ID NO: 11.
A vector comprising the nucleic acid according to any one of claims 10-12.
A host cell comprising the nucleic acid according to any one of claims 10-12 or the vector according to claim 13.
A composition comprising the antibody or the antigen binding fragment thereof according to any one of claims 1-9.
A kit comprising the antibody or the antigen binding fragment thereof according to any one of claims 1-9.
A conjugate, comprising the antibody or the antigen binding fragment thereof according to any one of claims 1-9, and a chemical moiety conjugated thereto.
The conjugate according to claim 17, wherein the chemical moiety is a detectable moiety.
The conjugate according to claim 18, wherein the detectable moiety is selected from the group consisting of biotins, streptavidin, enzymes or catalytically active fragments thereof, radionuclides, nanoparticles, paramagnetic metal ions, nucleic acid probes, contrast agents, and fluorescent, phosphorescent or chemiluminescent molecules.
A method for the diagnosis or auxiliary diagnosis of a cancer comprising:

(i) contacting a biological sample with the antibody or the antigen binding fragment thereof according to any one of claims 1-9 or the conjugate according to any one of claims 17-19; and

(ii) detecting the presence of a ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of the cancer,

wherein the cancer is ROR1 positive.
The method according to claim 20, wherein the subject is a human subject having or being suspected to have a cancer.
A method for staging a ROR1 positive cancer in a subject comprising:

(i) contacting a biological sample with the antibody or the antigen binding fragment thereof according to any one of claims 1-9 or the conjugate according to any one of claims 17-19; and

(ii) determining the level and/or location of ROR1 in the biological sample.
A method for determining the prognosis of a cancer in a subject comprising:

(i) contacting a biological sample with the antibody or the antigen binding fragment thereof according to any one of claims 1-9 or the conjugate according to any one of claims 17-19; and

(ii) detecting the presence of a ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of a poor prognosis.
A method for selecting a subject for treatment with an anti-ROR1 therapy comprising:

(i) contacting a biological sample with the antibody or the antigen binding fragment thereof according to any one of claims 1-9 or the conjugate according to any one of claims 17-19; 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, selecting the subject for treatment with an anti-ROR1 therapy.
A method for treating a cancer in a subject comprising:

(i) contacting a biological sample with the antibody or the antigen binding fragment thereof according to any one of claims 1-9 or the conjugate according to any one of claims 17-19; 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, administering to the subject an anti-ROR1 therapy;

wherein the cancer is an ROR1-positive.
A method for detecting the presence or determining the level of a ROR1 in a sample comprising:

(i) contacting the sample with the antibody or the antigen binding fragment thereof according to any one of claims 1-9 or the conjugate according to any one of claims 17-19; and

(ii) detecting the presence of ROR1 or the level of ROR1 bound by the antibody or the conjugate,

optionally the sample is a biological sample.
A method for imaging/identifying a cancer cell expressing ROR1 in a sample comprising:

(i) contacting the sample with the antibody or the antigen binding fragment thereof according to any one of claims 1-9 or the conjugate according to any one of claims 17-19; and

(ii) detecting the presence of a ROR1 in the biological sample, wherein the presence of the ROR1 is indicative of the cancer cell,

optionally the sample is a biological sample.
The method according to anyone of claims 20-27, wherein the biological sample is obtained from the subject.
The method according to any one of claims 20-28, wherein the biological sample is a tissue sample.
The method according to any one of claims 20-29, wherein the presence or level of the ROR1 in the biological sample is determined by a method selected from the group consisting of flow cytometry, immunohistochemical staining, ELISA assay, Western Blot, immunofluorescence assay, chemiluminescence assay, radioimmunoassay, and multiplex immunoassay.
The method according to any one of claim 22, wherein the location of the 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.
The method according to any one of claims 20-25, 27-31, wherein the cancer is ROR1-positve 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, cholangiocarcinoma, chondrosarcoma, kidney cancer, thyroid cancer, and skin cancer, or ROR1-positive hematological malignancies selected from the group consisting of lymphoma and myeloma.
The method according to 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.