WO2024012434A1

WO2024012434A1 - Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof

Info

Publication number: WO2024012434A1
Application number: PCT/CN2023/106728
Authority: WO
Inventors: Qing Li; Xinquan LIU; Larry Lo
Original assignee: Hansoh Bio Llc; Shanghai Hansoh Biomedical Co., Ltd.; Changzhou Hansoh Pharmaceutical Co., Ltd.
Priority date: 2022-07-11
Filing date: 2023-07-11
Publication date: 2024-01-18

Abstract

Provided are receptor tyrosine kinase like orphan receptor 1 antibodies or antigen-binding fragments, and their pharmaceutical use.

Description

ANTIBODY, ANTIGEN-BINDING FRAGMENT THEREOF, AND PHARMACEUTICAL USE THEREOF

FIELD OF THE INVENTION

The present invention relates to antibodies, and in particular, to antibodies exhibiting specificity for Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) , and to use thereof, for example in the treatment of cancer.

BACKGROUND

ROR (Receptor Tyrosine Kinase-Like Orphan Receptor) is a transmembrane protein of RTK (Receptor Tyrosine Kinase) family, and there are 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 called a Frizzled (Fz) domain, and a Kringle (Kr) domain. All three domains are involved in protein-protein interactions. Intracellularly, ROR1 and ROR2 possess a tyrosine kinase (TK) domain and a proline-rich domain (PRD) straddled by two serine/threonine-rich domains (Borcherding et al., 2014, Protein Cell, 5: 496; Rebagay et al., 2012, Prontiers in oncology, 2: 1) .

ROR1 is expressed in the process of embryo and fetal development, and it controls cell polarity, cell migration and neurite growth, etc. The expression is gradually reduced according to progress of development, and it is hardly expressed in adults, and it is temporarily expressed in the process of development of B cell, and only little expression has been reported in adipocytes (Hudecek et al., 2010, Blood 116: 4532; Matsuda et al., 2001, Mech. Dev. 105: 153) .

However, as the overexpression of ROR1 is observed in various cancer cells, it is classified as an oncofetal gene. In particular, ROR1 has received attention as an anti-cancer antibody target, as it is discovered that ROR1 is overexpressed in chronic lymphocytic leukemia (CLL) . It has been reported as overexpressed in not only hematologic malignancy such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML) , acute lymphoblastic leukemia (ALL) , etc. In addition to chronic lymphocytic leukemia (CLL) but also solid cancer including breast cancer, ovarian cancer, gastric cancer, lung cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer (NSCLC) , colon cancer, etc.

Such a cancer cell-specific expression of ROR1 shows that ROR1 can be an effective cancer target, and therefore the development of an antibody specifically recognizing it is required. This oncofetal protein is an attractive target for cancer therapy. Antibodies to ROR1 have been described in the literature W02014031174 (UC961) , WO2017127664 (XBR1-402) , etc. And a humanized murine anti-ROR1 antibody, UC961, has entered clinical trials for relapsed or refractory chronic lymphocytic leukemia.

Since antibodies against the same ROR1 antigen can be developed to various anti-cancer antibodies depending on properties or uses of each antibody, considering cancer-specific expression of ROR1, and its expression in various cancers, it is necessary to develop various antibodies that can replace or complement existing antibodies. Due to the low number of available ROR1 specific monoclonal antibodies, there is a need in the art for better anti-ROR1 antibodies that have efficient internalization or other functional properties not possessed by the known antibody. There is also a need for additional diagnostic tools for detecting ROR1 expressions in ROR1-related disease conditions. The instant invention is directed to addressing these and other needs.

SUMMARY OF THE INVENTION

The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen, so the insufficient internalization of ROR1-targeting monoclonal antibody is also an urgent problem to be solved.

The technical problem to be solved by the present invention is to overcome the defects of weak binding ability of ROR1-targeting antibodies to ROR1, cross-reactivity with ROR2 and insufficient internalization in a human cell.

Specifically, the present invention encompasses the following aspects:

The present disclosure provides an anti-ROR1 antibody or an antigen-binding fragment thereof, comprising one or more the CDR region sequences selected from the following sequence thereof: An anti-ROR1 antibody or an antigen-binding fragment thereof comprising: the antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: a) HCDR1 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 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 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; d) LCDR1 as 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 as 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 as shown in 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.

