Anti-ROR1 antibodies

Abstract

The present invention relates to an antibody that recognizes ROR1 and an antigen-binding fragment thereof. In some embodiments, these antibodies provide methods of treating ROR1 positive cancers. In some embodiments, these antibodies are used to diagnose or image ROR1 positive cancers.

Description

Technical Field

[0001] The present invention relates to antibodies and antigen-binding fragments thereof binding to ROR1 and uses thereof. Background Art

[0002] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed during embryonic and infant development, and this expression level is significantly reduced in children and adults. The expression of ROR1 is significantly increased in a variety of blood cancers and solid tumors. Blood cancers that highly express ROR1 include, for example, B-cell leukemias, lymphomas, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and marginal zone lymphoma (MZL). Among solid tumors, cancer types expressing ROR1 include, for example, breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, adrenal cancer, and many other cancers. Therefore, ROR1 has become a new tumor-specific target. Many data show that ROR1 plays an important role in promoting tumor growth and metastasis, inducing drug resistance, and inhibiting apoptosis.

[0003] The current consensus on ROR1 signal transduction is that ROR1 can play an important role in a variety of physiological processes by mediating signal transduction of non-classical wnt pathways (especially wnt5a), including regulating cell division, proliferation, migration and cell chemotaxis. Wnt5a is a typical non-classical activator of the Wnt signaling pathway, and is involved in the phosphorylation of NF-κB subunit p65, activating the NF-κB pathway in tumor cells, promoting cell migration and invasion, EMT, cancer metastasis, etc. Wnt5a / ROR1 is highly expressed in many cancers. ROR1, as a receptor for Wnt5a, is involved in the activation of the NF-κB channel in tumor cells.

[0004] ROR1 is considered a potential target because it is a tyrosine kinase receptor that is resistant to drugs and is expressed on the cell surface. More importantly, it is highly expressed in tumor cells but very low in healthy adult tissues.

[0005] There is a lack of antibodies, especially fully human antibodies, that have high affinity for ROR1, do not cross-react with ROR2, and have good binding ability and internalization activity. Summary of the invention

[0006] The present invention provides novel antibodies or antigen-binding fragments thereof that bind to ROR1. The novel antibodies may be in the form of heavy chain only antibodies (HCAbs).

[0007] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), and wherein the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in any one of SEQ ID NOs: 147-202.

[0008] In some embodiments, the VH comprises HCDRs 1-3 having the amino acid sequence shown below:

[0009] SEQ ID NO: 11, 40, 100, respectively,

[0010] SEQ ID NO: 12, 41, 101, respectively,

[0011] SEQ ID NO: 12, 42, 102, respectively,

[0012] SEQ ID NO: 12, 40, 103, respectively,

[0013] SEQ ID NO: 12, 43, 104, respectively,

[0014] SEQ ID NO: 13, 44, 105, respectively,

[0015] SEQ ID NO: 12, 40, 106, respectively,

[0016] SEQ ID NO: 12, 45, 107, respectively,

[0017] SEQ ID NO: 12, 40, 108, respectively,

[0018] SEQ ID NO: 14, 46, 109, respectively,

[0019] SEQ ID NO: 12, 47, 110, respectively,

[0020] SEQ ID NO: 12, 48, 111, respectively,

[0021] SEQ ID NO: 12, 45, 112, respectively,

[0022] SEQ ID NO: 15, 49, 113, respectively,

[0023] SEQ ID NO: 16, 50, 114, respectively,

[0024] SEQ ID NO: 12, 51, 115, respectively,

[0025] SEQ ID NO: 18, 56, 126, respectively,

[0026] SEQ ID NO: 21, 40, 130, respectively,

[0027] SEQ ID NO: 22, 60, 131, respectively,

[0028] SEQ ID NO: 12, 40, 132, respectively,

[0029] SEQ ID NO: 23, 61, 133, respectively,

[0030] SEQ ID NO: 19, 57, 127, respectively,

[0031] SEQ ID NO: 20, 58, 128, respectively,

[0032] SEQ ID NO: 14, 59, 129, respectively,

[0033] SEQ ID NO: 17, 53, 119, respectively,

[0034] SEQ ID NO: 17, 53, 116, respectively,

[0035] SEQ ID NO: 17, 53, 120, respectively,

[0036] SEQ ID NO: 17, 53, 121, respectively,

[0037] SEQ ID NO: 17, 53, 122, respectively,

[0038] SEQ ID NO: 17, 53, 123, respectively,

[0039] SEQ ID NO: 17, 53, 124, respectively,

[0040] SEQ ID NO: 17, 53, 125, respectively,

[0041] SEQ ID NO: 17, 52, 116, respectively,

[0042] SEQ ID NO: 17, 53, 117, respectively,

[0043] SEQ ID NO: 17, 52, 117, respectively,

[0044] SEQ ID NO: 17, 40, 116, respectively,

[0045] SEQ ID NO: 17, 53, 118, respectively,

[0046] SEQ ID NO: 17, 54, 116, respectively,

[0047] SEQ ID NO: 17, 55, 116, respectively,

[0048] SEQ ID NO: 17, 54, 117, respectively,

[0049] SEQ ID NO: 17, 40, 117, respectively,

[0050] SEQ ID NO: 17, 55, 117, respectively,

[0051] SEQ ID NO: 17, 54, 118, respectively,

[0052] SEQ ID NO: 17, 40, 118, respectively,

[0053] SEQ ID NO: 17, 55, 118, respectively,

[0054] SEQ ID NO: 17, 55, 103, respectively,

[0055] SEQ ID NO: 12, 55, 117, respectively, or

[0056] They are SEQ ID NO: 12, 55, and 118 respectively.

[0057] In some embodiments, VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 147-202.

[0058] In some embodiments, the antibody comprises an Fc region.

[0059] In some embodiments, the antibody comprises a heavy chain (HC), and wherein the HC 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 any one of SEQ ID NOs: 205-260.

[0060] In some embodiments, the antibody does not comprise a light chain.

[0061] In some embodiments, the antibody comprises two heavy chains.

[0062] In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0063] In some embodiments, the antibody is of an isotype selected from IgG, IgA, IgM, IgE, and IgD.

[0064] In some embodiments, the antibody is of a subtype selected from IgGl, IgG2, IgG3, and IgG4.

[0065] In some embodiments, the antigen binding fragment is selected from HCAb, VHH, nanobody, Fab, Fab', F(ab') 2 , Fd, Fd' and dAb.

[0066] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0067] In some embodiments, the antibodies are monovalent, bivalent, or multivalent.

[0068] In some embodiments, the antibody or antigen-binding fragment is linked to a fluorescent label, a radioactive label, or a cytotoxic agent.

[0069] In another aspect, the present invention provides a bispecific antibody comprising the antibody of the present invention or an antigen-binding fragment thereof and a second antigen-binding region that specifically binds to a tumor-associated antigen or an immune cell antigen; preferably, the second antigen-binding region specifically binds to CD3.

[0070] In yet another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention or the bispecific antibody of the present invention.

[0071] In yet another aspect, the present invention provides a vector comprising the nucleic acid of the present invention.

[0072] In another aspect, the present invention provides a host cell comprising the nucleic acid of the present invention or the vector of the present invention.

[0073] In another aspect, the present invention provides an antibody-drug conjugate (ADC), which comprises the antibody or antigen-binding fragment thereof of the present invention or the bispecific antibody of the present invention.

[0074] In yet another aspect, the present invention provides a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof of the present invention, a bispecific antibody of the present invention, a nucleic acid of the present invention, a vector of the present invention, a host cell of the present invention, or an antibody-drug conjugate of the present invention; and (ii) a pharmaceutically acceptable carrier or excipient.

[0075] In some embodiments, the composition further comprises a second therapeutic agent selected from an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0076] In another aspect, the present invention provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of the present invention, the bispecific antibody of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention, the antibody-drug conjugate of the present invention, or the pharmaceutical composition of the present invention.

[0077] In some embodiments, the cancer is a ROR1 -positive cancer, preferably selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0078] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

[0079] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0080] In another aspect, the present invention provides use of an antibody or antigen-binding fragment thereof of the present invention, a bispecific antibody of the present invention, a nucleic acid of the present invention, a vector of the present invention, a host cell of the present invention, an antibody-drug conjugate of the present invention, or a pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer in a subject.

[0081] In some embodiments, the cancer is a ROR1 -positive cancer, preferably selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0082] In some embodiments, the medicament further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

[0083] In some embodiments, the drug is administered in combination with a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

[0084] In another aspect, the present invention provides use of an antibody or antigen-binding fragment thereof of the present invention, a bispecific antibody of the present invention, a nucleic acid of the present invention, a vector of the present invention, a host cell of the present invention, an antibody-drug conjugate of the present invention, or a pharmaceutical composition of the present invention for treating cancer in a subject.

[0085] In some embodiments, the cancer is a ROR1 -positive cancer, preferably selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof of the invention, the bispecific antibody of the invention, the nucleic acid of the invention, the vector of the invention, the host cell of the invention, the antibody-drug conjugate of the invention, or the pharmaceutical composition of the invention is administered in combination with a second therapeutic agent, preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0087] In another aspect, the present invention provides a method for diagnosing ROR1-positive cancer in a subject, the method comprising:

[0088] (a) obtaining a biological sample from the subject,

[0089] (b) contacting the sample with an antibody or antigen-binding fragment thereof of the present invention; and

[0090] (c) detecting binding of the antibody to the sample,

[0091] Wherein increased binding of the antibody or antigen-binding fragment thereof to the sample compared to binding of the antibody or antigen-binding fragment thereof to a control sample identifies the subject as having ROR1 -positive cancer.

[0092] In another aspect, the present invention provides a method for imaging a ROR1 -positive cancer in a subject, the method comprising:

[0093] (a) administering to the subject an antibody or antigen-binding fragment thereof of the present invention, wherein the antibody is conjugated to a detectable marker; and

[0094] (b) detecting the presence of the marker.

[0095] In some embodiments, the detectable marker is 111 In, and preferably, the detection of the marker is performed by single photon emission computed tomography. In some embodiments, the detectable marker is 89 Zr, and preferably, detection of this marker is performed by positron emission tomography. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 . Workflow of screening strategies and methods for single B cell clone screening.

[0097] Figure 2 . Binding of HCAb antibody to CHO-K1-cynoROR1 cells.

[0098] Figure 3 . Binding of HCAb antibody to PANC-1 cells.

[0099] Figure 4 . Binding of HCAb antibody to HEK293T-hROR1 cells.

[0100] Figure 5 . Binding of HCAb antibody to A549 cells.

[0101] Figure 6. The binding of HCAb antibody to hu-ROR1-ECD-his was tested by ELISA.

[0102] Figure 7 . Binding of the PR005340 variant (VH-Flag-His) in monovalent form to PANC-1 cells.

[0103] Figure 8 . Binding of the bivalent form of PR005340 variants (HCAb) to PANC-1 cells.

[0104] Fig. 9 . The binding of HCAb antibody to hu-ROR2-his was tested by ELISA.

[0105] Fig.10 . Internalization of HCAb antibodies on HEK293T-hROR1 cells was tested by Ab-MMAF cytotoxicity method.

[0106] Fig.11 . Internalization of HCAb antibody on PANC-1 cells was measured by pHAb kit.

[0107] Sequence Listing

[0108] The sequences of the heavy chain, heavy chain variable region, and heavy chain CDRs of the anti-ROR1 HCAb antibodies of the present invention are shown in Tables 1 to 3 below. The sequences of the light chain, heavy chain, light chain variable region, heavy chain variable region, light chain, and heavy chain CDRs of the reference antibody PR000374 are shown in Table 4 below.

[0109] Table 1. Sequences of the heavy chains of HCAb antibodies

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Table 2. Sequences of the heavy chain variable regions of HCAb antibodies

[0121]

[0122]

[0123]

[0124]

[0125] Table 3. Sequences of heavy chain CDRs 1-3 of HCAb antibodies (Chothia numbering system)

[0126]

[0127]

[0128] Table 4. Sequence of reference antibody PR000374

[0129]

[0130] DETAILED DESCRIPTION

[0131] The above features and advantages as well as additional features and advantages of the present invention will be more clearly understood from the following detailed description of embodiments in conjunction with the accompanying drawings.

