Antibodies, antigen-binding fragments thereof and pharmaceutical uses thereof

Abstract

The invention relates to receptor tyrosine kinase-like orphan receptor 1 antibodies or antigen binding fragments and pharmaceutical uses thereof.

Description

Technical Field

[0001] The present invention relates to antibodies, in particular to antibodies showing specificity for receptor tyrosine kinase-like orphan receptor 1 (ROR1), and uses thereof, for example in cancer treatment. Background Art

[0002] ROR (receptor tyrosine kinase-like orphan receptor) is a transmembrane protein of the RTK (receptor tyrosine kinase) family, with ROR1 and ROR2, which are type I transmembrane receptor tyrosine kinases. The extracellular region of ROR1 and ROR2 contains an immunoglobulin (Ig) domain, a cysteine-rich domain (CRD) (also known as a Frizzled (Fz) domain), and a Kringle (Kr) domain. All three domains are involved in protein-protein interactions. Intracellularly, ROR1 and ROR2 have a tyrosine kinase (TK) domain and a proline-rich domain (PRD), spanned by two serine / threonine-rich domains (Borcherding et al., 2014, Protein Cell, 5:496; Rebagay et al., 2012, Prontiers inoncology, 2:1).

[0003] ROR1 is expressed during embryonic and fetal development, and controls cell polarity, cell migration, neurite outgrowth, etc. Expression gradually decreases as development progresses, it is hardly expressed in adults, is transiently expressed during B cell development, and only minimal expression has been reported in adipocytes (Hudecek et al., 2010, Blood 116:4532; Matsuda et al., 2001, Mech. Dev. 105:153).

[0004] However, since overexpression of ROR1 has been observed in various cancer cells, it is classified as an oncofetal gene. In particular, ROR1 has attracted attention as an anticancer antibody target because it was found to be overexpressed in chronic lymphocytic leukemia (CLL). It has been reported that in addition to chronic lymphocytic leukemia (CLL), it is not only overexpressed in hematological malignancies such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), etc., but also in solid cancers, including breast cancer, ovarian cancer, gastric cancer, lung cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colon cancer, etc.

[0005] This cancer cell-specific expression of ROR1 shows that ROR1 can be an effective cancer target, so it is necessary to develop antibodies that specifically recognize it. This oncofetal protein is an attractive target for cancer therapy. ROR1 antibodies have been described in documents WO2014031174 (UC961), WO2017127664 (XBR1-402), etc. And a humanized mouse anti-ROR1 antibody UC961 has entered clinical trials for relapsed or refractory chronic lymphocytic leukemia.

[0006] Considering the cancer-specific expression of ROR1 and its expression in various cancers, antibodies against the same ROR1 antigen can be developed into different anti-cancer antibodies according to the characteristics or uses of each antibody, so it is necessary to develop various antibodies that can replace or supplement existing antibodies. Due to the small number of available ROR1-specific monoclonal antibodies, there is a need in the art for better anti-ROR1 antibodies that have effective internalization or other functional properties that known antibodies do not have. Additional diagnostic tools are also needed to detect ROR1 expression in ROR1-related disease conditions. The present invention is intended to address these and other needs. Summary of the invention

[0007] The anticancer efficacy of antibody-drug conjugates is believed to rely on the uptake of surface-expressed antigens by cancer cells, and thus the insufficient internalization of monoclonal antibodies targeting ROR1 is an urgent issue to be addressed.

[0008] The technical problem to be solved by the present invention is to overcome the defects of the antibody targeting ROR1, such as weak binding ability with ROR1, cross-reactivity with ROR2 and insufficient internalization in human cells.

[0009] Specifically, the present invention covers the following aspects:

[0010] The present disclosure provides an anti-ROR1 antibody or an antigen-binding fragment thereof, which comprises one or more CDR region sequences selected from the following sequences: an anti-ROR1 antibody or an antigen-binding fragment thereof, which comprises: an antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and an antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: a) HCDR1 is as shown in SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05 or SEQ ID NO: 06; b) HCDR2 is as shown in SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; c) HCDR3 is as shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 NO:20; d) LCDR1 is shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26; e) LCDR2 is shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32; f) LCDR3 is shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQID NO:37, SEQ ID NO:38 or SEQ ID NO:39.

[0011] In some embodiments, the heavy chain variable region sequence comprises: a HCDR1 as shown in SEQ ID NO:01, a HCDR2 as shown in SEQ ID NO:07, and a HCDR3 as shown in SEQ ID NO:14; or a HCDR1 as shown in SEQ ID NO:02, a HCDR2 as shown in SEQ ID NO:08, and a HCDR3 as shown in SEQ ID NO:15; or a HCDR1 as shown in SEQ ID NO:03, a HCDR2 as shown in SEQ ID NO:09, and a HCDR3 as shown in SEQ ID NO:16; or a HCDR1 as shown in SEQ ID NO:04, a HCDR2 as shown in SEQ ID NO:10, and a HCDR3 as shown in SEQ ID NO:17; or a HCDR1 as shown in SEQ ID NO:05, a HCDR2 as shown in SEQ ID NO:11, and a HCDR3 as shown in SEQ ID NO:18; or a HCDR1 as shown in SEQ ID NO:05, a HCDR2 as shown in SEQ ID NO:12, and a HCDR3 as shown in SEQ ID NO:13. HCDR3 as shown in SEQ ID NO:19; or HCDR1 as shown in SEQ ID NO:06, HCDR2 as shown in SEQ ID NO:13 and HCDR3 as shown in SEQ ID NO:20.

[0012] In some embodiments, the light chain variable region sequence comprises: LCDR1 as shown in SEQ ID NO:21, LCDR2 as shown in SEQ ID NO:27, and LCDR3 as shown in SEQ ID NO:33; or LCDR1 as shown in SEQ ID NO:22, LCDR2 as shown in SEQ ID NO:28, and LCDR3 as shown in SEQ ID NO:34; or LCDR1 as shown in SEQ ID NO:23, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:35; or LCDR1 as shown in SEQ ID NO:24, LCDR2 as shown in SEQ ID NO:30, and LCDR3 as shown in SEQ ID NO:36; or LCDR1 as shown in SEQ ID NO:24, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:37; or LCDR1 as shown in SEQ ID NO:25, LCDR2 as shown in SEQ ID NO:32, and LCDR3 as shown in SEQ ID NO:35. LCDR3 as shown in SEQ ID NO:38; or LCDR1 as shown in SEQ ID NO:26, LCDR2 as shown in SEQ ID NO:32, and LCDR3 as shown in SEQ ID NO:39.