In some embodiments, 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, respectively; 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 respectively; 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 respectively; 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 respectively; 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 respectively; 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 respectively; 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 respectively.

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, respectively; 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, respectively; 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, respectively; 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, respectively; 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, respectively; 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, respectively; 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, respectively.

In a preferable embodiment, the anti-ROR1 antibody or antigen-binding fragment comprises: a) a 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 a 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 b) a 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 a 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) a 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 a 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) a 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 a 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 e) a 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 a 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) a 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 a 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) a 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 a 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.

In a preferable embodiment, the antibody or antigen-binding fragment thereof is selected from murine antibody, chimeric antibody, humanized antibody, human antibody or the antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 40, 42, 44, 46, 48, 50, 52, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 41, 43, 45, 47, 49, 51 and 53, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.

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

In some embodiments, the antibody or antigen-binding fragment thereof comprising: a) the heavy chain variable region as shown in SEQ ID NO: 40; and/or the light chain variable region as shown in SEQ ID NO: 41; b) the heavy chain variable region as shown in SEQ ID NO: 42; and/or 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/or the light chain variable region as shown in SEQ ID NO: 45; d) 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; e) the heavy chain variable region as shown in SEQ ID NO: 48; and/or 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/or 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/or the light chain variable region as shown in SEQ ID NO: 53.

In a preferable embodiment, the antibody or antigen-binding fragment thereof comprising: a) 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; 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: 46 and the light chain variable region as shown in SEQ ID NO: 47; 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.

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

In some embodiments, the antigen-binding fragment above is selected from the group consisting of Fab, Fab', F (ab') 2, variable fragment (Fv) , single chain variable fragment (scFv) , dimerized domain V (diabody) , disulfide stabilized Fv (dsFv) and CDR-containing peptides.

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

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

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 the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.

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

In one aspect, the present disclosure also provides a method for immunologically detecting or measuring ROR1, wherein the method comprises detecting the ROR1 by contacting with the above-mentioned antibody or the antigen-binding fragment.

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

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

In some embodiments, the present disclosure also provides a method of treatment or prevention of a disease related to overexpression of ROR1, comprising a step of administering a therapeutically effective amount of the antibody or its antigen-binding fragment above, or the pharmaceutical composition above, to a subject in need of treatment or prevention of the disease.

In some embodiments, the disease related to overexpression of ROR1 is cancer; preferably the cancer is 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.

The antibody or its antigen-binding fragment may (1) specifically recognize or bind to a ROR1 which is expressed on a cell surface derived from human, or (2) specifically recognize or bind to an extracellular domain of ROR1 which is not present on a cell surface, or (3) have sufficient internalization in a human cell.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

Figure 3. Internalization kinetics of anti-ROR1 recombinant antibodies in ROR1+ Jeko1 cells.

DETAILED DESCRIPTION

The present invention is based on the development of an antibody which can specifically bind to ROR1. The titles used in the present section are for convenience of specification only, and do not limit the present invention.

Unless otherwise defined herein, the scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. Further, unless the context specifically requires, the singular includes the plural, and the plural includes the singular.

DEFINITIONS

Before the present invention is detailed below, it is to be understood that the present invention is not limited to the particular methodologies, protocols and reagents described herein, as those may vary. It is also to be understood that the terminology used herein is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

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

The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin 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.

The term "antigen-binding fragment" of an antibody, as used herein, 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 performed by fragments 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 the VL, VH, CL and CHI domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting 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 complementarity determining region (CDR) , and (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ; 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.

The term "human antibody" , as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues 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, the term "human antibody" , as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "recombinant human antibody" , as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or trans chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further in Section I, below) , (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve 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. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus 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 within the human antibody germline repertoire in vivo.

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

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 the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883) , Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)) , AbM (University of Bath) , Contact (University College London) , International ImMunoGeneTics database (IMGT) (imgt. cines. fr/on the World Wide Web) , and North CDR definition based on the affinity propagation clustering using a large number of crystal structures. Those skilled in the art can easily identify the CDRs defined by each numbering system.