[0132] The embodiments described herein with reference to the accompanying drawings are illustrative, exemplary, and are used to generally understand the present invention. These embodiments should not be construed as limiting the scope of the present invention. Throughout the specification, identical or similar elements and elements with identical or similar functions are represented by the same reference numerals.

[0133] Unless otherwise noted or defined, all terms used have their ordinary meanings in the art, which are clear to those skilled in the art. For example, reference is made to standard manuals such as Leuenberger, HGW, Nagel, B. and Klbl, H., eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Volumes 1 to 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 edition), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th edition), Garland Science Publishing / Churchill Livingstone, New York (2005), as well as the general background technology cited above.

[0134] As used herein, the singular forms "a", "an", "and", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and in some embodiments reference to "an antibody" includes a plurality of antibodies, and so forth.

[0135] Unless otherwise stated or defined, the term "comprise" and variations thereof such as "include" and "comprising" should be understood to imply the inclusion of the stated elements or steps or groups of elements or steps, but not the exclusion of any other elements or steps or groups of elements or steps. The term "comprising" encompasses "including" as well as "consisting of", for example, a composition "comprising" X may consist of X alone, or may include some additional substances, such as X+Y.

[0136] The term "about" in relation to a value x is optional and means, for example, that x ± 10% or x±5%.

[0137] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies generally include a variable region and a constant region. The constant region of an antibody can mediate the binding of an 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 the first component CIq in the classical pathway of complement activation.

[0138] The "heavy chain variable region" (VH) consists of a "framework" region interrupted by three "complementarity determining regions" or "CDRs." The framework regions are used to align the CDRs for specific binding to an epitope of an antigen. The CDRs contain the amino acid residues of the antibody that are primarily responsible for antigen binding. The VH domain contains the following framework (FR) and CDR regions from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0139] The amino acid assignments for the VH domains are consistent 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 and Lesk, J. Mol. Biol. Vol. 196: pp. 901-917, 1987; Chothia et al., Nature, Vol. 342: pp. 878-883, 1989); a combination of the Chothia Kabat CDRs, where CDR-H1 is a combination of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the contact definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. Although the present disclosure can use CDRs defined according to any of these numbering systems, a preferred embodiment uses the Chothia defined CDRs.

[0140] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two different antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0141] The terms "heavy chain antibody only", "heavy chain antibody" and "HCAb" are used interchangeably herein and refer in a broad sense to an antibody or more or more parts of an antibody, such as one or more arms of an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to: homodimeric antibodies comprising a VH antigen binding domain and CH1, CH2 and CH3 constant domains; functional (antigen binding) variants of such antibodies, soluble VH variants, homodimeric Ig-NARs comprising one variable domain (V-NAR) and five C-like constant domains (C-NAR) and functional fragments thereof; and soluble single domain antibodies (sUniDabs™).

[0142] In one embodiment, only the heavy chain antibody consists of a variable region antigen binding domain, which consists of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In another embodiment, only the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and CH1, CH2 and CH3 domains. In another embodiment, only the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, only the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH1 domain. In another embodiment, only the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH2 domain. In another embodiment, only the heavy chain antibody consists of an antigen binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies in which CH1 and / or CH2 and / or CH3 domains are truncated are also included herein. In another embodiment, the heavy chain consists of an antigen binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but lacks a hinge region.

[0143] Only heavy chain antibodies can be in the form of dimers, wherein the two heavy chains are covalently or non-covalently linked to each other by disulfide bonds or otherwise. Only heavy chain antibodies can belong to the IgG subclass, but antibodies belonging to other subclasses such as IgM, IgA, IgD and IgE subclasses are also included herein. In a specific embodiment, the heavy chain antibody is an IgG1, IgG2, IgG3 or IgG4 subtype, particularly an IgG1 or IgG4 subtype.

[0144] In one embodiment, the heavy chain antibody is of IgG1 or IgG4 subtype, wherein one or more CH domains are modified to alter the effector function of the antibody. Modifications to the CH domain that alter effector function are further described herein.

[0145] 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 (e.g., ROR1). Studies have shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody.

[0146] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, i.e., a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a portion of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul, ed., Supplementary Version 3, 1993); (iv) a Fd fragment consisting of a VH domain and a CH1 domain; (v) a Fd' fragment having a VH domain and a CH1 domain and one or more cysteine ​​residues located at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody; (vii) dAb fragments (Ward et al. (1989), Nature, Vol. 341: pp. 544-546), which consist of a VH domain; (viii) an isolated complementarity determining region (CDR); and (ix) a nanobody, i.e., a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains of the Fv fragment (VL and VH) are encoded by separate genes, recombinant methods can be used to connect these domains via a synthetic linker, allowing these domains to be made into a single protein chain in which the VL region and the VH region pair to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al. (1988), Science, Vol. 242: pp. 423-426; and Huston et al. (1988), Proc. Natl. Acad. Sci. USA, Vol. 85: pp. 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) and a modified form of any of the aforementioned fragments, which together with the complementary light chain polypeptide form an antigen-binding region, and the modified forms of these fragments retain antigen-binding activity.

[0147] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments screened for utility in the same manner as intact antibodies.

[0148] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as a non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity, represented by the equilibrium constant (KD) of antigen-antibody dissociation, is a measure of the strength of binding between an antigenic determinant (epitope) and an antigen binding site on an antibody: the smaller the KD value, the stronger the binding strength between the antigenic determinant (epitope) and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD.

[0149] Avidity is a measure of the strength of binding between an antibody and its associated antigen. Avidity is related to both the affinity between an antigenic determinant (epitope) and its antigen binding site on the antibody, and the number of associated binding sites present on the antibody. -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or a dissociation constant (KD) of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 (such as at least 10 12 M -1 ) binding affinity. Any K D Value greater than 10 -4 M is generally considered to indicate non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), biomembrane interferometry (BLI) assays and sandwich competition assays, as well as different variations of these methods known per se in the art.

[0150] The term "epitope" refers to a site on an antigen to which an antibody binds. An epitope can be formed by continuous amino acids, or by non-continuous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also called linear epitopes) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding (also called conformational epitopes) are usually lost when treated with denaturing solvents. An epitope usually includes at least 3, more usually at least 5 or 8 to 10 amino acids in a unique spatial conformation. An epitope defines the minimum binding site of an antibody and is therefore a specific target for an antibody or its antigen-binding fragment.

[0151] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acids) have identical residues at the same position in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity of the amino acid sequence to SEQ ID NO: Y, and is detailed as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, computer programs are used to perform such calculations. Exemplary programs for comparing and aligning sequence pairs 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).

[0152] In addition, when determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called conservative amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure and has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example according to WO 04 / 037999, GB-A-2 357 768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and (preferred) types and / or combinations of such substitutions may be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0153] Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from 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.

[0154] Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0155] Any amino acid substitutions applied to the polypeptides described herein may also be based on the following analyses: analysis of the frequency of amino acid variations between homologous proteins from different species, studied by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978; analysis of structural potential, studied by Chou and Fasman, Biochemistry, Vol. 13: p. 211, 1974 and Adv. Enzymol., Vol. 47: p. 45-149, 1978; and analysis of structural potential, studied by Eisenberg et al., Proc. Nat. Acad Sci. USA, Vol. 81: p. 140-144, 1984, Kyte and Doolittle, J Mol. Biol. , Vol. 157: pp. 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem., Vol. 15: pp. 321-353, 1986, the entire contents of which are incorporated herein by reference.

[0156] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, each antibody constituting the population is identical except for a small amount of possible naturally occurring mutations. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology in this article, and should not be construed as requiring the production of antibodies by any particular method.

[0157] The term "bispecific antibody" in the context of the present invention is understood to mean an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as different target binding, but also includes binding to different epitopes in one target.

[0158] As used herein, the term "tumor-associated antigen" refers to an antigen that is differentially expressed in cancer cells compared to normal cells and, therefore, can be used to target cancer cells.

[0159] As used herein, the term "bispecific T cell engager" or "BiTE" refers to a monomeric polypeptide chain molecule having two antigen binding domains, one of which binds to a T cell antigen and the second binds to an antigen present on the surface of a target (see PCT Publication WO 05 / 061547; Baeuerle et al., 2008, Drugs of the Future, Vol. 33: 137-147; Bargou et al., 2008, Science, Vol. 321: 974-977, the entirety of which is incorporated herein by reference). Thus, the BiTE of the present disclosure has an antigen binding region that binds to ROR1 and a second antigen binding region for a T cell antigen.

[0160] 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.

[0161] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0162] The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and is substantially not deleterious to the recipient thereof, and / or such carrier or adjuvant is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0163] As used herein, the term "treatment" or "treating" or the like refers to the administration of an agent or the performance of a procedure in order to obtain an effect. The effect may be a preventive effect that completely or partially prevents a disease or its symptoms, and / or may be a therapeutic effect that partially or completely cures a disease and / or disease symptoms. As used herein, "treatment" may include treating a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or disease symptom in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease (e.g., including a disease that may be associated with or caused by a primary disease); (b) inhibiting the disease, i.e., preventing the disease from developing; and (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any marker of success in the treatment or improvement or prevention of cancer, including any objective or subjective parameter, such as mitigation; relief; reducing symptoms or making the patient more tolerant to the disease condition; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. The treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes administering an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or stop or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to reducing, eliminating or preventing a disease, disease symptoms or side effects of a disease in a subject.

[0164] As used herein, the term "effective amount" refers to an amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for the disease.

[0165] As used herein, the term "subject" refers to any mammalian subject in need of diagnosis, treatment or therapy. For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, livestock and farm animals, as well as laboratory animals, zoo animals, sports animals or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0166] The term "receptor tyrosine kinase-like orphan receptor 1" or "ROR1" includes any ROR1 variants, isoforms and species homologs that are naturally expressed by cells of any origin, or expressed on cells transferred with a gene or cDNA encoding ROR1 that is naturally expressed on cells of any origin.

[0167] The terms "rhesus monkey ROR1," "cynomolgus monkey ROR1," and "cynomolgus monkey ROR1" are used interchangeably herein and refer to cynomolgus monkey ROR1. The term includes any ROR1 variants, isoforms, and species homologs that are naturally expressed by cynomolgus monkey cells or expressed on cells of any source transfected with a gene or cDNA encoding cynomolgus monkey ROR1 that is naturally expressed on cynomolgus monkey cells.

[0168] The terms "human ROR1," "huROR1," and "hROR1" are used interchangeably herein and refer to any ROR1 variants, isoforms, and species homologs that are naturally expressed by human cells, or expressed on cells of any source that are transfected with a gene or cDNA encoding human ROR1 that is naturally expressed on human cells.

[0169] Anti-ROR1 Antibody

[0170] The present invention provides antibodies against receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0171] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), and wherein the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in any one of SEQ ID NOs: 147 to 202. In some preferred embodiments, the CDRs are determined by the Chothia numbering system.

[0172] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 11, 40 and 100, respectively.

[0173] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 41 and 101, respectively.

[0174] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 42 and 102, respectively.

[0175] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 40 and 103, respectively.

[0176] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 43 and 104, respectively.

[0177] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 13, 44 and 105, respectively.

[0178] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 40 and 106, respectively.

[0179] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 45 and 107, respectively.

[0180] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 40 and 108, respectively.

[0181] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 14, 46 and 109, respectively.

[0182] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 47 and 110, respectively.

[0183] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 48 and 111, respectively.

[0184] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 45 and 112, respectively.

[0185] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 15, 49 and 113, respectively.

[0186] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 16, 50 and 114, respectively.

[0187] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 51 and 115, respectively.

[0188] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 18, 56 and 126, respectively.

[0189] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 21, 40 and 130, respectively.

[0190] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 22, 60 and 131, respectively.

[0191] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 40 and 132, respectively.

[0192] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 23, 61 and 133, respectively.

[0193] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 19, 57 and 127, respectively.

[0194] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 20, 58 and 128, respectively.

[0195] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 14, 59 and 129, respectively.

[0196] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 119, respectively.

[0197] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 116, respectively.

[0198] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 120, respectively.

[0199] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 121, respectively.

[0200] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 122, respectively.

[0201] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 123, respectively.

[0202] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 124, respectively.

[0203] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 125, respectively.

[0204] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 52 and 116, respectively.

[0205] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 117, respectively.