[0013] In preferred embodiments, the anti-ROR1 antibody or antigen-binding fragment comprises: a) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 07 and SEQ ID NO: 14, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 33, respectively; or b) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 02, SEQ ID NO: 08 and SEQ ID NO: 15, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 34, respectively; or c) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 03, SEQ ID NO: 09 and SEQ ID NO: 10, respectively. NO: 16; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 29 and SEQ ID NO: 35, respectively; or d) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 10 and SEQ ID NO: 17, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 30 and SEQ ID NO: 36, respectively; or e) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 11 and SEQ ID NO: 18, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 31 and SEQ ID NO: or f) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 12 and SEQ ID NO: 19, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 25, SEQ ID NO: 32 and SEQ ID NO: 38, respectively;or g) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 13 and SEQ ID NO: 20, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 32 and SEQ ID NO: 39, respectively.;

[0014] In a preferred embodiment, the antibody or antigen-binding fragment thereof is selected from a murine antibody, a chimeric antibody, a humanized antibody, a human antibody or an antigen-binding fragment thereof.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 41, 43, 45, 47, 49, 51 and 53, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region as set forth in SEQ ID NO:40 or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as set forth in SEQ ID NO:41 or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or b) a heavy chain variable region as set forth in SEQ ID NO:42 or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as set forth in SEQ ID NO:43 or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or c) a heavy chain variable region as set forth in SEQ ID NO:44 or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as set forth in SEQ ID NO: NO:45, or at least 80%, 85%, 90%, 95% or 99% sequence identity thereof; or d) a heavy chain variable region as set forth in SEQ ID NO:46, or at least 80%, 85%, 90%, 95% or 99% sequence identity thereof; and / or a light chain variable region as set forth in SEQ ID NO:47, or at least 80%, 85%, 90%, 95% or 99% sequence identity thereof; or e) a heavy chain variable region as set forth in SEQ ID NO:48, or at least 80%, 85%, 90%, 95% or 99% sequence identity thereof; and / or a light chain variable region as set forth in SEQ ID NO:49, or at least 80%, 85%, 90%, 95% or 99% sequence identity thereof; or f) a heavy chain variable region as set forth in SEQ ID NO:41, or at least 80%, 85%, 90%, 95% or 99% sequence identity thereof; NO:50, or a sequence identity thereto of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as set forth in SEQ ID NO:51, or a sequence identity thereto of at least 80%, 85%, 90%, 95% or 99%; or g) a heavy chain variable region as set forth in SEQ ID NO:52, or a sequence identity thereto of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as set forth in SEQ ID NO:53, or a sequence identity thereto of at least 80%, 85%, 90%, 95% or 99%.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region as shown in SEQ ID NO:40; and / or a light chain variable region as shown in SEQ ID NO:41; b) a heavy chain variable region as shown in SEQ ID NO:42; and / or a light chain variable region as shown in SEQ ID NO:43; c) a heavy chain variable region as shown in SEQ ID NO:44; and / or a light chain variable region as shown in SEQ ID NO:45; d) a heavy chain variable region as shown in SEQ ID NO:46; and / or a light chain variable region as shown in SEQ ID NO:47; e) a heavy chain variable region as shown in SEQ ID NO:48; and / or a light chain variable region as shown in SEQ ID NO:49; f) a heavy chain variable region as shown in SEQ ID NO:50; and / or a light chain variable region as shown in SEQ ID NO:51; g) a heavy chain variable region as shown in SEQ ID NO:52; and / or a light chain variable region as shown in SEQ ID NO:53.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region as shown in SEQ ID NO:40 and a light chain variable region as shown in SEQ ID NO:41; b) a heavy chain variable region as shown in SEQ ID NO:42 and a light chain variable region as shown in SEQ ID NO:43; c) a heavy chain variable region as shown in SEQ ID NO:44 and a light chain variable region as shown in SEQ ID NO:45; d) a heavy chain variable region as shown in SEQ ID NO:46 and a light chain variable region as shown in SEQ ID NO:47; e) a heavy chain variable region as shown in SEQ ID NO:48 and a light chain variable region as shown in SEQ ID NO:49; f) a heavy chain variable region as shown in SEQ ID NO:50 and a light chain variable region as shown in SEQ ID NO:51; g) a heavy chain variable region as shown in SEQ ID NO:52 and a light chain variable region as shown in SEQ ID NO:53.

[0019] In some embodiments, the antibody is a full-length antibody, which further comprises a human antibody constant region; preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and conventional variants thereof, and the light chain constant region of the human antibody constant region is selected from the kappa chain and lambda chain constant regions of human antibodies and conventional variants thereof; more preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 68 and the human light chain constant region of SEQ ID NO: 69.

[0020] In some embodiments, the above antigen binding fragment is selected from: Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (diabody), disulfide stabilized Fv (dsFv) and CDR-containing peptide.

[0021] In some embodiments, the present disclosure provides an isolated nucleic acid molecule encoding any antibody or antigen-binding fragment.

[0022] In one aspect, the present disclosure also provides a recombinant vector comprising the above-mentioned isolated nucleic acid molecule.

[0023] In another aspect, the present disclosure also provides a host cell transformed with the above-mentioned recombinant vector, wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, and more preferably mammalian cells.

[0024] In one aspect, the present disclosure also provides a method for producing the above-mentioned antibody or antigen-binding fragment in a culture medium to produce and accumulate the antibody or antigen-binding fragment thereof, and harvest the antibody or antigen-binding fragment thereof from the culture.

[0025] In one aspect, the present disclosure also provides a method for immunodetection or measurement of ROR1, wherein the method comprises detecting ROR1 by contacting with the above-mentioned antibody or antigen-binding fragment.

[0026] In one aspect, the present disclosure also provides a method for diagnosing a disease associated with human ROR1-positive cells, wherein the method comprises detecting or measuring ROR1 or ROR1-positive cells by contacting with the above-mentioned antibody or antigen-binding fragment.

[0027] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned antibody or antigen-binding fragment, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0028] In some embodiments, the present disclosure also provides a method for treating or preventing a disease associated with overexpression of ROR1, comprising the step of administering a therapeutically effective amount of the above antibody or antigen-binding fragment thereof or the above pharmaceutical composition to a subject in need of treating or preventing the disease.