A useful comparison of CDR numbering is as below:

Note 1: some of these definitions (particularly for Chothia loops) vary depending on the individual publication examined; Note2: any of the numbering schemes can be used for these CDR defintions, except the contact definition uses the Chothia or Martin (enhanced Chothia) definition; Note 3: the end of the Chothia HCDR1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places the insertions at H35A and H35B.

The term “nucleic acid molecule” as used herein refers to a DNA molecule and a RNA molecule. The nucleic acid molecule may be single stranded or double stranded but is preferably a double stranded DNA. A nucleic acid is “effectively linked” when it is placed into functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.

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

Those skilled in the art know that the base sequence encoding the amino acid sequence of the protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog. The homolog 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 activity of the antibody.

The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked. In one embodiment, the vector is a “plasmid” that refers to a circular double stranded DNA loop into which additional DNA segment can be ligated. In another embodiment, the vector is a viral vector, wherein an additional DNA segment can be ligated into viral genome. The vectors disclosed herein are capable of self-replicating in a host cell into which they have been introduced (for example, a bacterial vector having a bacterial replication origin and a episomal mammalian vector) or can be integrated into the genome of a host cell upon introduction into host cell, thereby is replicated along with the host genome (e.g., a non-episomal mammalian vector) .

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, thus 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, phage or virus vectors and the like.

The term "transfectoma" , as used herein, includes recombinant eukaryotic host cell expressing the antibody, such as CHO cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells.

The sequence of the DNA molecule for the antibody or a fragment thereof according to the present invention can be obtained by conventional techniques, for example, methods such as PCR amplification or genomic library screening. In addition, the sequences encoding light chain and heavy chain can be fused together, to form a single-chain antibody.

Once a relevant sequence is obtained, the relevant sequence can be obtained in bulk using a recombination method. This is usually carried out by cloning the sequence into a vector, transforming a cell with the vector, and then separating the relevant sequence from the proliferated host cell by conventional methods.

In addition, a relevant sequence can be synthesized artificially, especially when the fragment is short in length. Usually, several small fragments are synthesized first, and then are linked together to obtain a fragment with a long sequence.

At present, it is possible to obtain a DNA sequence encoding the antibody of the present invention (or fragments thereof, or derivatives thereof) completely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or, for example, vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.

In general, under conditions suitable for expression of the antibody according to the present invention, the host cell obtained is cultured. Then, the antibody of the present invention is purified by using conventional immunoglobulin purification steps, for example, the conventional separation and purification means well known to those skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

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

The antibody according to the present invention can be expressed in a cell or on the cell membrane, or is secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods according to its physical, chemical, and other properties. These methods are well known to those skilled in the art. The examples of these methods comprise, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation) , centrifugation, cell lysis by osmosis, ultrasonic treatment, supercentrifugation, molecular sieve chromatography (gel chromatography) , adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) , and any other liquid chromatography, and the combination thereof.

The term "variant " of a polypeptide such as for example, 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, as compared to another polypeptide sequence, and includes a fusion polypeptide. In addition, a protein variant includes one modified by protein enzyme cutting, phosphorylation or other posttranslational modification, but maintaining biological activity of the antibody disclosed herein, for example, binding to ROR1 and specificity. 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 its antigen-binding fragment disclosed herein. The percent identity (%) or homology may be calculated with reference to the following description.

In one embodiment, the percent homology or identity may be calculated as 100 x [ (identical position) /min (TGA, TGB) ] , and in the formula, TGA, TGB are the sum of the number of residues of sequences A and B compared and the internal gap position (Russell et al., J. Mol Biol., 244: 332-350 (1994) .

In the present invention, the antibody of the present invention also includes a conservative variant thereof, which means that, compared to the amino acid sequence of the antibody of the present invention, there are up to 10, preferably up to 8 and more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

Table A

The term "K_D " (M) , as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. “K_D” refers to the dissociation constant, which is obtained from the ratio of K_d to K_a (i.e., K_d/K_a) and is expressed as a molar concentration (M) . K_D values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the K_D of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a system, or by using bio-layer interferometry technology, such as an Octet RED96 system.

The term "affinity" is the strength of interaction between an antibody or its antigen-binding fragment and an antigen, and it is determined by properties of the antigen such as size, shape and/or charge of antigen, and CDR sequences of the antibody or antigen-binding fragment. The methods for determining the affinity are known in the art, and the followings can be referred.