[0206] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 52 and 117, respectively.

[0207] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 40 and 116, respectively.

[0208] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 53 and 118, respectively.

[0209] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 54 and 116, respectively.

[0210] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 55 and 116, respectively.

[0211] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 54 and 117, respectively.

[0212] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 40 and 117, respectively.

[0213] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 55 and 117, respectively.

[0214] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 54 and 118, respectively.

[0215] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 40 and 118, respectively.

[0216] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 55 and 118, respectively.

[0217] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 17, 55 and 103, respectively.

[0218] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 55 and 117, respectively.

[0219] In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), wherein the VH comprises HCDRs 1-3 having amino acid sequences as shown in SEQ ID NOs: 12, 55 and 118, respectively.

[0220] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), and wherein

[0221] (1) 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: 147;

[0222] (2) 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: 148;

[0223] (3) 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: 149;

[0224] (4) 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: 150;

[0225] (5) 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: 151;

[0226] (6) 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: 152;

[0227] (7) 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: 153;

[0228] (8) 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: 154;

[0229] (9) 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: 155;

[0230] (10) 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: 156;

[0231] (11) 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: 157;

[0232] (12) 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: 158;

[0233] (13) 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: 159;

[0234] (14) 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: 160;

[0235] (15) 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: 161;

[0236] (16) 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: 162;

[0237] (17) 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: 163;

[0238] (18) 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: 164;

[0239] (19) 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: 165;

[0240] (20) 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: 166;

[0241] (21) 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: 167;

[0242] (22) 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: 168;

[0243] (23) 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: 169;

[0244] (24) 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: 170;

[0245] (25) 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: 171;

[0246] (26) 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: 172;

[0247] (27) 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: 173;

[0248] (28) 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: 174;

[0249] (29) 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: 175;

[0250] (30) 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: 176;

[0251] (31) 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: 177;

[0252] (32) 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: 178;

[0253] (33) 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: 179;

[0254] (34) 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: 180;

[0255] (35) 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: 181;

[0256] (36) 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: 182;

[0257] (37) 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: 183;

[0258] (38) 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: 184;

[0259] (39) 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: 185;

[0260] (40) 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: 186;

[0261] (41) 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: 187;

[0262] (42) 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: 188;

[0263] (43) 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: 189;

[0264] (44) 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: 190;

[0265] (45) 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: 191;

[0266] (46) 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: 192;

[0267] (47) 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: 193;

[0268] (48) 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: 194;

[0269] (49) 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: 195;

[0270] (50) 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: 196;

[0271] (51) 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: 197;

[0272] (52) 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: 198;

[0273] (53) 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: 199;

[0274] (54) 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: 200;

[0275] (55) 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: 201; or

[0276] (56) 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: 202.

[0277] In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region can be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or is derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0278] In one embodiment, the Fc region is an effector function defective type. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3 or IgG4, which has been mutated so that the ability to mediate effector functions such as ADCC has been reduced or even eliminated. Such mutations have been described in, for example, Dall'Acqua WF et al., J Immunol. Vol. 177, No. 2: pp. 1129-1138 (2006) and Hezareh M, J Virol., Vol. 75, No. 24: pp. 12161-12168 (2001). In some embodiments, the Fc region of the antibody comprises a wild-type IgG1 Fc with L234A, L235A and G237A mutations.

[0279] In some embodiments, the antibody is mutated at one or more post-translational modification sites. In one embodiment, the Fc region comprises a mutation that removes an acceptor site for Asn-linked glycosylation, or the Fc region is manipulated to eliminate the effector function of the antibody.

[0280] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain (HC), and wherein:

[0281] (1) the HC 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: 205;

[0282] (2) the HC 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: 206;

[0283] (3) the HC 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: 207;

[0284] (4) the HC 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: 208;

[0285] (5) the HC 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: 209;

[0286] (6) the HC 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: 210;

[0287] (7) the HC 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: 211;

[0288] (8) the HC 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: 212;

[0289] (9) the HC 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: 213;

[0290] (10) the HC 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: 214;

[0291] (11) the HC 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: 215;

[0292] (12) the HC 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: 216;

[0293] (13) the HC 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: 217;

[0294] (14) the HC 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: 218;

[0295] (15) the HC 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: 219;

[0296] (16) the HC 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: 220;

[0297] (17) the HC 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: 221;

[0298] (18) the HC 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: 222;

[0299] (19) the HC 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: 223;

[0300] (20) the HC 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: 224;

[0301] (21) the HC 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: 225;

[0302] (22) the HC 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: 226;

[0303] (23) the HC 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: 227;

[0304] (24) the HC 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: 228;

[0305] (25) the HC 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: 229;

[0306] (26) the HC 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: 230;

[0307] (27) the HC 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: 231;

[0308] (28) the HC 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: 232;

[0309] (29) the HC 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: 233;

[0310] (30) the HC 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: 234;

[0311] (31) the HC 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: 235;

[0312] (32) the HC 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: 236;

[0313] (33) the HC 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: 237;

[0314] (34) the HC 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: 238;

[0315] (35) the HC 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: 239;

[0316] (36) the HC 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: 240;

[0317] (37) the HC 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: 241;

[0318] (38) the HC 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: 242;

[0319] (39) the HC 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: 243;

[0320] (40) the HC 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: 244;

[0321] (41) the HC 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: 245;

[0322] (42) the HC 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: 246;

[0323] (43) the HC 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: 247;

[0324] (44) the HC 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: 248;

[0325] (45) the HC 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: 249;

[0326] (46) the HC 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: 250;

[0327] (47) the HC 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: 251;

[0328] (48) the HC 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: 252;

[0329] (49) the HC 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: 253;

[0330] (50) the HC 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: 254;

[0331] (51) the HC 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: 255;

[0332] (52) the HC 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: 256;

[0333] (53) the HC 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: 257;

[0334] (54) the HC 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: 258;

[0335] (55) the HC 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: 259; or

[0336] (56) The HC 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: 260.

[0337] In some embodiments, the antibodies of the invention comprise a heavy chain and a light chain. In some embodiments, the antibodies of the invention comprise two heavy chains and two light chains.

[0338] Based on the amino acid sequence of the constant region of the heavy chain of the antibody, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of the antibody can be classified as lambda (λ) chain or kappa (κ) chain. The antibodies disclosed herein can be any of the above types or subtypes.

[0339] In some embodiments, the antibody can be an isotype selected from IgG, IgA, IgM, IgE and IgD. In some embodiments, the antibody can be a subtype selected from IgG1, IgG2, IgG3 and IgG4. In a preferred embodiment, the antibody is an IgG1 antibody.

[0340] In some embodiments, the antibodies of the present invention do not comprise a light chain. In some specific embodiments, the antibodies comprise only one or two heavy chains. In some preferred embodiments, the antibodies of the present invention consist of one heavy chain. In some preferred embodiments, the antibodies of the present invention consist of two heavy chains.

[0341] In some embodiments, an antibody of the invention is a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains.

[0342] In one embodiment, an antibody of the invention is a heavy chain-only antibody consisting of a variable region antigen binding domain consisting of Framework 1, CDR1, Framework 2, CDR2, Framework 3, CDR3 and Framework 4.

[0343] In another embodiment, the antibody of the present invention is a heavy chain-only antibody, which consists of an antigen binding domain, at least a portion of a hinge region, and CH1, CH2, and CH3 domains. In another embodiment, the heavy chain-only antibody consists of an antigen binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, the antibody of the present invention is a heavy chain-only antibody, which consists of an antigen binding domain, at least a portion of a hinge region, and a CH1 domain. In another embodiment, the antibody of the present invention is a heavy chain-only antibody, which consists of an antigen binding domain, at least a portion of a hinge region, and a CH2 domain. In another embodiment, the antibody of the present invention is a heavy chain-only antibody, which consists of an antigen binding domain, at least a portion of a hinge region, and a CH3 domain. In another embodiment, the antibody of the present invention is a heavy chain, which consists of an antigen binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but without a hinge region.

[0344] In some embodiments, an antibody of the invention is a heavy chain-only antibody in the form of a dimer in which the two heavy chains are covalently or non-covalently linked to each other by a disulfide bond or otherwise.

[0345] In some embodiments, the antibodies of the invention are heavy chain only antibodies belonging to the IgG, IgM, IgA, IgD or IgE subclass of antibodies.

[0346] In some embodiments, the antibodies of the invention are heavy chain-only antibodies of the IgG1, IgG2, IgG3 or IgG4 subtype, in particular of the IgG1 or IgG4 subtype.

[0347] In one embodiment, the antibody of the invention is a heavy chain antibody of the IgG1 or IgG4 subtype, wherein one or more CH domains are modified to alter the effector function of the antibody.

[0348] The antibodies disclosed herein may be complete antibodies or antigen-binding fragments thereof. The antigen-binding fragment may be any fragment of an antibody that retains the ability to specifically bind to ROR1. Examples of antigen-binding fragments include, but are not limited to, Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; dAb fragments; isolated complementarity determining regions (CDRs); nanobodies; linear antibodies comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) and a modified form of any of the aforementioned fragments, wherein the modified forms of these fragments retain antigen-binding activity.

[0349] In some embodiments, the antigen binding fragment can be selected from HCAb, VHH, nanobody, Fab, Fab', F(ab') 2 , Fd, Fd' and dAb.

[0350] In some embodiments, the antibodies of the invention are chimeric, humanized, or human antibodies.

[0351] In some embodiments, the antibodies of the invention are monoclonal antibodies, bispecific antibodies, or multispecific antibodies.

[0352] In some embodiments, the antibodies are monovalent, bivalent, or multivalent.

[0353] In some embodiments, an antibody or antigen-binding fragment of the invention is linked to a fluorescent label, a radioactive label, or a cytotoxic agent.

[0354] Bispecific Antibodies

[0355] In a second aspect, the present application provides a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific antibody, which further comprises a second antigen binding region that binds a second antigen. In some embodiments, the second antigen can be a tumor-associated antigen, an immune checkpoint molecule, or an immune cell antigen.

[0356] Many tumor-associated antigens associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that can potentially stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but not necessarily expressed by normal cells. These antigens can be characterized as those that are usually silent (i.e., not expressed) in normal cells, those that are only expressed at certain stages of differentiation, and those that are expressed over time, such as embryonic antigens and fetal antigens. Other tumor antigens are encoded by mutant cell genes, such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., mutant p53), and fusion proteins produced by internal deletions or chromosomal translocations. Other tumor antigens can be encoded by viral genes, such as those carried by RNA tumor viruses and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be produced by those skilled in the art.

[0357] Examples of tumor-associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, ROR1, MUC-1, mutant p53, mutant Ras, ROR1, VEGFR2, and combinations thereof.

[0358] In some embodiments, the second antigen is an immune cell antigen. In some embodiments, the T cell antigen can be selected from T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD44, CD62L, CD69, ICOS, 41-BB (CD137) and NKG2D, or any combination thereof. Preferably, the second antigen is CD3.

[0359] In some embodiments, the second antigen is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule can be selected from PD-1, PD-L1, CTLA-4, etc.

[0360] In some embodiments, the bispecific antibody comprises a monomeric peptide chain comprising a first antigen binding region and a second antigen binding region and optionally an Fc region.

[0361] The Fc region may be of any isotype, including but not limited to IgG1, IgG2, IgG3 and IgG4, and may comprise one or more mutations or modifications. In one embodiment, the Fc region is of or derived from the IgG1 isotype, optionally with one or more mutations or modifications.

[0362] In one embodiment, the Fc region is an effector function defective type. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3 or IgG4, which has been mutated so that the ability to mediate effector functions such as ADCC has been reduced or even eliminated. Such mutations have been described, for example, in Dall'Acqua WF et al., J Immunol. Vol. 177 No. 2: pp. 1129-1138 (2006) and Hezareh M, J Virol., Vol. 75 No. 24: pp. 12161-12168 (2001).

[0363] In one embodiment, the Fc region comprises a mutation that removes an Asn-linked glycosylated acceptor site, or the Fc region is otherwise manipulated to change the glycosylation properties. For example, in an IgG1 Fc region, an Asn-linked glycosylation site can be removed using an N297Q mutation. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence having an N297Q mutation. For example, in an IgG1 Fc region, an Asn-linked glycosylation site can be removed using an N297Q mutation. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence having an N297Q mutation.