[0029] In some embodiments, the disease associated with overexpression of ROR1 is cancer; preferably, the cancer is chronic lymphocytic leukemia (CLL); mantle cell lymphoma (MCL); B-cell acute lymphoblastic leukemia (B-ALL); marginal zone lymphoma (MZL); neuroblastoma; renal cancer; lung cancer; or breast cancer.

[0030] The antibody or antigen-binding fragment thereof can (1) specifically recognize or bind to ROR1 expressed on the surface of human-derived cells, or (2) specifically recognize or bind to the extracellular domain of ROR1 on a non-cell surface, or (3) have sufficient internalization in human cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 . In vitro binding characterization of ROR1 hybridoma clones to HT29 (A), HCC1187 (B) and T47D (C) cell lines using flow cytometric analysis. MFI: median fluorescence intensity. Purified anti-human ROR1 antibody was used as a positive control (BioLegend, Cat#357802). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, Cat#400102).

[0032] Figure 2 . In vitro binding characterization of anti-ROR1 recombinant antibodies to ROR1+ Jeko-1 cells (A) and ROR1- T47D cells (B) determined by flow cytometric analysis.

[0033] Figure 3 .Internalization kinetics of anti-ROR1 recombinant antibody in ROR1+Jeko1 cells. DETAILED DESCRIPTION

[0034] The present invention is based on the development of an antibody that can specifically bind to ROR1. The headings used in this section are for convenience only and do not limit the present invention.

[0035] Unless otherwise defined herein, the scientific and technical terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, unless the context specifically requires otherwise, the singular includes the plural and the plural includes the singular.

[0036] definition

[0037] Before describing the present invention in detail below, it will be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may be different. It will also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0038] For interpretation of the specification, the following definitions will apply, and where appropriate, terms used in the singular may also include the plural, and vice versa. It will be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0039] The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH region and the VL region can be further subdivided into hypervariable regions called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0040] 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). It has been shown that the antigen binding function of an antibody can be achieved by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a Fd fragment, which consists of VH and CHI domains; (iv) a Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; (vi) an isolated complementary determining region (CDR); and (vii) a combination of two or more isolated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, enabling them to be made into a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.

[0041] As used herein, the term "human antibody" is intended to include antibodies having variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted to human framework sequences.

[0042] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) of human immunoglobulin genes or hybridomas prepared therefrom (further described in Section 1 below), (b) antibodies isolated from host cells transformed to express antibodies, such as from transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire in vivo.

[0043] The term "CDR" refers to one of the six hypervariable regions within an antibody variable domain that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (LCDR1, LCDR2, LCDR3 or L1, L2, L3) of a light chain variable domain, and CDR1, CDR2, and CDR3 (HCDR1, HCDR2, HCDR3 or H1, H2, H3) of a heavy chain variable domain.

[0044] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within a given HCVR and / or LCVR amino acid sequence disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, Chothia (Chothia et al. (1989) Nature 342:877-883) based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics Database (IMGT) (imgt.cines.fr / on the World Wide Web), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. One skilled in the art can readily identify the CDRs defined by each numbering system.

[0045] A useful comparison of CDR numbers is as follows:

[0046] CDR IMGT Kabat AbM <![CDATA[Chothia 1 ]]> <![CDATA[Contact 2 ]]> LCDR1 27-32 24-34 24-34 24-34 30-36 LCDR2 50-51 50-56 50-56 50-56 46-55 LCDR3 89-97 89-97 89-97 89-97 89-96 HCDR1 26-35B <![CDATA[31 - 35B (Kabat number) 3 > 26-35B 26-32..34 30-35B HCDR2 51-56 50-65 50-58 52-56 47-58 HCDR3 93-102 95-102 95-102 95-102 93-101 HCDR1 26-33 31-35 (Chothia numbering) 26-35 26-32 30-35

[0047] Note 1: Some of these definitions (particularly for the Chothia loop) vary depending on the individual publications examined; Note 2: Any numbering scheme can be used for these CDR definitions, except for the contact definition, which uses the Chothia or Martin (enhanced Chothia) definition; Note 3: When numbering using the Kabat numbering convention, the ends of the Chothia HCDR1 loop vary between H32 and H34, depending on the length of the loop. This is because the Kabat numbering scheme places the insertion at H35A and H35B.

[0048] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, it is "operably linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to a coding sequence.

[0049] The method for preparing nucleic acid is a conventional method in the art. Preferably, it comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full-length sequence synthesis.

[0050] Those skilled in the art know that the base sequence encoding the protein amino acid sequence can be appropriately substituted, deleted, changed, inserted or added to provide a polynucleotide homologue. The homologue of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the antibody activity.

[0051] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA fragments can be connected. In another embodiment, the vector is a viral vector, in which additional DNA fragments can be connected to the viral genome. Vectors disclosed herein can replicate themselves in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication), or can be incorporated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome (e.g., non-episomal mammalian vectors).

[0052] The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors, thereby being constructed. The expression vector is one of a variety of conventional vectors in the art, as long as it can carry the above-mentioned nucleic acid molecule. The vector preferably includes: various plasmids, cosmids, phages or viral vectors, etc.

[0053] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293 cells, plant cells, or fungi (including yeast cells), that express the antibody.

[0054] The sequence of the DNA molecule of the antibody according to the present invention or its fragment can be obtained by conventional techniques (for example, as the method of PCR amplification or genomic library screening).In addition, the sequence encoding light chain and heavy chain can be fused together to form a single-chain antibody.

[0055] Once the related sequence is obtained, it can be obtained in bulk using recombinant methods. This is usually done by cloning the sequence into a vector, transforming cells with the vector, and then isolating the related sequence from the propagated host cells by conventional methods.

[0056] In addition, related sequences can be synthesized artificially, especially when the fragment length is short. Usually, several small fragments are synthesized first and then connected together to obtain a long sequence fragment.

[0057] At present, the DNA sequence encoding the antibody of the present invention (or its fragment or derivative thereof) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or, for example, vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0058] Typically, the host cells obtained are cultured under conditions suitable for expressing the antibodies according to the invention. The antibodies of the invention are then purified using conventional immunoglobulin purification steps, such as conventional separation and purification means well known to those skilled in the art, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

[0059] The obtained monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined by, for example, Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)).

[0060] The antibodies according to the present invention can be expressed in cells or on cell membranes, or can be secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods according to the physical, chemical and other characteristics of the recombinant protein. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and any other liquid chromatography, and combinations thereof.