The antibody or its antigen-binding fragment is called "specifically binding " to its target such as an antigen, when a dissociation constant (K_D) is < l0⁶ M. The antibody specifically binds to a target with "high affinity " , when K_D is < l0⁹ M.

The term "Pharmaceutical composition" , as used herein, is intended to refer to a mixture containing one or more of the compounds or a physiological/pharmaceutically acceptable salt or prodrug thereof described herein with other chemical components, such as physiological /pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and exerts the biological activity.

“Administration” and “treatment” , when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact with an exogenous pharmaceutical, therapeutic, diagnostic reagent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. “Administration” and “treatment” can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contacting the cell with a reagent, as well as contacting a fluid with a reagent, wherein the fluid is in contact with the cell. “Administration” and “treatment” also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition or by another cell. “Treatment, ” when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

In addition, the present disclosure includes a medicament for treating a disease associated with ROR1, comprising an antibody or an antigen-binding fragment thereof of the present disclosure as an active ingredient.

There is no limitation on the diseases related to 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 human ROR1. Therefore, the molecules of the present disclosure are very useful for those who suffer a tumor, cancer or infectious disease when in preparations and formulations suitable for therapeutic applications.

In addition, the present disclosure relates to a method for immunologically detecting or measuring ROR1, a reagent for immunologically detecting or measuring ROR1, a method for immunologically detecting or measuring cells expressing ROR1, and a diagnostic reagent for diagnosis of disease related to ROR1 positive cells, comprising the antibody or antigen-binding fragment of the present disclosure that specifically recognizes human ROR1, as an active ingredient.

In the present disclosure, the method for detecting or determining the amount of ROR1 may be any known method. For example, it includes immunodetection or assay.

The immunodetection or assay is a method of detecting or determining the amount of antibody or antigen by using labeled antigen or antibody. Examples of immunodetection or assay include a radioactive substance labeled immunological antibody method (RIA) , an enzyme immunoassay (EIA or ELISA) , a fluorescent immunoassay (FIA) , a luminescent immunoassay, a western blotting method, physicochemical methods, etc.

The above-mentioned diseases related to ROR1 positive cells can be diagnosed by detecting or measuring cells expressing ROR1 by using the antibodies or antibody fragments thereof of the present invention.

In order to detect cells expressing the polypeptide, a known immunodetection can be used, and preferably immunoprecipitation, fluorescent cell staining or immunohistochemically staining etc. can be used. Furthermore, a fluorescent antibody staining method etc. using FMAT8100HTS system (Applied Bio system) can be used.

EXAMPLE

The invention is further illustrated by the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the 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. "Guide to Molecular Cloning Laboratory" (translated by Huang Peitang et al., Beijing: Science Press, 2002) , or in accordance with the conditions recommended by the manufacturer (for example, product manuals) . Percentages and parts are by weight unless otherwise stated. The experimental materials and reagents used in the following examples are commercially available unless otherwise specified.

The room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30℃.

EXAMPLE 1: Immunization and screening of antibody

EXAMPLE 1-1: Preparation of immunogen

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

EXAMPLE 1-2: Immunization

Immune protocol:

Anti-ROR1 antibodies were obtained by immunizing genetically modified mouse encoding human immunoglobulin heavy 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 cell, and HT-29 cells (ATCC, catalog number HTB38) . The antibody immune response was monitored by a ROR1-specific immunoassay. When a desired immune response was achieved splenocytes were harvested from each mouse and fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for ROR1 specificity.

EXAMPLE 1-3: Spleen cell fusion

The spleen lymphocytes and myeloma cells Sp2/0 (CRL-158) were fused to obtain hybridoma cells by electrofusion or PEG fusion. PEG fusion was performed using Clonacell^TM HY technology (STEMCELL technologies) , following manufacturer’s instructions. The primary cell: mouse myeloma cell line ratio was 1: 1 for electrofusion, and 10: 1 for PEG fusion.