[0364] In another embodiment, the Fc region is glycoengineered to reduce fucose, thereby enhancing ADCC, for example by adding compounds to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010), Biotechnol. Bioeng. Vol. 105: p. 350, or by using FUT8 knockout cells, for example, as described in Yamane-Ohnuki et al. (2004), Biotechnol. Bioeng, Vol. 87: p. 614. ADCC can alternatively be optimized using the method described in Umaña et al. (1999), Nature Biotech, Vol. 17: p. 176. In another embodiment, the Fc region has been engineered to enhance complement activation, for example, as described in Natsume et al. (2009), Cancer Sci., Vol. 100: p. 2411.

[0365] Nucleic Acids

[0366] In a third aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the anti-ROR1 antibody or antigen-binding fragment thereof disclosed herein or the bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0367] The term "polynucleotide" or "nucleic acid" includes both single-stranded nucleotide polymers and double-stranded nucleotide polymers. The nucleotides comprising the nucleic acid can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. The modifications include base modifications (such as bromouridine and inosine derivatives), ribose modifications (such as 2', 3'-dideoxyribose) and internucleotide linkage modifications (such as phosphorothioates, phosphorodithioates, selenophosphates, diselenphosphates, anilinothioates (phosphoroanilothioate), anilinophosphates (phoshoraniladate) and phosphoramidates.

[0368] For example, the present invention provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein.

[0369] For example, the present invention provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of the light chain variable region sequences disclosed herein.

[0370] For example, the present invention provides nucleic acid molecules encoding the following items: (i) any of the heavy chain variable region sequences disclosed herein; and (ii) any of the light chain variable region sequences disclosed herein. The present 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 following items: (i) any of the heavy chain variable region sequences disclosed herein; and (ii) any of the light chain variable region sequences disclosed herein.

[0371] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding an anti-ROR1 antibody or an antigen-binding fragment thereof disclosed herein or a bispecific antibody or an antigen-binding fragment thereof disclosed herein. In some embodiments, the present invention provides a deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an anti-ROR1 antibody or an antigen-binding fragment thereof disclosed herein or a bispecific antibody or an antigen-binding fragment thereof disclosed herein.

[0372] Therefore, a deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an anti-ROR1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein is used to treat a disease. In some embodiments, the disease is cancer, preferably a ROR1-positive cancer. In some embodiments, the cancer is selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0373] Therefore, ribonucleic acid (RNA) comprising a deoxynucleotide sequence encoding an anti-ROR1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein can be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from ROR1 -positive cancers, preferably selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0374] In some embodiments, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of the cell.

[0375] Carrier

[0376] In a fourth aspect, the present invention also provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding the anti-ROR1 antibody or antigen-binding fragment thereof disclosed herein or the bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0377] In some embodiments, the vector is a recombinant expression vector capable of expressing a polypeptide comprising a heavy chain variable region or a light chain variable region of an anti-ROR1 antibody. For example, the present invention provides a recombinant expression vector comprising any of the above nucleic acid molecules.

[0378] Any vector may be suitable for use in 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, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papillomatosis virus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0379] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses. For example, the vector can be selected from pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors useful in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0380] Recombinant expression vectors can be prepared using standard recombinant DNA techniques, which are described, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0381] Therefore, the vector can be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from ROR1 positive cancers, preferably selected from B cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer. The vector of the present invention can be introduced into cells. In some embodiments, the vector of the present invention can be introduced into cells in vitro or in vitro. Optionally, the cells introduced with the vector can then be administered to the subject. In some embodiments, the vectors of the invention can be introduced into cells in vivo.

[0382] For example, the vector can be an adenovirus vector comprising a nucleotide sequence encoding the anti-ROR1 antibody or antigen-binding fragment thereof disclosed herein or the bispecific antibody or antigen-binding fragment thereof disclosed herein. The vector can be administered to a subject and then enter the subject's cells in vivo, thereby integrating the nucleotide sequence encoding the anti-ROR1 antibody or antigen-binding fragment thereof disclosed herein or the bispecific antibody or antigen-binding fragment thereof disclosed herein into the genome of the cell, and then the cell expresses the anti-ROR1 antibody or antigen-binding fragment thereof disclosed herein, so as to treat the diseases disclosed herein.

[0383] Host cells

[0384] In a fifth aspect, the present invention also provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

[0385] Any cell can be used as a host cell for the nucleic acids or vectors of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, e.g., 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, the host cell includes, for example, a CHO cell, such as a CHOS cell and a CHO-K1 cell, or a HEK293 cell, such as HEK293A, HEK293T, and HEK293FS.

[0386] The host cells of the present invention are prepared by introducing the vector disclosed herein or the nucleic acid disclosed herein in vitro or ex vivo. The host cells of the present invention can be administered to a subject, and the host cells express the anti-ROR1 antibody or antigen-binding fragment thereof disclosed herein in vivo to treat the diseases disclosed herein.

[0387] The present invention also provides a host cell into which any of the above vectors has been introduced. The present invention also provides a method for producing the antibodies and antibody fragments of the present invention by culturing the host cell under conditions that allow the production of the antibodies or antibody fragments and recovering the antibodies and antibody fragments thus produced.

[0388] Antibody-drug conjugates

[0389] In a sixth aspect, the present invention provides an antibody-drug conjugate (ADC), which comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention.

[0390] In the context of the present disclosure, a "conjugate" refers to an antibody or antibody fragment (such as an antigen binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, a toxin, a therapeutic agent, a detectable marker, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates". 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". Other antibody conjugates include, for example, multispecific (such as bispecific or trispecific) antibodies.

[0391] In some embodiments, the effector molecule can be a detectable marker or an immunotoxin. Specific non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38 and PE40), diphtheria toxin (DT), botulinum toxin or its modified toxins, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that usually cause death through hepatotoxicity. However, PE and DT can be modified to be used as immunotoxins by removing the natural targeting components of the toxins (such as domain 1a of PE and B chain of DT) and replacing them with different targeting parts (such as antibodies). The term "conjugated" or "connected" can refer to making two polypeptides into a continuous polypeptide molecule. In one embodiment, the antibody is connected to the effector molecule. In another embodiment, the antibody connected to the effector molecule is further connected to a lipid or other molecule, to a protein or peptide, to increase its half-life in vivo. The connection can be carried out chemically or recombinantly. In one embodiment, the linkage is chemical, wherein the reaction between the antibody portion and the effector molecule has resulted in a covalent bond formed between the two molecules, thereby forming one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule.

[0392] The present invention provides immunoconjugates comprising a monoclonal antibody or antigen binding fragment disclosed herein and an effector molecule. In some embodiments, the effector molecule is a toxin, such as but not limited to Pseudomonas exotoxin or a variant thereof. In other embodiments, the effector molecule is a detectable label, such as but not limited to a fluorophore, an enzyme, or a radioisotope.

[0393] The disclosed monoclonal antibodies may be conjugated to therapeutic agents or effector molecules. Immunoconjugates include, but are not limited to, molecules in which a therapeutic agent is covalently linked to an antibody. A therapeutic agent is an agent having a specific biological activity against a specific target molecule or a cell carrying a target molecule. Those skilled in the art will appreciate that therapeutic agents may include various drugs, such as vinblastine, daunomycin, etc.; cytotoxins such as natural or modified Pseudomonas exotoxin or diphtheria toxin; encapsulating agents (such as liposomes) containing pharmacological compositions; radioactive agents such as 125 I. 32 P. 14 C. 3 H and 35 S and other labels; target moieties; and ligands.

[0394] The choice of a particular therapeutic agent depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the therapeutic agent may be a cytotoxin used to cause the death of a particular target cell, such as a tumor cell. Conversely, when it is desired to cause a non-lethal biological response, the therapeutic agent may be conjugated to a non-lethal pharmaceutical agent or a liposome containing a non-lethal pharmaceutical agent.

[0395] Using the therapeutic agents and antibodies described herein, a skilled artisan can easily construct multiple clones containing functionally equivalent nucleic acids, such as nucleic acids that differ in sequence but encode the same effector portion or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.

[0396] The effector molecule can be attached to the antibody of interest using any number of means known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. The method of attaching the effector molecule to the antibody varies depending on the chemical structure of the effector. Polypeptides generally contain a variety of functional groups; such as carboxylic acids (COOH), free amines (-NH 2 ) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to cause the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or connect additional reactive functional groups. Derivatization may involve connecting any of a number of known linker molecules. The linker may be any molecule used to connect the antibody to the effector molecule. The linker is capable of forming a covalent bond with both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art, and include but are not limited to straight or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecule are polypeptides, the linker may be connected to the constituent amino acids through their side groups (such as through a disulfide bond with cysteine), or to the alpha carbon amino group and carboxyl group of the terminal amino acid.

[0397] In some cases, when the immunoconjugate has reached its target site, it is desirable to release the effector molecule from the antibody. Therefore, in these cases, the immunoconjugate will contain a bond that is cleavable near the target site.

[0398] Cleavage of the linker can be facilitated by enzymatic activity or by conditions to which the immunoconjugate is subjected within the target cell or near the target site to release the effector molecule from the antibody.

[0399] In view of the numerous methods 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 the appropriate method for attaching a given agent to an antibody or other polypeptide.

[0400] The antibodies disclosed herein may be derivatized or linked to another molecule (such as another peptide or protein). Typically, the antibody or portion thereof is derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, the antibody functionality may be linked (by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific 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 streptavidin core region or a polyhistidine tag).

[0401] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to produce bispecific antibodies). Suitable cross-linking agents include those with heterobifunctional groups having two distinct reactive groups separated by a suitable spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester), or those with homobifunctional groups (such as disuccinimidyl suberate). Such linkers are commercially available.

[0402] The antibody can be conjugated to a detectable marker; for example, the detectable marker can be detected by ELISA, spectrophotometry, flow cytometry, 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 inspection, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). The antibody or antigen binding fragment can also be conjugated to an enzyme for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen binding fragment is conjugated to a detectable enzyme, detection can be performed by adding an additional reagent that uses the additional reagent to produce a distinguishable reaction product. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. The antibody or antigen binding fragment can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin protein binding. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0403] The antibody can be fused to a self-labeled protein tag (e.g., HaloTag). For example, a protein tag can be cloned at the end of a constant region. HaloTag is a self-labeled protein tag derived from a bacterial enzyme (haloalkane dehalogenase) that is designed to be covalently bound to a synthetic ligand. In some cases, the synthetic ligand comprises a chloroalkane linker connected to a fluorophore such as a near-infrared fluorophore (Los et al. (2008), ACS Chem Biol., Vol. 3, No. 6: pp. 373-82).

[0404] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0405] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter gene (such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag). In some embodiments, the labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0406] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect expression of the target antigen by X-ray, emission spectroscopy or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I.

[0407] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological characteristics of antibodies, such as increasing serum half-life or increasing tissue binding.

[0408] Toxins can be used with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxins and their subunits, and botulinum toxins A to F. These toxins are readily available from commercial sources (Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins described herein (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095).

[0409] The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing tumor or viral antigens on the surface. Therefore, the antibodies disclosed herein can be directly or via a joint connected to a drug that will be delivered directly to cells expressing cell surface antigens. This can be used for treatment, diagnosis or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates or recombinant viruses. Nucleic acid therapy and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent crosslinking with single-stranded or double-stranded DNA, and oligonucleotides that form triplexes.

[0410] Alternatively, the molecule attached to the antibody can be an encapsulation system, such as a nanoparticle, a liposome or a micelle, which contains a therapeutic composition, such as a drug, a nucleic acid (e.g., an antisense nucleic acid), or another therapeutic moiety that is preferably protected from direct exposure to the circulatory system. Methods for preparing liposomes attached to antibodies are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther., Vol. 28: pp. 341-365, 1985).

[0411] The antibodies described herein may also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such uses include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. Useful labels include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, etc.) beads.

[0412] The means for detecting such labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent markers can be detected using photodetectors to detect emitted illumination. Enzyme labels are usually detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, while colorimetric labels are detected by simply visualizing the colored label.

[0413] The ADCs disclosed herein can be used alone or in combination with another therapeutic agent to treat cancer, and / or in combination with any standard therapy (such as surgical removal of the tumor, chemotherapy or radiation therapy) to treat cancer, wherein the cancer is responsive to reduction, inhibition and / or blocking of ROR1-mediated immunomodulatory function or activity.