[0061] The term "variant" of a polypeptide, such as a variant of an antigen-binding fragment, a protein or an antibody, is a polypeptide in which one or more amino acid residues are inserted, deleted, added and / or substituted compared to another polypeptide sequence, and includes fusion polypeptides. In addition, protein variants include protein variants that are modified by proteinase cleavage, phosphorylation or other post-translational modifications, but retain the biological activity of the antibodies disclosed herein, such as binding and specificity to ROR1. The variant may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% identical to the sequence of the antibody or antigen-binding fragment thereof disclosed herein. The identity or homology percentage (%) can be calculated with reference to the following description.

[0062] In one embodiment, the homology or identity percentage can be calculated as 100×[(same positions) / min(TGA, TGB)], and in this formula, TGA, TGB are the sum of the residue numbers and internal gap positions of the compared sequences A and B (Russell et al., J. Mol Biol., 244:332-350 (1994)).

[0063] In the present invention, the antibodies of the present invention also include conservative variants thereof, which means that compared with the amino acid sequence of the antibodies of the present invention, up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are substituted by amino acids with similar or similar properties to form polypeptides. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0064] Table A

[0065] Original residue Representative replacement Preferred substitution Ala Val; Leu; Ile Val Arg Lys; Gln; Asn Lys Asn Gln; His; Lys; Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn Asn Glu Asp Asp Gly Pro; Ala Ala His Asn; Gln; Lys; Arg Arg Ile Leu; Val; Met; Ala; Phe Leu Leu Ile; Val; Met; Ala; Phe Ile Lys Arg; Gln; Asn Arg Met Leu; Phe; Ile Leu Phe Leu; Val; Ile; Ala; Tyr Leu Pro Ala Ala Ser Thr Thr Thr Ser Ser Trp Tyr; Phe Tyr Tyr Trp; Phe; Thr; Ser Phe Val Ile; Leu; Met; Phe; Ala Leu

[0066] As used herein, the term "K D "(M) is intended to refer to the dissociation equilibrium constant for a particular antibody-antigen interaction. "K D ” refers to the dissociation constant, which is composed of K d With K a The ratio of K d / K a ) and expressed as molar concentration (M). In view of the present disclosure, the K of an antibody can be determined using methods in the art. D For example, the K D It can be determined by using surface plasmon resonance, such as by using a biosensor system (eg, the RAPID® system) or using bio-layer interferometry (eg, the Octet RED96 system).

[0067] The term "affinity" is the strength of the interaction between an antibody or its antigen-binding fragment and an antigen, which is determined by the characteristics of the antigen (such as the size, shape and / or charge of the antigen) and the CDR sequence of the antibody or antigen-binding fragment. Methods for determining affinity are known in the art, and reference may be made to the following.

[0068] When the dissociation constant (K D ) <l0 6 M, an antibody or antigen-binding fragment thereof is said to "specifically bind" to its target (e.g., antigen). D <l0 9 When M, the antibody binds specifically to its target with "high affinity".

[0069] As used herein, the term "pharmaceutical composition" is intended to refer to a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs thereof and other chemical components (such as physiologically / pharmaceutically acceptable carriers and excipients). The purpose of the pharmaceutical composition is to facilitate administration to an organism, which is beneficial to the absorption of the active ingredient and the exertion of biological activity.

[0070] "Administering" and "treating" when applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid refers to contacting the animal, human, subject, cell, tissue, organ, or biological fluid with an exogenous pharmaceutical agent, therapeutic agent, diagnostic agent, or composition. "Administering" and "treating" can refer, for example, to therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treatment of cells encompasses contacting cells with agents, as well as contacting fluids with agents, wherein the fluids are in contact with cells. "Administering" and "treating" also refer to in vitro and ex vivo treatments, such as in vitro and ex vivo treatments of cells by reagents, diagnostic agents, in combination with compositions, or by another cell. When applied to humans, veterinary medicine, or research subjects, "treating" refers to therapeutic treatments, preventive or prophylactic measures, research and diagnostic applications.

[0071] Furthermore, the present disclosure includes a drug for treating a disease associated with ROR1, comprising the antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.

[0072] There is no limitation on the disease associated with ROR1, as long as it is a disease associated with ROR1, for example, the therapeutic response induced by the molecules disclosed in the present disclosure can be reduced by binding to human ROR1. Therefore, when in a formulation suitable for therapeutic application, the molecules disclosed in the present disclosure are very useful for those suffering from tumors, cancer or infectious diseases.

[0073] In addition, the present disclosure relates to a method for immunodetection or measurement of ROR1, an agent for immunodetection or measurement of ROR1, a method for immunodetection or measurement of cells expressing ROR1, and a diagnostic agent for diagnosing a disease associated with ROR1-positive cells, which comprises the antibody or antigen-binding fragment of the present disclosure that specifically recognizes human ROR1 as an active ingredient.

[0074] In the present disclosure, the method for detecting or determining the amount of ROR1 may be any known method. For example, it includes immunoassay or determination.

[0075] Immunoassay or assay is a method for detecting or determining the amount of an antibody or antigen by using a labeled antigen or antibody. Examples of immunoassay or assay include radioactive labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, Western blotting, physicochemical methods, etc.

[0076] The above-mentioned diseases associated with ROR1-positive cells can be diagnosed by detecting or measuring cells expressing ROR1 using the antibodies or antibody fragments thereof of the present invention.

[0077] In order to detect cells expressing the polypeptide, known immunoassays can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc. can be used. In addition, a fluorescent antibody staining method or the like can be used, which uses the FMAT8100HTS system (Applied Bio system).

[0078] Example

[0079] The present invention is further illustrated by the following specific examples. It will be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods without detailed conditions in the following examples are generally in accordance with the conditions described in the conventional conditions, such as Sambrook.J et al. "Molecular Cloning Experiment Guide" (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer (such as product instructions). Unless otherwise stated, percentages and parts are all by weight. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0080] The room temperature described in the examples is a conventional room temperature in the art, and is usually 10-30°C.

[0081] Example 1: Immunization and antibody screening

[0082] Example 1-1: Preparation of immunogen

[0083] A combination of the recombinant protein antigen huROR1-Fc (R&D systems, catalog number 9490-RO), engineered huROR1-HEK293 cells and HT-29 cells (ATCC, catalog number HTB38) was used to immunize mice. For the engineered huROR1-HEK293 cell line, HEK293 cells were transduced with a lentivirus containing a gene sequence encoding the extracellular domain of human ROR1 and a puromycin resistance gene. A single clone with the highest huROR1 expression was selected from the transduced HEK293 cells.