EXAMPLE 1-4: Screening of hybridoma clones specifically binding to human ROR1 protein by ELISA

ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 1μg/ml of human ROR1-Fc (9490-R0-050) , human ROR2-Fc (8609-RO-050) or BSA overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50μl ROR1 hybridoma supernatant was added in duplicate wells and incubated for 1 hour. Excess unbound antibodies were washed off and 50μl of 1: 30000 diluted secondary antibody Goat anti-mouse IgG Fc-HRP (ab5870) was added to each well for another 1 hour. Plates were washed before the addition 50 μL of chemiluminescence agents (color A and color B) according to manufacturer's protocol. The reactions were stopped using 25μL of 2N sulfuric acid. Optical density at 450nm of samples was measured by a microplate reader (PerkinElmer) . All tested clones bound selectively to human ROR1 but not to human ROR2 nor BSA, demonstrating ROR1 specificity.

Table 1. Binding characterization of hybridoma clones to human ROR1 and human ROR2 by ELISA assay (OD₄₅₀)

EXAMPLE 1-5: Screening of hybridoma clones specifically binding to ROR1 expressing cancer cells by Flow cytometry

Hybridoma supernatants was subjected to binding tests on ROR1+ cell line HT29 (ATCC, HTB-38) , HCC1187 (ATCC, CRL-2322) , and ROR1-ROR2+ cell line T47D (ATCC, HTB-133) using flow cytometry analysis. Briefly, 50 μL of cells in cell staining buffer (2×10⁶ cells/mL) was mixed with 50 μL undiluted hybridoma supernatants. The mixture was incubated on ice for 20 min and then washed with ice cold staining buffer twice. The cells were subsequently stained with 50 μL of PE labelled secondary antibody (1: 400 dilution, BioLegend, Cat#405307) for 20 min. After washing by staining buffer and fixing by 4%PFA, cells were analyzed by flow cytometry. A purified anti-human ROR1 antibody was used as positive control (BioLegend, Cat#357802) . A purified mouse IgG1 antibody was used as isotype control (Biolegend, Cat#400102) . Figure 1 shows examples of selected cell binding signals measured by flow cytometry. The clones 1E9, 8B10, 15G4, 29D4, 31G8, 37F9, 38H10 were identified with enhanced binding profile to HT29 cells and HCC1187 cells compared to T47D cells.

EXAMPLE 2 Sequencing of positive hybridoma clones

The process of cloning sequences from positive hybridomas are as follows. The logarithmic growth phase hybridoma cells were collected, RNA was extracted, and reverse transcription was performed then followed by VDJ region amplification. Amplified cDNA library from each clone were subjected to next-generation sequencing. The amino acid sequences of the heavy and light chain variable region DNA sequences corresponding to the antibodies 1E9, 8B10, 15G4, 29D4, 31G8, 37F9, and 38H10 were obtained. Upon the manufacturability assessment evaluation, several mutations were made in the FR region, and the amino acid sequence of heavy chain variable and light chain variable regions and CDR sequence of each antibody are as the following tables. The amino acid residues of the CDRs in VH/VL are numbered and annotated according to the Kabat &Wu numbering system.

Table 2. CDR sequence of heavy chain variable domain for ROR1 hybridoma clones

Table 3. CDR sequence of light chain variable domain for ROR1 hybridoma clones

Table 4. Sequences of heavy chain and light chain variable domains for ROR1 hybridoma clones

Example 3 Construction and Expression of anti-ROR1 Recombinant Antibody

Example 3-1: Molecular cloning of recombinant antibodies

The cDNA sequences that encode VH and VL regions of selected clones were directly synthesized as DNA fragments with 5’ -end in-frame leader sequence (MGWSCIILFLVATATGVHS) . These DNA fragments were cloned into selected vectors using NEBuilder DNA Assembly Cloning Kit (New England Biolabs) . VH region was cloned into pFUSE-CHIg_hG1 vector (InvivoGen #pfuse-hchg1) , which in-frame with constant region of hIgG1 heavy chain in the vector. VL region was cloned into pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk) , which in-frame with constant region of hIg kappa light chain in the vector.

The IgG form of antibodies were disclosed as the following heavy chain and light chain full-lengths in order of 1E9, 8B10, 15G4 (N65S) , 29D4, 31G8, 37F9 and 38H10 (N81K, T83S) : 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) , respectively.