[0414] Pharmaceutical composition

[0415] In a seventh aspect, the present invention provides a pharmaceutical composition comprising (i) the antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the bispecific antibody of the second aspect of the present invention, or the nucleic acid of the third aspect of the present invention, or the vector of the fourth aspect of the present invention, or the host cell of the fifth aspect of the present invention, or the ADC of the sixth aspect of the present invention; and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0416] The present invention provides pharmaceutical compositions comprising antibodies of the present invention. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). For example, in some embodiments, the composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer.

[0417] The antibodies or agents of the present invention (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologues can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise an antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. that are compatible with drug administration. Suitable carriers are described in the latest edition of "Remington's Pharmaceutical Sciences", which is a standard reference text in this field, and is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous solvents such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, these media or agents are considered to be used in the composition. Supplementary active compounds may also be incorporated into the composition.

[0418] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or glucose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic.

[0419] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of preparation and storage; and it must be preserved to resist the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing the following substances: for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Preventive effects on microorganisms can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Sustained absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0420] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into an appropriate solvent with one or more of the components listed above, as required, followed by sterilization filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from the components listed above. For sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.

[0421] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier used as a mouthwash, wherein the compound in the fluid carrier is orally administered and spit out or swallowed after rinsing the mouth. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants such as colloidal dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate or orange flavoring.

[0422] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0423] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream well known in the art.

[0424] The compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0425] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid discharge from the body, such as controlled release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such preparations are obvious to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These materials can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811, a method for producing antibodies of interest such as human antibodies is disclosed.

[0426] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect when associated with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the art of compounding such active compounds for the treatment of individuals, and are directly dependent on these factors.

[0427] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0428] The present invention provides therapeutic compositions comprising an anti-ROR1 antibody or antigen-binding fragment thereof of the present invention. The therapeutic compositions according to the present invention will be administered with suitable carriers, excipients and other agents, which are incorporated into formulations to improve transfer, delivery, tolerance, etc. A large number of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations", PDA (1998), J PharmSci Technol, Vol. 52: pp. 238-311.

[0429] Generation method

[0430] Monoclonal antibodies can be prepared using hybridoma methods such as those described by Kohler and Milstein, Nature, Vol. 256: 495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are generally immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0431] Immunizing agents generally include protein antigens, fragments thereof, or fusion proteins thereof. Typically, if human cells are required, peripheral blood lymphocytes are used, or if non-human mammalian sources are required, spleen cells or lymph node cells are used. Lymphocytes are then fused with immortalized cell lines using suitable fusion agents such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103). Immortalized cell lines are generally transformed mammalian cells, particularly myeloma cells of rodent, cattle, and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will generally contain hypoxanthine, aminopterin, and thymidine ("HAT culture medium"), which prevent the growth of HGPRT-deficient cells.

[0432] Preferred immortalized cell lines are those that can effectively fuse, support the stable high-level expression of antibodies by the selected antibody-producing cells, and are sensitive to culture media such as HAT culture media. A more preferred immortalized cell line is a mouse myeloma cell line, which can be obtained, for example, from the Salk Institute Cell Distribution Center in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human interspecies myeloma cell lines for producing human monoclonal antibodies have also been described. (See Kozbor, J. Immunol., Vol. 133: p. 3001 (1984); Brodeur et al., "Monoclonal Antibody Production Techniques and Applications", Marcel Dekker, Inc., New York (1987), p. 51-63)).

[0433] The presence of monoclonal antibodies against antigens in the culture medium of the hybridoma cells can then be determined. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is measured by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). These techniques and assays are known in the art. The binding affinity of the monoclonal antibodies can be measured, for example, by "Scatchard analysis of Munson and Pollard", Anal. Biochem., Vol. 107: p. 220 (1980). In addition, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity to the target antigen.

[0434] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods. (See Goding, "Monoclonal Antibodies: Principles and Practice, Academic Press", (1986), pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in mammals as ascites.

[0435] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0436] Monoclonal antibodies can also be produced by recombinant DNA methods such as U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the present invention can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). The hybridoma cells of the present invention are used as a preferred source of such DNA. After separation, the DNA can be placed in expression vectors, which are then transfected into host cells such as ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins to achieve the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing homologous mouse sequences with coding sequences of human heavy and light chain constant domains (see U.S. Patent No. 4,816,567; Morrison, Nature, Vol. 368: pp. 812-813 (1994)) or by covalently linking immunoglobulin coding sequences to all or part of the coding sequences of non-immunoglobulin polypeptides. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen combining site of an antibody of the invention to create a chimeric bivalent antibody.

[0437] A fully human antibody is an antibody molecule in which the entire sequence of both the light chain and the heavy chain (including CDR) is derived from human genes. Such antibodies are referred to herein as "humanized antibodies", "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared using tri-hybridoma technology; human B cell hybridoma technology (see Kozbor et al., 1983, Immunol Today, Vol. 4: p. 72); and EBV hybridoma technology to produce human monoclonal antibodies (see Cole et al., 1985, in: "MONOCLONAL ANTIBODIES AND CANCER THERAPY", Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote et al., 1983, Proc Natl Acad Sci USA, Vol. 80: pp. 2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole et al., 1985, in: "MONOCLONAL ANTIBODIES AND CANCERTHERAPY", Alan R. Liss, Inc., pp. 77-96).

[0438] In addition, humanized antibodies can be produced in transgenic plants as a low-cost production alternative to existing mammalian systems. For example, transgenic plants can be tobacco plants, i.e., Nicotiania benthamiana and Nicotiana tabaccum. Antibodies are purified from plant leaves. Stable transformation of plants can be achieved by using Agrobacterium tumefacien or particle bombardment. For example, via transformation, a nucleic acid expression vector containing at least heavy and light chain sequences is expressed in a bacterial culture, i.e., Agrobacterium tumefaciens strain BLA4404. Infiltration of plants can be accomplished via injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Isolation and purification of antibodies can be easily performed by many methods known to those skilled in the art. Other methods for producing antibodies in plants are described in, for example, Fischer et al., Vaccine, 2003, Vol. 21: 820-825; and Ko et al., Current Topics in Microbiology and Immunology, Vol. 332, 2009, 55-78. Therefore, the present invention also provides any cell or plant comprising a vector encoding an antibody of the present invention or producing an antibody of the present invention.

[0439] Furthermore, the (human) antibodies of interest may be produced in fungi. For example, the fungus may be Myceliophthora thermophila (e.g., Myceliophthora thermophila strain C1; Visser et al. (2011), Industrial Biotechnology, Vol. 7, No. 3: pp. 214-223). Other examples include Aspergillus species (e.g., A. oryzae (Huynh et al. (2020), Fungal Biology and Biotechnology, Vol. 7: p. 7), A. niger (Ward et al. (2004), Environ. Microbiol., Vol. 70: pp. 2567-2576), or A. awamori (Joosten et al. (2003), Microb. Cell Fact, Vol. 2: p. 1)) and Trichoderma species (e.g., T. reesei (Nyyssönen et al. (1993), Biotechnology, Vol. 11: pp. 591-595)). In other cases, the fungus can be a yeast, such as Saccharomyces cerevisiae, Candida boidinii, Hansenula polymorpha, Pichia methanolica, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces lactis, or Ogataeaminuta (Joosten et al. (2003); Suzuki et al. (2017), J Biosci Bioeng., Vol. 124: pp. 156-163).

[0440] In addition, human antibodies can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., Vol. 227: p. 381 (1991); Marks et al., J. Mol. Biol., Vol. 222: p. 581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which is very similar to that observed in humans in all aspects including gene rearrangement, assembly, and antibody repertoires. This method is described, for example, in WO 2006 / 008548, WO 2007 / 096779, WO 2010 / 109165, WO 2010 / 070263, WO 2014 / 141189 and WO 2014 / 141192.

[0441] A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat. No. 5,916,771. The method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into a mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing a heavy chain and a light chain.

[0442] In a further improvement on this procedure, PCT Publication WO 99 / 53049 discloses methods for identifying clinically relevant epitopes on immunogens and associated methods for selecting antibodies that immunospecifically bind to the relevant epitopes with high affinity.

[0443] The antibody can be expressed using a vector containing a DNA fragment encoding the above-mentioned single-chain antibody.

[0444] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include: chemical conjugates, such as those described in WO 93 / 64701, which have a targeting portion (e.g., a ligand for a cell surface receptor) and a nucleic acid binding portion (e.g., polylysine); viral vectors (e.g., DNA or RNA viral vectors); fusion proteins, such as those described in PCT / US 95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting portion (e.g., an antibody specific for a target cell) and a nucleic acid binding portion (e.g., protamine); plasmids; phages, etc. Vectors may be chromosomal, non-chromosomal or synthetic.

[0445] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. Preferred are DNA viral vectors. These vectors include poxvirus vectors such as orthopoxvirus or avian poxvirus vectors, herpesvirus vectors such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al., J. Neurochem, Vol. 64: p. 487 (1995); Lim, F. et al., in: DNA Cloning: Mammalian Systems, D. Glover, ed. (Oxford Univ. Press, Oxford, England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA, Vol. 90: p. 7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci USA, Vol. 87: p. 1149 (1990), adenovirus vectors (see LeGal LaSalle et al., Science, Vol. 259: p. 988 (1993); Davidson et al., Nat. Genet, Vol. 3: p. 219 (1993); Yang et al., J. Virol., Vol. 69: p. 2004 (1995) and adeno-associated virus vectors (see Kaplitt, MG et al., Nat. Genet., Vol. 8: p. 148 (1994).

[0446] Poxvirus vectors introduce genes into the cytoplasm. Fowlpox virus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferably used to introduce nucleic acids into neural cells. Adenovirus vectors result in shorter-term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The specific vector selected will depend on the target cell and the condition being treated. Introduction can be performed by standard techniques such as infection, transfection, transduction, or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0447] The vector can be used to target substantially any desired target cell. For example, stereotactic injection can be used to guide the vector (e.g., adenovirus, HSV) to the desired location. In addition, micropump infusion systems such as the SynchroMed infusion system can be used to deliver particles by intracerebroventricular (icv) infusion. It has also been shown that the bulk flow-based method called convection is effective in delivering macromolecules to extended areas of the brain and can be used to deliver the vector to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA, Vol. 91: pp. 2076-2080 (1994); Morrison et al., Am. J. Physiol., Vol. 266: pp. 292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injection, as well as oral or other known routes of administration.

[0448] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of ROR1 in a sample. The antibodies can also be used to try to bind to ROR1.

[0449] Methods for screening antibodies with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known in the art.

[0450] Treatment

[0451] The antibodies provided herein can be administered to slow or inhibit the progression of ROR1-positive cancers, and / or inhibit the metastasis of ROR1-positive cancers. In these applications, a therapeutically effective amount of the composition is administered to a subject in an amount sufficient to inhibit the growth, replication or metastasis of cancer cells or to inhibit signs or symptoms of cancer. Suitable subjects can include those diagnosed with a cancer expressing ROR1, such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0452] Administration of the antibodies disclosed herein may also be accompanied by administration of other anticancer agents or therapeutic treatments (such as surgical resection of a tumor). Any suitable anticancer agent may be administered in combination with the antibodies disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutic agents such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvival agents, biological response modifiers, antihormones (e.g., antiandrogens), and antiangiogenic agents. Other anticancer treatments include radiation therapy and other antibodies that specifically target cancer cells.

[0453] In some embodiments, the antibodies or antigen-binding fragments thereof, bispecific antibodies, nucleic acids, vectors, host cells, antibody-drug conjugates or pharmaceutical compositions of the invention can be administered in combination with antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

[0454] Another common treatment for some types of cancer is surgery, such as surgical removal of metastatic tumors. Another example of treatment is radiation therapy, such as the administration of radioactive material or energy (such as external beam radiation therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical removal.