[0084] Example 1-2: Immunization

[0085] Immunization regimen:

[0086] Anti-ROR1 antibodies were obtained by immunizing genetically modified mice encoding human immunoglobulin heavy chain and kappa light chain variable regions with a combination of recombinant protein antigen huROR1-Fc (R&D systems, catalog number 9490-RO), engineered huROR1-HEK293 cells and HT-29 cells (ATCC, catalog number HTB38). Antibody immune responses were monitored by ROR1-specific immunoassays. When the desired immune response was achieved, splenocytes were harvested from each mouse and fused with mouse myeloma cells to maintain their viability and form hybridoma cells, and screened for ROR1 specificity.

[0087] Example 1-3: Spleen cell fusion

[0088] By electrofusion or PEG fusion, spleen lymphocytes and myeloma cells Sp2 / 0 ( CRL-158) to obtain hybridoma cells. TM HY technology (STEMCELL technologies) was used for PEG fusion. The ratio of primary cells: mouse myeloma cell line for electrofusion was 1:1, and that for PEG fusion was 10:1.

[0089] Example 1-4: Screening of hybridoma clones specifically binding to human ROR1 protein by ELISA

[0090] ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated with 1 μg / ml human ROR1-Fc (9490-R0-050), human ROR2-Fc (8609-RO-050) or BSA overnight. Excess unbound protein was washed away by washing the plate three times with wash buffer and then blocked at room temperature for 1 hour. 50 μl of ROR1 hybridoma supernatant was added to duplicate wells and incubated for 1 hour. Excess unbound antibody was washed away, and 50 μl of a 1:30000 diluted secondary goat anti-mouse IgG Fc-HRP (ab5870) was added to each well for 1 hour. According to the manufacturer's protocol, the plate was washed and 50 μL of chemiluminescent agent (color A and color B) was added. The reaction was stopped using 25 μL of 2N sulfuric acid. The optical density of the sample at 450 nm was measured by an ELISA reader (PerkinElmer). All clones tested selectively bound to human ROR1 but not to human ROR2 or BSA, demonstrating ROR1 specificity.

[0091] Table 1. ELISA determination (OD 450) Characterization of the binding of hybridoma clones to human ROR1 and human ROR2

[0092]

[0093] Example 1-5: Screening of hybridoma clones that specifically bind to cancer cells expressing ROR1 by flow cytometry

[0094] Hybridoma supernatants were tested for binding on ROR1+ cell lines HT29 (ATCC, HTB-38), HCC1187 (ATCC, CRL-2322) and ROR1-ROR2+ ​​cell lines T47D (ATCC, HTB-133) using flow cytometry analysis. Briefly, 50 μL of cells (2×10 6 Cells / mL) were mixed with 50 μL of undiluted hybridoma supernatant. The mixture was incubated on ice for 20 min and then washed twice with ice-cold staining buffer. The cells were then stained with 50 μL of PE-labeled secondary antibody (1:400 dilution, BioLegend, Cat#405307) for 20 min. After washing with staining buffer and fixing with 4% PFA, the cells were analyzed by flow cytometry. Purified anti-human ROR1 antibody was used as a positive control (BioLegend, Cat#357802). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, Cat#400102). Figure 1 Examples of selected cell binding signals measured by flow cytometry are shown. Clones 1E9, 8B10, 15G4, 29D4, 31G8, 37F9, 38H10 were identified to have enhanced binding to HT29 cells and HCC1187 cells compared to T47D cells.

[0095] Example 2: Sequencing of positive hybridoma clones

[0096] The process of cloning sequences from positive hybridomas is as follows. Hybridoma cells in the logarithmic growth phase are collected, RNA is extracted, reverse transcribed, and then the VDJ region is amplified. Next-generation sequencing is performed on the cDNA library amplified from each clone. The amino acid sequences of the heavy chain and light chain variable region DNA sequences corresponding to antibodies 1E9, 8B10, 15G4, 29D4, 31G8, 37F9 and 38H10 were obtained. After manufacturability evaluation, several mutations were made in the FR region, and the amino acid sequences and CDR sequences of the heavy chain variable region and light chain variable region of each antibody are shown in the following table. The amino acid residues of the CDR in VH / VL are numbered and annotated according to the Kabat and Wu numbering systems.

[0097] Table 2. CDR sequences of heavy chain variable domains of ROR1 hybridoma clones

[0098]

[0099] Table 3. CDR sequences of light chain variable domains of ROR1 hybridoma clones

[0100]

[0101]

[0102] Table 4. Sequences of the heavy and light chain variable domains of ROR1 hybridoma clones

[0103]

[0104]

[0105] Example 3: Construction and expression of anti-ROR1 recombinant antibodies

[0106] Example 3-1: Molecular cloning of recombinant antibodies

[0107] The cDNA sequence of VH and VL district encoding the selected clone is directly synthesized as a DNA fragment with a 5' end in-frame leader sequence (MGWSCIILFLVATATGVHS). These DNA fragments are cloned into the selected vector using NEBuilder DNA assembly cloning kit (New England Biolabs). The VH district is cloned into the pFUSE-CHIg_hG1 vector (InvivoGen#pfuse-hchg1), and the hIgG1 heavy chain constant region in the VH district and the vector is in frame. The VL district is cloned into the pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk), and the hIgκ light chain constant region in the VL district and the vector is in frame.

[0108] The IgG form of the antibody is disclosed as the following full length heavy and light chains, in the order of 1E9, 8B10, 15G4 (N65S), 29D4, 31G8, 37F9 and 38H10 (N81K, T83S), respectively: SEQ ID NO: 54 (heavy chain) and 55 (light chain); SEQ ID NO: 56 (heavy chain) and 57 (light chain); SEQ ID NO: 58 (heavy chain) and 59 (light chain); SEQ ID NO: 60 (heavy chain) and 61 (light chain); SEQ ID NO: 62 (heavy chain) and 63 (light chain); SEQ ID NO: 64 (heavy chain) and 65 (light chain); SEQ ID NO: 66 (heavy chain) and 67 (light chain).