Table 5. Sequences of recombinant antibodies IgG constant regions

Table 6. Sequences of heavy chain and light chain full lengths for anti-ROR1 recombinant antibodies

Example 3-2: Expression and purification of recombinant antibodies

The heavy chain expression plasmid and light chain plasmids were co-transfected into Expi293F cells (ThermoFisher, #A14527) using ExpiFectamine 293 Transfection Kit (ThermoFisher, A14524) , or into ExpiCHO-S cells (ThermoFisher #A29127) using ExpiFectamine CHO Transfection Kit (ThermoFisher, A29129) . Based on the manufacturer’s instructions, plasmid DNA concentration reached 1.0 ug per ml of suspended cells, with LC: HC vector ratio 1: 1. The transfected cells were cultured 5 to 7 days on an orbital shaker at 37 C, 8%CO₂. Conditioned medium was collected and antibodies were purified using HiTrap MabSelect SuRe column (Cytiva, #17549112) on AKTA Pure 25 machine (Cytiva) . Eluted antibodies were neutralized with Tris Buffer (pH 9.0) and subjected to PBS buffer exchange. Product concentration was measured by UV absorption, and quality was determined by SDS-PAGE and HPLC.

Example 4: Binding characterization of anti-ROR1 recombinant antibody to ROR1+ and ROR1-cell lines by flow cytometry

Binding of the hlgGl mAbs to the 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 cell.

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

To determine the binding of hIgG1 mAbs to cell surface ROR1 receptors, cells were first harvested and resuspended in cell staining buffer (BioLegend) at 2×10⁶ cells/mL. Then, the cells were treated with human Fc receptor blocking reagent (BioLegend) on the ice for 10 min. The resulting cell suspension was aliquoted into 50 μL aliquots. 50 μL of hIgG1 mAbs at various concentrations were mixed with the cell aliquots, the final concentration of hIgG1 mAbs in the mixture ranged from 1.1 pM to 200 nM. The cells were incubated on the ice for 1 hour and then washed with cell staining buffer twice. 50 μL of secondary antibody (PE conjugated goat anti-human Fc, eBioscience^TM, 1: 200 dilution) was added to each sample to resuspend the cells. The cells were incubated on the ice for another 30 min. Cells were subsequently 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 corresponding channels. The result was disclosed in Figure 2 and Table 7. As a result, it was confirmed that the anti-ROR1 antibody of the present invention specifically binds to the human ROR1 as originally expressed in cells in a concentration-dependent pattern. In addition, it was confirmed that it does not bind to a family member protein, human ROR2.

Table 7. K_D values of anti-ROR1 recombinant antibodies binding to ROR1+ Jeko-1 cells

Example 5: Characterization of anti-ROR1 recombinant antibody cell internalization in ROR1 expressing cells

To explore the possibility of developing ADC strategy with the ROR1 antibodies of the invention, the inventors evaluated endocytosis of the antibodies induced by ROR1-antibody binding. In the assay, Jeko-1 cells were collected and suspended in ice cold staining buffer (PBS with 0.5%FBS) at 2×10⁶ cells/mL, followed by treating with human Fc receptor blocking reagent (BioLegend, 422302) . The cells were then pre-incubated with Alexa Fluor 488 labelled antibodies on ice for 1 hour. After incubation, cells were transfer to 37℃ incubator. At predetermined time point (0 min, 15 min, 30 min, 45 min, 1 hour, 2 hour, 4 hour, 6 hour) , 50 μL of the cell suspension were aliquoted and transferred to incubate on ice till end of the incubation for internalization. Then, all aliquoted samples were washed twice with ice cold staining buffer. Cell surface bound Alexa Fluor 488 signal was quenched by anti-Alexa Fluor antibody (Invitrogen, A-11094) . The remaining signals inside the cells, resulting from internalization, were then analyzed by flow cytometry. An additional 50 μL aliquot of the cells at 0 min was collected kept on ice and analyzed by flow cytometry without quenching. The sample was served as control for 100%cell surface binding signal. The amount of internalized antibodies were calculated and normalized to 100%cell surface binding signal of each antibody, as calculated by the following equation:
Normalized Internalization (%) = (MFI_t-MFIt₀) / (MFI_total-MFIt₀) ×100%

Internalization results based on median fluorescence intensity (MFI) . MFIt is the MFI of samples collected at time t; MFIt₀) is the MFI of samples collected at time 0 minute. MFI_total is the MFI of the additional sample collected and served as control for 100%cell surface binding signal.