[0455] Diagnosis and testing methods

[0456] In one aspect, the invention provides a method for determining a subject having cancer or at risk of developing a ROR1 -positive cancer, wherein the method comprises:

[0457] (a) obtaining a biological sample from the subject,

[0458] (b) contacting the sample with an antibody or antigen-binding fragment thereof of the present invention; and

[0459] (c) detecting binding of the antibody to the sample,

[0460] Wherein increased binding of the antibody or antigen-binding fragment thereof to the sample compared to binding of the antibody or antigen-binding fragment thereof to a control sample identifies the subject as having ROR1 -positive cancer.

[0461] In another aspect, the present invention provides a method for imaging a ROR1 -positive cancer in a subject, wherein the method comprises:

[0462] (a) administering to the subject an antibody or antigen-binding fragment thereof of the present invention, wherein the antibody is conjugated to a detectable marker; and

[0463] (b) Detecting the presence of the marker

[0464] Provided herein are methods for detecting ROR1 protein in vitro or in vivo. In some cases, ROR1 expression is detected in a biological sample. The sample can be any sample, including but not limited to a blood sample, a tumor sample, a tissue from a biopsy, an autopsy, and a pathological specimen. Biological samples also include tissue sections, for example, frozen sections for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0465] Provided herein is a method for determining whether a subject has cancer by contacting a sample from the subject with a ROR1-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample identifies the subject as having cancer.

[0466] In another embodiment, a method of diagnosing cancer in a subject is provided by contacting a sample from a subject diagnosed with cancer with a ROR1-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0467] In some examples of the disclosed methods, the monoclonal antibody is directly labeled.

[0468] In other examples, the method further includes contacting the sample with a second antibody that specifically binds to the monoclonal antibody; and detecting binding of the second antibody. Increased binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects cancer in the subject or confirms a diagnosis of cancer in the subject.

[0469] In some cases, the cancer is a ROR1 -positive cancer, preferably selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

[0470] In some examples, the control sample is a sample from a subject without cancer. In certain examples, the sample is a blood or tissue sample.

[0471] In some embodiments of the diagnostic and detection methods, the anti-ROR1 antibody is directly labeled with a detectable label. In another embodiment, the anti-ROR1 antibody (first antibody) is unlabeled, and the second antibody or other molecule that can bind to the first antibody is labeled. As is well known to those skilled in the art, a secondary antibody of a particular type and class that can specifically bind to the first antibody is selected. For example, if the first antibody is human IgG, the secondary antibody can be anti-human IgG. Other molecules that can bind to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available.

[0472] Suitable labels for antibodies or secondary antibodies 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, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I. 131 I. 35 S or 3 H.

[0473] In another embodiment, ROR1 in a biological sample can be measured by competitive immunoassay using ROR1 protein standards labeled with a detectable substance and unlabeled anti-ROR1 antibodies. In this assay, the biological sample, the labeled ROR1 protein standard, and the anti-ROR1 antibody are combined, and the amount of the 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 the labeled ROR1 protein standard bound to the anti-ROR1 antibody.

[0474] The immunoassays and methods disclosed herein can be used for many purposes. In one embodiment, an anti-ROR1 antibody can be used to detect the production of ROR1 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of ROR1 in a biological sample (such as a tumor sample, a tissue sample, or a blood or serum sample). In some examples, ROR1 is a cell surface ROR1. In other examples, the ROR1 protein is soluble (e.g., in a cell culture supernatant, or in a body fluid sample such as a blood or serum sample).

[0475] In one embodiment, a kit for detecting ROR1 in a biological sample (such as a tumor sample, a blood sample, or a tissue sample) is provided. For example, in order to verify a cancer diagnosis in a subject, a biopsy may be performed to obtain a tissue sample for histological examination. The kit for detecting a polypeptide will generally include a monoclonal anti-ROR1 antibody, such as any of the monoclonal antibodies disclosed herein. In another embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzyme label).

[0476] In one embodiment, the kit includes instructional materials that disclose methods of using the anti-ROR1 antibody. The instructional materials may be written in electronic form (such as a computer disk or CD), or may be visual (such as a video file). The kit may also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may additionally contain a device for detecting the label (such as an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, an appropriate secondary label (such as a secondary antibody), etc.). The kit may additionally include buffers and other reagents routinely used to implement a particular method. These kits and appropriate contents are well known to those skilled in the art.

[0477] In one embodiment, the diagnostic kit includes an immunoassay. Although the details of the immunoassay may vary depending on the particular format used, the method of detecting ROR1 in a biological sample generally includes the step of contacting the biological sample with an anti-ROR1 antibody. The antibody is allowed to specifically bind under immunoreactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0478] The antibodies disclosed herein can also be used in immunoassays, such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemical assays. The antibodies can also be used in fluorescence activated cell sorting (FACS). FACS uses multiple color channels, low-angle and obtuse light scatter detection channels, and impedance channels, as well as other more complex detection levels, to separate or sort cells (see U.S. Patent No. 5,061,620). As disclosed herein, any monoclonal antibody that binds to ROR1 can be used in these assays. Therefore, the antibodies can be used in conventional immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting, or immunoprecipitation.

[0479] Example

[0480] Example 1 Preparation of antigens and stable cell lines

[0481] 1.1. Preparation of antigens and other proteins

[0482] Recombinant human ROR1 ECD Fc-tagged protein (Catalog No. RO1-H5250) and human ROR1-ECD-his (abbreviated as hROR1-ECD-his) were purchased from Acro Biosystems (Catalog No.: RO1-H522y).

[0483] 1.2. Preparation of stable cell lines

[0484] HEK293T-hROR1 (human ROR1) was purchased from Kyinno (Catalog No.: KC-1018). CHO-K1-cynoROR1 (cynomolgus monkey ROR1) and CHO-K1-huROR1 (human ROR1) stable cell lines were prepared as follows. CHO-K1 cell suspension was seeded into 6-well plates at a density of 50,000 / mL at 2 mL / well, incubated overnight, and then Polybrene (Shanghai Jikai Gene, REVG0001, 10 mg / mL) was added to a final concentration of 4 μg / mL, mixed and 10 μL of virus (LV-CynoROR1, purchased from Shanghai Genechem, 42582-1, titer 1E9 / mL, 50 μL / vial) was added, mixed and incubated at 37°C for 8 hours, and then the supernatant was discarded and replaced with fresh medium and cultured at 37°C. After 24 hours of digestion, the cells were resuspended in complete medium containing 8 μg / mL puromycin (Thermo, A1113803), diluted to 5 / mL, and cultured in 10 96-well plates at 37°C for 10 days with 100 μL / well. Single clones were selected for FACS verification. After verification, the verified clones were further expanded and cultured for frozen storage.

[0485] Example 2 Animal Immunization Scheme

[0486] To obtain ROR1-specific antibodies, Harbour HCAb transgenic mice (https: / / harbourantibodies.com / ) were immunized by different methods. After immunization, multiple HCAb antibodies that bind to the ROR1 extracellular (ECD) protein or ROR1-expressing cells were obtained.

[0487] 2.1. Immunization by injection of ROR1 protein

[0488] Recombinant human ROR1 ECD Fc-tagged protein (Acro Biosystem, catalog number RO1-H5250) was used as an immunogen to immunize Harbour HCAb transgenic mice.

[0489] The immunization schedule for the Harbour HCAb mouse immunization group is listed in Table 5 below. Briefly, each mouse was given a first boost immunization with 50 μg of immunogen and adjuvant (Sigma, F5881) via the peritoneal cavity, and subsequent boost immunizations were given with 25 μg of immunogen and adjuvant (Sigma, S6322) via the peritoneal cavity. Immunizations were performed every two weeks for a total of 5 times. The final immunization was performed intraperitoneally with the immunogen diluted in PBS. Serum titers against human recombinant human ROR1 ECD His-tag protein (Acro Biosystem, catalog number RO1-H522y) were tested using ELISA, and serum titers against cell lines expressing ROR1 were tested using FACS.

[0490] Table 5. Immunization schedule

[0491]

[0492] 2.2. Immunization by injection of ROR1 cells

[0493] HEK293T-hROR1 was used as an immunogen to immunize Harbour HCAb transgenic mice. The immunization schedule for the Harbour HCAb mouse immunization group is listed in Table 6 below. Briefly, each mouse was injected intraperitoneally with 1.0×10 7 HEK293T-hROR1 cells were used for primary immunization. For booster immunization, 1.0×10 7 HEK293T-hROR1 cells were resuspended in PBS and injected intraperitoneally into mice. The interval between the primary immunization and the first booster immunization was 2 weeks. For subsequent boosters, 1.0×10 7 HEK293T-hROR1 cells were injected intraperitoneally into mice for a total of 5 times. Seven days after each booster immunization, mice were bled and tested for serum titers against recombinant human ROR1 ECD His-tagged protein (Acro Biosystem, catalog number RO1-H522y) using ELISA and against cell lines expressing ROR1 using FACS.

[0494] Table 6. Immunization schedule

[0495]

[0496] Example 3 Screening of ROR1-specific HCAb antibodies

[0497] 3.1. Screening of HCAb antibody directional cloning using HEK293-pCAG-HCAb

[0498] In this example, lymph nodes were collected from mice with high antibody titers for cDNA preparation. The variable region of HCAb cDNA was amplified by PCR using specific primers (5'-GGTGTCCAGTGTSAGGTGCAGCTG-3' (SEQ ID NO: 262), 5'-AATCCCTGGGCACTGAAGAGACGGTGACC-3' (SEQ ID NO: 263)) and cloned into a mammalian expression vector (pCAG) containing the Fc portion of the human immunoglobulin heavy chain of the IgG1 subclass, named pCAG-HCAb library. Plasmids of the pCAG-HCAb library were prepared and transfected into HEK293 cells (ATCC, CRL-1573) on 96-well plates for expression, and then the supernatants of HEK293-pCAG-HCAb were harvested and transferred to different 96-well plates for screening by in vitro binding assays. Binding to CHO-K1-huROR1, a stable cell line expressing human ROR1, and to CHO-K1-cynoROR1, a stable cell line expressing cynomolgus monkey ROR1, were tested by Mirrorball (SPT Labtech). HEK293 cell supernatants that showed binding to both CHO-K1-huROR1 and CHO-K1-cynoROR1 were selected for subsequent FACS screening. Finally, multiple HCAb clones were selected for further characterization.

[0499] 3.2. Single B cell screening for HCAb antibodies

[0500] The Beacon® Optofluidic system is used for single B cell screening. The system uses optoelectronic positioning (OEPTM) technology to move single cells and allows for simultaneous biological function testing, experimental analysis, positive clone selection, and other operations under cell culture conditions. The Beacon platform can perform these tasks in a massively parallel, automated manner for thousands of cells.

[0501] In this example, a plasma cell discovery workflow is used. In each experiment, up to 14,000 individual plasma cells are screened for secretion of ROR1-specific antibodies. Plasma cells secreting antigen-specific antibodies are then transferred to 96-well plates for subsequent single B cell sequencing to identify the heavy chain of the antibody produced by a single B cell (monoclonal). Figure 1 The screening strategy and process are shown.

[0502] This embodiment uses a single B cell sequencing method to obtain the sequence of the heavy chain of the antibody from a single plasma cell. The general procedure includes extracting and purifying total RNA from a single plasma cell lysate, reverse transcription to synthesize cDNA, amplifying and purifying cDNA, amplifying the DNA sequence encoding the heavy chain of the antibody, cloning and transfection, and Sanger sequencing. The obtained sequence is subjected to uniqueness and cluster analysis, and then the DNA sequence encoding the heavy chain of the antibody is synthesized.

[0503] Example 4. Antibody production and purification

[0504] The recombinant plasmid encoding the target antibody was transiently transfected into HEK293-6E cells (National Research Council) using PEI (Polyscience, 24885). After transfection, the cells were incubated at 37°C with 5% CO 2 The cells were incubated at 4 °C and shaken at 120 rpm. Six to seven days after transfection, the cell culture supernatant containing the target antibody was harvested by centrifugation and filtration. Monoclonal antibodies were purified using protein A magnetic beads (AmMag protein A magnetic beads, Genscript, L00695).

[0505] The purity of the antibody was tested by SEC-HPLC (Agilent 1260 Infinity II HPLC system, Welch Xtimate SEC-300 column, 1× PBS at pH 7.4 as mobile phase) and SDS-PAGE (SurePAGE, Bis-Tris, 10×8, 4%-12%, 12-well, Genscript, M00653). The recombinant antibody was successfully expressed and purified for further characterization.