[0109] Table 5. Sequences of the constant regions of recombinant antibody IgG

[0110]

[0111] Table 6. Full-length sequences of heavy and light chains of anti-ROR1 recombinant antibodies

[0112]

[0113]

[0114]

[0115] Example 3-2: Expression and purification of recombinant antibodies

[0116] Heavy chain expression plasmids and light chain plasmids were co-transfected into Expi293F cells (ThermoFisher, #A14527) using the ExpiFectamine 293 transfection kit (ThermoFisher, A14524) or into ExpiCHO-S cells (ThermoFisher, #A29127) using the ExpiFectamine CHO transfection kit (ThermoFisher, A29129). Plasmid DNA concentrations reached 1.0 μg per ml of suspended cells based on the manufacturer's instructions, with a LC:HC vector ratio of 1:1. Transfected cells were cultured on an orbital shaker at 37°C, 8% CO2 for 5 to 7 days. Conditioned media were collected and antibodies were purified using HiTrap MabSelect SuRe columns (Cytiva, #17549112) on an AKTA Pure 25 machine (Cytiva). The eluted antibody was neutralized with Tris buffer (pH 9.0) and subjected to PBS buffer exchange. The product concentration was measured by UV absorption, and the quality was determined by SDS-PAGE and HPLC.

[0117] Example 4: Characterization of binding of anti-ROR1 recombinant antibodies to ROR1+ and ROR1- cell lines by flow cytometry

[0118] Binding of hIgG1 mAb to cell surface ROR1 was determined by FACS analysis using different cancer cell lines positive for ROR1 or ROR2 expression, including Jeko-1 and T-47D cells.

[0119] Jeko-1 cells (ROR1 positive) were maintained in RPMI-1640 medium supplemented with 20% FBS and 1% penicillin and streptomycin. T-47D cells (ROR2 positive) were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin. Both cell lines were cultured at 37°C in a humidified atmosphere of 5% CO2.

[0120] To determine the binding of hIgG1 mAb to the cell surface ROR1 receptor, cells were first harvested and cultured at 2 × 10 6 Cells were resuspended in cell staining buffer (BioLegend) at 100 cells / mL. The cells were then treated with human Fc receptor blocking reagent (BioLegend) on ice for 10 min. The resulting cell suspension was divided into 50 μL aliquots. 50 μL of different concentrations of hIgG1 mAb were mixed with the cell aliquots, and the final concentration of hIgG1 mAb in the mixture ranged from 1.1 pM to 200 nM. The cells were incubated on ice for 1 hour and then washed twice with cell staining buffer. 50 μL of secondary antibody (PE-conjugated goat anti-human Fc, eBioscience TM The cells were resuspended in 4% PFA (1:200 dilution). The cells were incubated on ice for another 30 min. The cells were then washed twice with cell staining buffer and resuspended in 4% PFA to fix the cells. The samples were analyzed using iQue3 to measure the median fluorescence intensity using the corresponding channels. The results are published in Figure 2 and Table 7. The results confirmed that the anti-ROR1 antibody of the present invention can specifically bind to the ROR1 protein expressed in situ in human cells in a concentration-dependent manner, and confirmed that the antibody does not bind to the ROR1 family member protein, human ROR2 protein.

[0121] Table 7. K of anti-ROR1 recombinant antibodies binding to ROR1+Jeko-1 cells D value

[0122] Antibody <![CDATA[K D (nM)]]> 1E9 0.26 8B10 1.93 15G4(N65S) 2.49 29D4 1.08 31G8 0.47 37F9 1.17 38H10(N81K / T83S) 0.20

[0123] Example 5: Characterization of cellular internalization of anti-ROR1 recombinant antibodies in cells expressing ROR1

[0124] To explore the possibility of developing an ADC strategy using the ROR1 antibodies of the present invention, the inventors evaluated the endocytosis of antibodies induced by ROR1 antibody binding. In the assay, Jeko-1 cells were collected and cultured at 2×10 6Cells / mL were suspended in ice-cold staining buffer (PBS with 0.5% FBS) and then treated with human Fc receptor blocking reagent (BioLegend, 422302). The cells were then pre-incubated on ice with antibodies labeled with Alexa Fluor 488 for 1 hour. After incubation, the cells were transferred to a 37°C incubator. At predetermined time points (0min, 15min, 30min, 45min, 1 hour, 2 hours, 4 hours, 6 hours), 50 μL of cell suspension was aliquoted and transferred to ice for incubation until the end of incubation for internalization. Then, all aliquots were washed twice with ice-cold staining buffer. The Alexa Fluor 488 signal bound to the cell surface was quenched by anti-Alexa Fluor antibody (Invitrogen, A-11094). The remaining intracellular signal caused by internalization was then analyzed by flow cytometry. Another 50 μL aliquot of cells was collected at 0min, stored on ice, and analyzed by flow cytometry without quenching. This sample served as a control for 100% cell surface binding signal. The amount of internalized antibody was calculated and normalized to 100% cell surface binding signal for each antibody as calculated by the following formula:

[0125] Normalized internalization (%) = (MFI t -MFI t0 ) / (MFI 总 -MFI t0 )×100%

[0126] Internalization results are based on median fluorescence intensity (MFI). t is the MFI of the sample collected at time t; MFI t0 is the MFI of the sample collected at time 0 minutes. 总 is the MFI of an additional sample collected and used as a control for 100% cell surface bound signal.

[0127] Table 8. Internalization kinetics of anti-ROR1 recombinant antibodies in ROR1+Jeko1 cells.

[0128]

[0129] like Figure 3 As shown in Table 8, sufficient internalization signals were observed for the anti-ROR1 antibodies 1E9, 15G4 (N65S), 29D4, 31G8, 37F9, and 38H10 (N81K, T83S) in Jeko-1 cells.

[0130] Example 6: Epitope Characterization of Anti-ROR1 Recombinant Antibodies

[0131] Example 6-1: ELISA binding to human ROR1 protein subdomain

[0132] ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated overnight with 1 μg / ml human ROR1 protein (RO1-H522y) or human ROR1 subdomain-Ig-like (RO1-H5221), Frizzled (RO1-H5222) and Kringle (RO1-H5223). Excess unbound protein was washed off by washing the plate three times with wash buffer and then blocked at room temperature for 1 hour. 50 μl of 100 μg / mL ROR1 recombinant antibody was added to duplicate wells and incubated for 1 hour. Excess unbound antibody was washed away, and 50 μl of a 1:2000 diluted secondary antibody goat anti-human IgG Fc-HRP (ab6858) was added to each well for 1 hour. According to the manufacturer's protocol, the plate was washed and 50 μL of chemiluminescent agent (color A and color B) was added. The reaction was stopped using 25 μL of 2N sulfuric acid. The optical density of the samples was measured at 450 nm by a microplate reader (PerkinElmer). All recombinant antibodies tested bound to the full-length human ROR1 protein. 1E9, 8B10, 15G4 (N65S), 29D4, 31G8, 37F9, and UC-961 bound to the Ig-like subdomain of the human ROR1 protein, while 38H10 (N81K / T83S) bound to the Kringle subdomain of the human ROR1 protein.