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

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

Example 6: Epitope characterization of anti-ROR1 recombinant antibodies

Example 6-1: ELISA binding to human ROR1 subdomain proteins

ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 1μg/ml of human ROR1 protein (RO1-H522y) or human ROR1 subdomains -Ig-like (RO1-H5221) , Frizzled (RO1-H5222) and Kringle (RO1-H5223) overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50 μl of 100 μg/mL ROR1 recombinant antibodies was added in duplicate wells and incubated for 1 hour. Excess unbound antibodies were washed off 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. Plates were washed before the addition of 50μL of chemiluminescence agents (color A and color B) according to manufacturer's protocol. The reactions were stopped using 25 μL of 2 N sulfuric acid. Optical density at 450nm of samples were measured by microplate reader (PerkinElmer) . All recombinant antibodies tested bound to full length human ROR1 protein. 1E9, 8B10, 15G4 (N65S) , 29D4, 31G8, 37F9 and UC-961 bound to the human ROR1 Ig-like subdomain protein, while 38H10 (N81K/T83S) bound to the human ROR1 Kringle subdomain protein.

Table 9. Binding characterization of ROR1 recombinant antibodies to human ROR1 full length or subdomain proteins by ELISA (OD₄₅₀)

Example 6-2: Octet binning

A total of five clones (15G4 (N65S) , 29D4, 31G8, 37F9 and 38H10 (N81K/T83S)) were subjected to epitope binning and compared to anti-ROR1 antibodies UC961. Antibody epitope binning was performed using an Octet Red384 system equipped with anti-species and Ni-NTA sensors from Pall Life Sciences (Menlo Park, CA) . The experiment was performed as an in-tandem binning assay. The assay is comprised of a five-step binding cycle: 1) A buffer baseline was established for 30 seconds, 2) 50 nM ROR1 antigen (RO1-H522y) was coupled to Ni-NTA octet sensors using a standard 1x assay buffer (PBS + 0.02%Tween20, 0.1%BSA, 0.05%sodium azide) diluted from a 10x kinetic buffer stock (ForteBio) for 5 minutes, 3) 250 nM of each antibody (saturating mAb) was loaded to saturate the immobilized antigen for 10 minutes, 4) 250 nM of each antibody (competing mAb) was bound for 5 minutes, and 5) capture sensors were regenerated for 30 seconds. As shown in Table 9 and Table 10, 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. In addition, UC-961 binding does not compete with any of the five anti-ROR1 recombinant antibodies (15G4 (N65S) , 29D4, 31G8, 37F9 and 38H10 (N81K/T83S) ) , indicating UC-961 binding epitope is in a separate epitope bin 3.

Table 10. Epitope binning for recombinant antibodies binding to human ROR1 protein determined by BLI using an Octet Red384 system.

Signal shift unit: nm

Table 11. Epitope bins of anti-ROR1 recombinant antibodies to human ROR1

Example 7: Cross reactivity of anti-ROR1 recombinant antibodies to murine ROR1 proteins

ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 1μg/ml of mouse ROR1 protein (RO1-M5221) , rat ROR1 protein (RO1-R5221) or control BSA overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50 μl of 25 μg/mL ROR1 recombinant antibodies was added in duplicate wells and incubated for 1 hour. Excess unbound antibodies were washed off 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. Plates were washed before the addition of 50 μL of chemiluminescence agents (color A and color B) according to manufacturer's protocol. The reactions were stopped using 25 μL of 2 N sulfuric acid. Optical density at 450nm of samples were measured by microplate reader (PerkinElmer) . Recombinant antibodies 15G4 (N65S) , 29D4, 31G8, 37F9 and 38H10 (N81K/T83S) are capable of binding to both mouse ROR1 protein and rat ROR1 protein. In comparison, UC961 does not show cross reactivity to mouse or rat ROR1 proteins.