[0506] By Examples 1 to 4, HCAb antibodies PR005337, PR005338, PR005339, PR005340, PR005341, PR005342, PR005343, PR005344, PR005345, PR005346, PR005347, PR005348, PR005349, PR005350, PR005351, PR005352, PR303125, PR303189, PR303191, PR303199, PR303201, PR303145, PR303147 and PR303155 were obtained. The amino acid sequences of these antibodies are listed in Tables 1 to 3 above.

[0507] Meanwhile, the anti-ROR1 antibody PR000374 was prepared according to the above method using the sequence information of International Patent Application No. WO2016 / 094873 (incorporated herein as a reference). PR000374 is a rabbit anti-human ROR1 Ab from patent WO2016 / 094873.

[0508] Example 5. Binding activity of antibodies

[0509] 5.1. Binding activity to cells expressing ROR1

[0510] The binding of the recombinant anti-ROR1 antibodies to cells overexpressing human or cynomolgus monkey ROR1 was tested by flow cytometry. In this example, the cell line expressing ROR1 was a HEK293T cell line that had been transfected to express human ROR1 on the surface (HEK293T-hu ROR1, KYINNO BIOTECHNOLOGY Co., Ltd., catalog number KC-1018), a CHO-K1 cell line that had been transfected to express cynomolgus monkey ROR1 (CHOK1-cyno ROR1), PANC-1 (ATCC, catalog number: CRL-1469), or an A549 cell line (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences).

[0511] Briefly, anti-ROR1 antibody was serially diluted in staining buffer (PBS buffer containing 2% FBS). 5 Cells were incubated for 1 hour at 4°C. The cells were washed twice with staining buffer (PBS containing 2% FBS), and 100 μL of a 1:1000 dilution of fluorescently labeled anti-human IgG antibody (Alexa Fluor 647 AffiniPure goat anti-human IgG Fc, Jackson ImmunoResearch, catalog 109-605-098, or Alexa Fluor® 488 AffiniPure goat anti-human IgG (H+L), Jackson ImmunoResearch, catalog 109-545-088) was added to each well. After incubation for 1 hour at 4°C, the cells were washed twice with staining buffer and subjected to flow cytometry. PR000374 (Bench marker 1) and non-relevant IgG isotype control (Crownbio) were used as positive and negative controls, respectively.

[0512] The results of PR005337, PR005338, PR005339, PR005340, PR005341, PR005342, PR005343, PR005344, PR005345, PR005346, PR005347, PR005348, PR005349, PR005350, PR005351 and PR005352 are shown in Figure 2 A. Figure 2 B. Figure 3 A. Figure 3 B. Figure 3 C and the following Tables 7 to 8. The results showed that the HCAb antibodies had good binding activity to PANC-1 cells and strong binding activity to CHO-K1-cynoROR1 cells, indicating that these HCAb antibodies had cross-reactivity with cynoROR1.

[0513] Table 7. Binding of anti-ROR1 HCAb antibodies to CHO-K1-cyno ROR1 cells

[0514]

[0515] Table 8. Binding of anti-ROR1 HCAb antibodies to PANC-1 cells

[0516]

[0517] The results of HCAb antibodies PR303125, PR303189, PR303191, PR303199, PR303201, PR303145, PR303147 and PR303155 are shown in Figure 2 C. Figure 3 D. Figure 4 , Figure 5 and in Table 9 below. The results indicate that the HCAb antibody exhibits strong binding activity to cells expressing both human ROR1 and cynomolgus monkey ROR1. These results indicate that the anti-ROR1 HCAb antibody is able to bind to human ROR1 and cynomolgus monkey ROR1 on the cell membrane with high affinity.

[0518] Table 9. Anti-ROR1 HCAb binding to cell surface ROR1 tested by FACS

[0519]

[0520] 5.2. Binding activity to hu-ROR1-ECD-his

[0521] Binding of recombinant anti-ROR1 antibodies to hu-ROR1-ECD-his was tested by ELISA. Briefly, 1 μg / mL of hu-ROR1-ECD-his was added to a 96-well plate at 100 μL / well and incubated overnight at 4°C, the plate was washed three times with 1× PBST at 300 μL / well, the plate was blocked with 2% BSA in 1× PBST at 200 μL / well and incubated at 37°C for 1 hour, and the anti-ROR1 antibody was serially diluted in staining buffer (PBS containing 2% BSA). The antibody solution was added to the plate and incubated at 37°C for 1 hour. The plate was washed 3 times with 1× PBST, 300 μl / well. Secondary antibody 100 μl / well was added and incubated at 37°C for 1 hour. The plate was washed 3 times with 1× PBST, 300 μl / well. Then 100 μl / well of TMB was added for about 5 minutes. The plate was blocked by adding 100 μl / well of 2M H 2 SO 4 The reaction was stopped and the plate was read at 450 nm and 570 nm using a Molecular Device Spectra Max Plus 384.

[0522] The results of HCAb antibody are shown in Figure 6 and in Table 10 below. The results show that the HCAb antibody exhibits strong binding activity to hu-ROR1-ECD-his. These results indicate that the anti-ROR1 HCAb antibody is able to bind to human ROR1 with high affinity.

[0523] Table 10. Anti-ROR1 HCAb binding to hu-ROR1-ECD-his tested by ELISA

[0524]

[0525] Example 6 PR005340 affinity maturation

[0526] The affinity maturation of PR005340 is carried out by carrying out yeast surface display with BD FACS AriaIII sorter.First, the sequence of PR005340 was analyzed by Kabat numbering, and the CDR district was also determined. Site saturation and CDR walking strategy are applied to this HCAb affinity maturation.Build four mutagenesis libraries, and be named as 5340-H1L, 5340-H2L, 5340-H3L and 5340-H2WL respectively.Carry out two rounds of sorting and screening as follows.

[0527] In the first round of sorting and screening, 5340-H1L, 5340-H2L and 5340-H3L were sorted and screened by BD FACS AriII; 5340-H2WL library was MACS enriched and FACS sorted and screened. For each library, gates were performed and populations with high binding were picked; sorted yeast cells were then cultured and picked for sequencing and analysis; unique hits were also characterized by FACS. Based on the FACS results, several hot spots were selected and combined into combinatorial mutagenesis libraries.

[0528] In round 2, combinatorial libraries were designed and constructed. The libraries were also gated and sorted; the sorted population with high binding was also cultured and picked for sequencing and analysis, and all unique hits were characterized by FACS.

[0529] Finally, 15 variants (PR009810, PR009811, PR009812, PR009813, PR009814, PR009815, PR009816, PR009817, PR009818, PR009819, PR009820, PR009821, PR009822, PR009823, and PR009824) and 1 monovalent form (VH-Flag-His) were screened and synthesized. 7 bivalent forms (HCAb) variants (PR007408, PR007409, PR007410, PR007411, PR007412, PR007413, PR007414, PR007415, PR007416, PR007417, PR007418, PR007419, PR007420, PR007421, PR007422, PR007423 and PR007424). The amino acid sequences of these variants are listed in Tables 1 to 3 above.

[0530] The binding activity of the variants to PANC-1 cells was tested using the same method described in Example 5.1. The results are shown in Figures 7 and 8 and in Tables 11 and 12. As shown, most variants showed significant binding enhancement compared to PR005340.

[0531] Table 11. Binding of PR005340 monovalent variants to PANC-1 cells

[0532]

[0533] Table 12. Binding of PR005340 bivalent variants to PANC-1 cells

[0534]

[0535] Example 7 Analysis of the binding activity of antibodies to soluble ROR1 protein by BLI method

[0536] In this example, the binding kinetics of anti-ROR1 antibodies to soluble ROR1 protein were analyzed by using biomembrane interferometry (BLI) analysis on a Fortebio Octet Red384 instrument (Fortebio).

[0537] 7.1. Measuring the binding affinity of antibodies to ROR1 protein by using the HIS1K sensor

[0538] For BLI analysis, recombinant anti-ROR1 antibodies were serially diluted with 10× kinetic buffer (Fortebio). Human ROR1-His protein was diluted to 20 nM. The diluted antibodies, ROR1 protein, and regeneration buffer (10 mM glycine HCl, pH 1.5) were then added to a 96-well plate (Greiner). The rate constants of association and dissociation were measured using a HIS1K sensor (Fortebio). The sensor surface was regenerated with regeneration buffer after each binding experiment. Traces were processed using Octet data analysis software (version 11.0, Pall ForteBio, CA, USA).

[0539] The binding kinetic parameters of the anti-ROR1 HCAb antibodies binding to human ROR1 are summarized in Table 13. As shown in Table 13, the HCAb antibodies showed high binding affinity to soluble ROR1 in the Octtec assay.

[0540] Table 13. Binding of anti-ROR1 HCAb antibodies to soluble human ROR1

[0541]

[0542] 7.2. Measuring the binding affinity of antibodies to ROR1 protein by using AHC sensor

[0543] In the BLI analysis, antibodies were diluted to 5 μg / mL using freshly prepared 1× kinetic buffer (10× kinetic buffer (ForteBio, #18-1105) diluted with PBS (BBI Life Sciences, # E607016-0500)) and captured on the surface of anti-human Fc (AHC) Octet biosensors (ForteBio, #18-5060) to achieve capture levels between 0.6 nm and 1.0 nm. The captured biosensors were then immersed in wells containing 2-fold serial dilutions of antigen protein to detect association signals, followed by a dissociation step in wells containing 1× kinetic buffer. Human ROR1hit-tagged protein (Acrobiosystems, #RO1-H522y) was diluted from 80 nM to 5 nM; the association phase was 180 s, and the dissociation phase was 600 s. Sensorgrams were recorded and reference signals were subtracted before curve fitting using ForteBio Data Analysis 11.0 software. The association rates (k) were calculated using a simple one-to-one Langmuir binding model. on ) and dissociation rate (k dis ). The equilibrium dissociation constant (K D ) is calculated as k dis / k on The binding kinetic parameters of the anti-ROR1 HCAb antibodies binding to human ROR1 are summarized in Table 14.

[0544] Table 14. Binding of anti-ROR1 antibodies to soluble human ROR1

[0545]

[0546] Example 8. Antibody specificity verification

[0547] 8.1. Antibodies were tested for binding to human ROR2 by ELISA.

[0548] 1 μg / mL hu-ROR2-ECD-his (Acrobiocystems, RO2-H52E5) was added to a 96-well plate, 100 μL / well, incubated overnight at 4°C, the plate was washed 3 times with 1× PBST, 300 μL / well, the plate was blocked with 2% BSA in 1× PBST, 200 μL / well, incubated at 37°C for 1 hour, the anti-ROR1 antibody was serially diluted in staining buffer (PBS containing 2% BSA). The antibody solution was added to the plate and incubated at 37°C for 1 hour, the plate was washed 3 times with 1× PBST, 300 μl / well, the secondary antibody 100 μl / well was added and incubated at 37°C for 1 hour, the plate was washed 3 times with 1× PBST, 300 μl / well, and then 100 μl / well of TMB was added for about 5 minutes, and the plate was blocked by adding 100 μl / well of 2M H 2 SO 4 The reaction was stopped and the plate was read at 450 nm and 570 nm using a Molecular Device Spectra Max Plus 384.

[0549] The results of HCAb antibody are shown in Fig. 9 The results showed that the HCAb antibody did not show binding activity to hu-ROR2-ECD-his. These results indicate that the anti-ROR1 HCAb antibody specifically binds to ROR1.

[0550] 8.2. Testing the binding of antibodies to human ROR2 by BLI

[0551] In this example, the binding kinetics of anti-ROR1 antibodies to soluble ROR2 protein were analyzed by using biomembrane interferometry (BLI) analysis on a Fortebio Octet Red384 instrument (Fortebio).

[0552] In the BLI analysis, antibodies were diluted to 5 μg / mL using freshly prepared 1× kinetic buffer (10× kinetic buffer (ForteBio, #18-1105) diluted with PBS (BBI Life Sciences, # E607016-0500)) and captured on the surface of anti-human Fc (AHC) Octet biosensors (ForteBio, #18-5060) to achieve capture levels between 0.6 nm and 1.0 nm. The captured biosensors were then immersed in wells containing 2-fold serial dilutions of antigen protein to detect association signals, followed by a dissociation step in wells containing 1× kinetic buffer. Human ROR2hit-tagged protein (Acrobiosystems, #RO2-H52E5) was diluted from 600 nM to 37.5 nM; the association phase was 180 seconds, and the dissociation phase was 180 seconds. Sensorgrams were recorded and reference signals were subtracted before curve fitting using ForteBio Data Analysis 11.0 software. Association rates (k) were calculated using a simple one-to-one Langmuir binding model. on ) and dissociation rate (k dis ). The equilibrium dissociation constant (K D ) is calculated as k dis / k on ratio.