[0133] Table 9. Results of ELISA (OD 450 ) Characterization of binding of ROR1 recombinant antibodies to full-length or subdomain human ROR1 protein

[0134]

[0135] Example 6-2: Octet typing (binning)

[0136] A total of five clones (15G4 (N65S), 29D4, 31G8, 37F9 and 38H10 (N81K / T83S)) were epitope typed and compared with the anti-ROR1 antibody UC961. Antibody epitope typing was performed using the Octet Red384 system equipped with anti-species and Ni-NTA sensors from Pall Life Sciences (Menlo Park, CA). The experiment was performed as a tandem typing assay. The assay consisted of a five-step binding cycle: 1) establish a buffer baseline for 30 seconds, 2) couple 50 nM ROR1 antigen (RO1-H522y) to the Ni-NTA octet sensor for 5 minutes using standard 1× assay buffer (PBS + 0.02% Tween 20, 0.1% BSA, 0.05% sodium azide) diluted from a 10× kinetic buffer stock (ForteBio), 3) load 250 nM of each antibody (saturated mAb) to saturate the immobilized antigen for 10 minutes, 4) allow 250 nM of each antibody (competing mAb) to bind for 5 minutes, 5) regenerate the capture sensor for 30 seconds. As shown in Tables 9 and 10, the five anti-ROR1 recombinant antibodies can be divided into 2 different epitope bins. 15G4 (N65S), 29D4, 31G8, and 37F9 are in the same bin 1, while 38H10 (N81K / T83S) is in a different bin 2. Furthermore, UC-961 binding did not compete with any of the five anti-ROR1 recombinant antibodies (15G4 (N65S), 29D4, 31G8, 37F9, and 38H10 (N81K / T83S)), indicating that the UC-961 binding epitope is located in a separate epitope subtype 3.

[0137] Table 10. Epitope typing of recombinant antibodies binding to human ROR1 protein by BLI using the Octet Red384 system.

[0138]

[0139] Signal displacement unit: nm

[0140] Table 11. Epitope typing of anti-ROR1 recombinant antibodies against human ROR1

[0141] Epitope typing Anti-ROR1 Antibody 1 15G4(N65S), 29D4, 31G8, 37F9 2 38H10(N81K / T83S) 3 UC961

[0142] Example 7: Cross-reactivity of anti-ROR1 recombinant antibodies with mouse ROR1 protein

[0143] ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated with 1 μg / ml mouse ROR1 protein (RO1-M5221), rat ROR1 protein (RO1-R5221) or control BSA overnight. Excess unbound protein was washed off by washing the plate three times with wash buffer and then blocked at room temperature for 1 hour. 50 μl of 25 μg / ml ROR1 recombinant antibody was added to duplicate wells and incubated for 1 hour. Excess unbound antibody was washed away, and 50 μl of 1:2000 diluted secondary antibody goat anti-human IgG Fc-HRP (ab6858) was added to each well for another 1 hour. According to the manufacturer's protocol, the plate was washed and 50 μL of chemiluminescent agent (color A and color B) was added. The reaction was stopped using 25 μL of 2 N sulfuric acid. The optical density of the sample at 450 nm was measured by a microplate reader (PerkinElmer). Recombinant antibodies 15G4 (N65S), 29D4, 31G8, 37F9 and 38H10 (N81K / T83S) were able to bind to both mouse and rat ROR1 proteins. In contrast, UC961 showed no cross-reactivity to either mouse or rat ROR1 proteins.

[0144] Table 12. Results of ELISA (OD 450 ) Characterization of binding of ROR1 recombinant antibody clones to mouse ROR1 or rat ROR1 protein

[0145]

[0146]

Claims

1. An anti-ROR1 antibody or an antigen-binding fragment thereof, comprising: an antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and an antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: a) HCDR1 is represented by SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05 or SEQ ID NO: 06; b) HCDR2 is represented by SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; c) HCDR3 is represented by SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20; d) LCDR1 is represented by SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26; e) LCDR2 is represented by SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32; f) LCDR3 is represented by SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:

39.

2. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region sequence comprises: HCDR1 as shown in SEQ ID NO: 01, HCDR2 as shown in SEQ ID NO: 07, and HCDR3 as shown in SEQ ID NO: 14; or HCDR1 as shown in SEQ ID NO: 02, HCDR2 as shown in SEQ ID NO: 08, and HCDR3 as shown in SEQ ID NO: 15; or HCDR1 as shown in SEQ ID NO: 03, HCDR2 as shown in SEQ ID NO: 09, and HCDR3 as shown in SEQ ID NO: 16; or HCDR1 as shown in SEQ ID NO: 04, HCDR2 as shown in SEQ ID NO: 10, and HCDR3 as shown in SEQ ID NO: 17; or HCDR1 as shown in SEQ ID NO: 05, HCDR2 as shown in SEQ ID NO: 11, and HCDR3 as shown in SEQ ID NO: 18; or HCDR1 as shown in SEQ ID NO: 05, HCDR2 as shown in SEQ ID NO: 12, and HCDR3 as shown in SEQ ID NO: 19; or HCDR1 as shown in SEQ ID NO: 06, HCDR2 as shown in SEQ ID NO: 13, and HCDR3 as shown in SEQ ID NO:

20.

3. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region sequence comprises: LCDR1 as shown in SEQ ID NO: 21, LCDR2 as shown in SEQ ID NO: 27, and LCDR3 as shown in SEQ ID NO: 33; or LCDR1 as shown in SEQ ID NO: 22, LCDR2 as shown in SEQ ID NO: 28, and LCDR3 as shown in SEQ ID NO: 34; or LCDR1 as shown in SEQ ID NO: 23, LCDR2 as shown in SEQ ID NO: 29, and LCDR3 as shown in SEQ ID NO: 35; or LCDR1 as shown in SEQ ID NO: 24, LCDR2 as shown in SEQ ID NO: 30, and LCDR3 as shown in SEQ ID NO: 36; or LCDR1 as shown in SEQ ID NO: 24, LCDR2 as shown in SEQ ID NO: 31, and LCDR3 as shown in SEQ ID NO: 37; or LCDR1 as shown in SEQ ID NO: 25, LCDR2 as shown in SEQ ID NO: 32, and LCDR3 as shown in SEQ ID NO: 38; or LCDR1 as shown in SEQ ID NO: 26, LCDR2 as shown in SEQ ID NO: 32, and LCDR3 as shown in SEQ ID NO:

39.

4. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1, wherein: a) the heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 10 and SEQ ID NO: 17, respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 30 and SEQ ID NO: 36, respectively; or b) the heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 02, SEQ ID NO: 08 and SEQ ID NO: 15, respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 34, respectively; or c) the heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 03, SEQ ID NO: 09 and SEQ ID NO: 16, respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 29 and SEQ ID NO: 35, respectively; or d) the heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:01, SEQ ID NO:07 and SEQ ID NO:14, respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:21, SEQ ID NO:27 and SEQ ID NO:33, respectively; or e) the heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 11 and SEQ ID NO: 18, respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 31 and SEQ ID NO: 37, respectively; or f) the heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:05, SEQ ID NO:12 and SEQ ID NO:19, respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:25, SEQ ID NO:32 and SEQ ID NO:38, respectively; or g) the heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 13 and SEQ ID NO: 20, respectively; and the light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 32 and SEQ ID NO: 39, respectively. 5 . The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 , wherein the antibody or antigen-binding fragment thereof is selected from a murine antibody, a chimeric antibody, a humanized antibody, a human antibody or an antigen-binding fragment thereof.

6. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 46, 40, 42, 44, 48, 50, 52, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 47, 41, 43, 45, 49, 51 and 53, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

7. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region as shown in SEQ ID NO:46, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:47, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or b) a heavy chain variable region as shown in SEQ ID NO:42, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:43, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or c) a heavy chain variable region as shown in SEQ ID NO:44, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:45, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or d) a heavy chain variable region as shown in SEQ ID NO:40, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:41, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or e) a heavy chain variable region as shown in SEQ ID NO:48, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:49, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or f) a heavy chain variable region as shown in SEQ ID NO:50, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:51, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or g) a heavy chain variable region as shown in SEQ ID NO:52, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:53, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

8. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody or antigen-binding fragment thereof comprises: a) the heavy chain variable region as shown in SEQ ID NO: 46; and / or the light chain variable region as shown in SEQ ID NO: 47; b) a heavy chain variable region as shown in SEQ ID NO: 42; and / or a light chain variable region as shown in SEQ ID NO: 43; c) a heavy chain variable region as shown in SEQ ID NO: 44; and / or a light chain variable region as shown in SEQ ID NO: 45; d) a heavy chain variable region as shown in SEQ ID NO: 40; and / or a light chain variable region as shown in SEQ ID NO: 41; e) a heavy chain variable region as shown in SEQ ID NO: 48; and / or a light chain variable region as shown in SEQ ID NO: 49; f) the heavy chain variable region as shown in SEQ ID NO: 50; and / or the light chain variable region as shown in SEQ ID NO: 51; g) a heavy chain variable region as shown in SEQ ID NO: 52; and / or a light chain variable region as shown in SEQ ID NO:

53.

9. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody or antigen-binding fragment thereof comprises: a) the heavy chain variable region as shown in SEQ ID NO:46 and the light chain variable region as shown in SEQ ID NO:47; b) the heavy chain variable region as shown in SEQ ID NO:42 and the light chain variable region as shown in SEQ ID NO:43; c) the heavy chain variable region as shown in SEQ ID NO:44 and the light chain variable region as shown in SEQ ID NO:45; d) the heavy chain variable region as shown in SEQ ID NO:40 and the light chain variable region as shown in SEQ ID NO:41; e) the heavy chain variable region as shown in SEQ ID NO:48 and the light chain variable region as shown in SEQ ID NO:49; f) the heavy chain variable region as shown in SEQ ID NO:50 and the light chain variable region as shown in SEQ ID NO:51; g) the heavy chain variable region as shown in SEQ ID NO: 52 and the light chain variable region as shown in SEQ ID NO:

53.

10. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is a full-length antibody, which further comprises a human antibody constant region; Preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and conventional variants thereof, and the light chain constant region of the human antibody constant region is selected from the constant regions of κ chain and λ chain of human antibodies and conventional variants thereof; More preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 68 and the human light chain constant region of SEQ ID NO:

69.

11. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof comprises: a) the heavy chain as shown in SEQ ID NO:60 and the light chain as shown in SEQ ID NO:61; b) the heavy chain as shown in SEQ ID NO:56 and the light chain as shown in SEQ ID NO:57; c) the heavy chain as shown in SEQ ID NO:58 and the light chain as shown in SEQ ID NO:59; d) the heavy chain as shown in SEQ ID NO:54 and the light chain as shown in SEQ ID NO:55; e) the heavy chain as shown in SEQ ID NO:62 and the light chain as shown in SEQ ID NO:63; f) the heavy chain as shown in SEQ ID NO:64 and the light chain as shown in SEQ ID NO:65; g) the heavy chain as shown in SEQ ID NO: 66 and the light chain as shown in SEQ ID NO:

67.

12. An anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antigen-binding fragment is selected from: Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (diabody), disulfide-stabilized Fv (dsFv) and a peptide containing CDR.

13. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. A recombinant vector comprising the isolated nucleic acid molecule of claim 13.

15. A host cell transformed with the recombinant vector according to claim 14, wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells.

16. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the method comprises: The host cell according to claim 15 is cultured in a culture medium to produce and accumulate the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and the antibody or antigen-binding fragment thereof is harvested from the culture. 17 . A method for immunologically detecting or measuring ROR1, wherein the method comprises detecting ROR1 by contacting with the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 .

18. A method for diagnosing a disease associated with human ROR1-positive cells, wherein the method comprises detecting or measuring the ROR1 or ROR1-positive cells by contacting with the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

19. A pharmaceutical composition comprising a therapeutically effective amount of the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and one or more pharmaceutically acceptable carriers, diluents or excipients.

20. A method for treating or preventing a disease associated with overexpression of ROR1, comprising A therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 19 is administered to a subject in need of treatment or prevention of the disease.

21. The method for treating or preventing a disease associated with overexpression of ROR1 according to claim 20, wherein the disease associated with overexpression of ROR1 is cancer; preferably, the cancer is chronic lymphocytic leukemia (CLL); mantle cell lymphoma (MCL); B-cell acute lymphoblastic leukemia (B-ALL); marginal zone lymphoma (MZL); neuroblastoma; renal cancer; lung cancer; or breast cancer.

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