Table 12. Binding characterization of ROR1 recombinant antibodies clones to mouse ROR1 or rat ROR1 proteins by ELISA (OD₄₅₀)

Claims

An anti-ROR1 antibody or an antigen-binding fragment thereof comprising: the antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein:

a) HCDR1 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 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 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;

d) LCDR1 as 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 as 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 as shown in 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.
An 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, respectively;

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 respectively;

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 respectively;

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 respectively;

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 respectively;

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 respectively;

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 respectively.
An 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, respectively;

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, respectively;

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, respectively;

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, respectively;

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, respectively;

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, respectively;

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, respectively.
An anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1, wherein:

a) a 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 a 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) a 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 a 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) a 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 a 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) a 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 a 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) a 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 a 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) a 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 a 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) a 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 a 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.
An anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is selected from murine antibody, chimeric antibody, humanized antibody, human antibody or the antigen-binding fragment thereof.
An anti-ROR1 antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 46, 40, 42, 44, 48, 50, 52, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 47, 41, 43, 45, 49, 51 and 53, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.
An anti-ROR1 antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprising:

a) the heavy chain variable region as shown in SEQ ID NO: 46, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 47, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;

or

b) the heavy chain variable region as shown in SEQ ID NO: 42, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO:43, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;

or

c) the heavy chain variable region as shown in SEQ ID NO: 44, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 45, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;

or

d) the heavy chain variable region as shown in SEQ ID NO: 40, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 41, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;

or

e) the heavy chain variable region as shown in SEQ ID NO: 48, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 49, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;

or

f) the heavy chain variable region as shown in SEQ ID NO: 50, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 51, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith;

or

g) the heavy chain variable region as shown in SEQ ID NO: 52, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 53, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.
An anti-ROR1 antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody or antigen-binding fragment thereof comprising:

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) the heavy chain variable region as shown in SEQ ID NO: 42; and/or 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/or 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/or 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/or 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/or 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/or the light chain variable region as shown in SEQ ID NO: 53.
An anti-ROR1 antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody or antigen-binding fragment thereof comprising:

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.
An anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claim 1-9, wherein the antibody is a full-length antibody, further comprising human antibody constant regions; preferably, the heavy chain constant region of the human antibody constant regions is selected from constant regions of human IgG1, IgG2, IgG3 and IgG4 and conventional variants thereof, and the light chain constant region of the human antibody constant regions is selected from κ and λ chain constant regions of human antibody and conventional variants thereof;

more preferably the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 68 and a human light chain constant region of SEQ ID NO: 69.
An anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1-10, wherein the antibody or antigen-binding fragment thereof comprising:

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.
An anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1-9, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F (ab') 2, variable fragment (Fv) , single chain variable fragment (scFv) , dimerized domain V (diabody) , disulfide stabilized Fv (dsFv) and CDR-containing peptides.
An isolated nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof according to any one of claims 1-12.
A recombinant vector comprising the isolated nucleic acid molecule according to claim 13.
A host cell transformed with the recombinant vector according to claim 14, wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
A method for producing the antibody or the antigen-binding fragment thereof according to any one of claims 1-12, wherein the method comprises culturing the host cell according to claim 15 in a medium to produce and accumulate the antibody or the antigen-binding fragment thereof according to any one of claims 1-12, and harvesting the antibody or the antigen-binding fragment thereof from the culture.
A method for immunologically detecting or measuring ROR1, wherein the method comprises detecting the ROR1 by contacting with the anti-ROR1 antibody or antigen-binding fragment thereof of any one of claim 1-12.
A method for diagnosing a disease related to a human ROR1 positive cell, wherein the method comprises a detecting or measuring the ROR1 or ROR1 positive cell by contacting with the anti-ROR1 antibody or antigen-binding fragment thereof of any one of claim 1-12.
A pharmaceutical composition, which comprises a therapeutically effective amount of the anti-ROR1 antibody or the antigen-binding fragment thereof according to any one of claim 1-12, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
A method of treatment or prevention of a disease related to overexpression of ROR1, comprising a step of administering a therapeutically effective amount of the antibody or its antigen-binding fragment according to any one of claim 1-12, or the pharmaceutical composition of claim 19, to a subject in need of treatment or prevention of the disease.
The method of treatment or prevention of a disease related to overexpression of ROR1 according to claim 20, wherein the disease related to overexpression of ROR1 is cancer; preferably the cancer is 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.