[0553] The binding kinetic parameters of the anti-ROR1 HCAb antibody binding to human ROR2 are summarized in Table 15. The results showed that the HCAb antibody had no binding activity to ROR2, indicating that the anti-ROR1 HCAb antibody specifically binds to ROR1.

[0554] Table 15. Binding of anti-ROR1 antibodies to soluble human ROR2

[0555]

[0556] Example 9. Determination of epitope binding of antibodies by competition assay

[0557] To determine whether the anti-ROR1 antibodies bind to human ROR1 at different or similar binding epitopes, epitope competition experiments were performed on anti-ROR1 antibodies using the ForteBio Octet® RED96e platform. Human ROR1 protein (Acrobiosystems, #RO1-H522y) was biotinylated and then captured on the SA biosensor (ForteBio, #18-5020) to achieve a loading level of 0.3nm to 0.4nm. A tandem competition assay format was applied and it contained two association steps. First, the biosensor loaded with antigen bound to each antibody (also called the first antibody, 1st Ab) at a saturation concentration of 200nM for 180 seconds to reach equilibrium, and then bound to 200nM of the competing antibody (also called the second antibody, 2nd Ab) for 180 seconds. When the first antibody was replaced by the dynamic buffer, the second binding signal was recorded as 100% signal for each antibody. All binding data were analyzed using ForteBioData Analysis 11.0 software. The inhibition rate was calculated by the following formula:

[0558] Inhibition rate (%) = (AB) / A*100

[0559] A: 100% signal for each antibody;

[0560] B: Signal from the second antibody binding step.

[0561] If the inhibition rate obtained is greater than 80 (%), it means that the epitopes of the two antibodies completely overlap; if the inhibition rate is less than 40 (%), it means that the epitopes of the two antibodies are different or far away from each other.

[0562] As shown in Tables 16 and 17, HCAb antibodies PR005338, PR005340, PR007417 and PR007424 share almost the same binding epitope on human ROR1, but are completely different from reference antibody PR000374. HCAb antibodies PR303189, PR303199, PR303145, PR303147, PR303155, PR303125, PR303191, PR303201 show different binding epitopes from PR000374; among them, PR303125, PR303191, PR303201 share similar or overlapping epitopes, but are different from other HCAb antibodies.

[0563] Table 16. Inhibition rate of antibodies in epitope competition assay

[0564]

[0565] Table 17. Inhibition rate of antibodies in epitope competition assay

[0566]

[0567] Example 10. Antibodies are internalized by cells expressing ROR1

[0568] 10.1. Testing of antibody internalization by HEK293T-hROR1 cells using the Ab-MMAF cytotoxicity assay

[0569] HEK293T-hROR1 cells were added to a flat-bottom 96-well plate at 12,000 cells / 90 μl and incubated overnight at 37°C in a 5% CO2 incubator. Antibodies were prepared at a 10× concentration (100 nM) in complete medium. The dilution factor was 5 and 6 doses were prepared (10, 2, 0.4, 0.08, 0.016, 0.0032). 10 μl of each complex dilution was transferred to the cells in duplicate, and the final volume of all assay wells was 100 μl. aHFc-CL-MMAF was prepared at a concentration of 50 μg / ml (50×) and 2 μl was added to the wells to a final concentration of 1 μg / ml. Cells were incubated at 37°C in a 5% CO2 incubator. 2 Culture for 120 hours. Add 100 μl of CellTiter-Glo reagent, which is equal to the volume of cell culture medium present in each well. Mix the contents on an orbital shaker for 2 minutes to induce cell lysis. Incubate the plate at room temperature for 10 minutes to stabilize the luminescent signal. Luminescence is recorded using PE Enspire.

[0570] The internalization rate of anti-ROR1 HCAb is shown in Fig.10 Middle. HCAb showed better internalization activity than PR000374 towards 293T cells overexpressing ROR1.

[0571] 10.2. Antibody internalization by PANC-1 cells measured by pHAb kit

[0572] In this example, pHAb amine reactive dye (Promega, catalog number G9845) was used to determine the antigen-based internalization of anti-ROR1 antibodies into PANC-1 cells. PHAb dye is a pH sensor dye that has very low fluorescence at pH>7, and the fluorescence increases significantly when the pH of the solution becomes acidic. When antibodies labeled with pHAb dyes are bound outside the cell membrane at neutral pH, no fluorescence or very low fluorescence is monitored. After internalization, the lower the environmental pH in endosomes and lysosomes, the stronger the fluorescence will be.

[0573] The antibody was labeled with pHAb dye, and the DAR was calculated according to the kit instructions. The labeled antibody was then incubated with PANC-1 for 24 hours at 4 °C (internalization activity at this temperature was very low, which was used as a background control) or 37 °C. The fluorescence with an excitation maximum (Ex) at 532 nm and an emission maximum (Em) at 560 nm was then detected. The final normalized result was shown as the fluorescence intensity at 37 °C minus the fluorescence intensity of the background at 4 °C, and then divided by the DAR of the pHAb dye of the antibody. Higher values ​​indicate higher internalization activity.

[0574] The results of the internalization rates of the anti-ROR1 HCAb antibodies detected at 7 hours and 24 hours are shown in Fig.11 A to Fig.11 In B. The results showed that HCAb clones PR303155, PR303191 and PR303199 showed good internalization by PANC-1 cells.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to ROR1, wherein the antibody comprises a heavy chain variable region (VH), and wherein the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in any one of SEQ ID NOs: 147-202.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises HCDRs 1-3 having the amino acid sequence shown below: SEQ ID NO: 11, 40, 100, respectively, SEQ ID NO: 12, 41, 101, respectively, SEQ ID NO: 12, 42, 102, respectively, SEQ ID NO: 12, 40, 103, respectively, SEQ ID NO: 12, 43, 104, respectively, SEQ ID NO: 13, 44, 105, respectively, SEQ ID NO: 12, 40, 106, respectively, SEQ ID NO: 12, 45, 107, respectively, SEQ ID NO: 12, 40, 108, respectively, SEQ ID NO: 14, 46, 109, respectively, SEQ ID NO: 12, 47, 110, respectively, SEQ ID NO: 12, 48, 111, respectively, SEQ ID NO: 12, 45, 112, respectively, SEQ ID NO: 15, 49, 113, respectively, SEQ ID NO: 16, 50, 114, respectively, SEQ ID NO: 12, 51, 115, respectively, SEQ ID NO: 18, 56, 126, respectively, SEQ ID NO: 21, 40, 130, respectively, SEQ ID NO: 22, 60, 131, respectively, SEQ ID NO: 12, 40, 132, respectively, SEQ ID NO: 23, 61, 133, respectively, SEQ ID NO: 19, 57, 127, respectively, SEQ ID NO: 20, 58, 128, respectively, SEQ ID NO: 14, 59, 129, respectively, SEQ ID NO: 17, 53, 119, respectively, SEQ ID NO: 17, 53, 116, respectively, SEQ ID NO: 17, 53, 120, respectively, SEQ ID NO: 17, 53, 121, respectively, SEQ ID NO: 17, 53, 122, respectively, SEQ ID NO: 17, 53, 123, respectively, SEQ ID NO: 17, 53, 124, respectively, SEQ ID NO: 17, 53, 125, respectively, SEQ ID NO: 17, 52, 116, respectively, SEQ ID NO: 17, 53, 117, respectively, SEQ ID NO: 17, 52, 117, respectively, SEQ ID NO: 17, 40, 116, respectively, SEQ ID NO: 17, 53, 118, respectively, SEQ ID NO: 17, 54, 116, respectively, SEQ ID NO: 17, 55, 116, respectively, SEQ ID NO: 17, 54, 117, respectively, SEQ ID NO: 17, 40, 117, respectively, SEQ ID NO: 17, 55, 117, respectively, SEQ ID NO: 17, 54, 118, respectively, SEQ ID NO: 17, 40, 118, respectively, SEQ ID NO: 17, 55, 118, respectively, SEQ ID NO: 17, 55, 103, respectively, SEQ ID NO: 12, 55, 117, respectively, or They are SEQ ID NO: 12, 55, and 118 respectively.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, 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 any one of SEQ ID NOs: 147-202.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody comprises an Fc region.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody comprises a heavy chain (HC), and wherein the HC 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 any one of SEQ ID NOs: 205-260.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody does not comprise a light chain.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody comprises two heavy chains.

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

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is of an isotype selected from IgG, IgA, IgM, IgE and IgD.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is of a subtype selected from IgG1, IgG2, IgG3 and IgG4.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is selected from HCAb, VHH, nanobody, Fab, Fab', F(ab')2, Fd, Fd' and dAb. 12 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antibody is monovalent, divalent or multivalent.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody or antigen-binding fragment is linked to a fluorescent label, a radioactive label or a cytotoxic agent.

15. A bispecific antibody, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 and a second antigen-binding region that specifically binds to a tumor-associated antigen or an immune cell antigen; preferably, the second antigen-binding region binds to CD3. 16 . A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the bispecific antibody according to claim 15 . A vector comprising the nucleic acid according to claim 16 .

18. A host cell comprising the nucleic acid according to claim 16 or the vector according to claim 17.

19. An antibody-drug conjugate (ADC), comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the bispecific antibody according to claim 15.

20. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, a bispecific antibody according to claim 15, a nucleic acid according to claim 16, a vector according to claim 17, a host cell according to claim 18, or an antibody-drug conjugate according to claim 19; and (ii) a pharmaceutically acceptable carrier or excipient.

21. The pharmaceutical composition of claim 20, wherein the composition further comprises a second therapeutic agent selected from the group consisting of an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

22. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the bispecific antibody according to claim 15, the nucleic acid according to claim 16, the vector according to claim 17, the host cell according to claim 18, the antibody-drug conjugate according to claim 19, or the pharmaceutical composition according to claim 20 or 21.

23. The method of claim 22, wherein the cancer is a ROR1 -positive cancer, preferably selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer.

24. The method of claim 22 or 23, further comprising administering to the subject a second therapeutic agent.

25. The method of claim 24, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

26. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the bispecific antibody according to claim 15, the nucleic acid according to claim 16, the vector according to claim 17, the host cell according to claim 18, the antibody-drug conjugate according to claim 19, or the pharmaceutical composition according to claim 20 or 21 in the preparation of a medicament for treating cancer in a subject.

27. The use according to claim 26, wherein the cancer is a ROR1-positive cancer, preferably selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

28. The use according to any one of claims 26 or 27, wherein the medicament further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

29. The use according to any one of claims 26 or 27, wherein the drug is administered in combination with a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

30. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the bispecific antibody according to claim 15, the nucleic acid according to claim 16, the vector according to claim 17, the host cell according to claim 18, the antibody-drug conjugate according to claim 19, or the pharmaceutical composition according to claim 20 or 21, for use in treating cancer in a subject.

31. The antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate or pharmaceutical composition for use according to claim 30, wherein the cancer is a ROR1-positive cancer, preferably selected from B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

32. The antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate or pharmaceutical composition for use according to claim 30 or 31 is administered in combination with a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

33. A method for diagnosing a ROR1-positive cancer in a subject, the method comprising: (a) obtaining a biological sample from the subject, (b) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14; and (c) detecting binding of the antibody to the sample, Wherein increased binding of the antibody or antigen-binding fragment thereof to the sample as compared to binding of the antibody or antigen-binding fragment thereof to a control sample identifies the subject as having ROR1 -positive cancer.

34. A method for imaging a ROR1 -positive cancer in a subject, the method comprising: (a) administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody is conjugated to a detectable marker; and (b) detecting the presence of the marker.

35. The method of claim 34, wherein: (a) The detectable marker is 111 In, and preferably, the detection of the marker is performed by single photon emission computed tomography, or (b) The detectable marker is 89 Zr, and preferably, the detection of said marker is performed by positron emission tomography.

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