Anti-ror1 antibodies and uses thereof

Abstract

The present application belongs to the field of biology, and relates to an antibody or antigen-binding fragment thereof specifically binding to ROR1 and application thereof. The anti-ROR1 antibody of the present application has significant specific binding to ROR1, high binding affinity, and good drugability.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to antibodies that specifically bind to ROR1 or their antigen-binding fragments and their applications. Background Technology

[0002] ROR1 is a transmembrane receptor tyrosine kinase protein that is not expressed or expressed at low levels in most normal adult tissues, but is highly expressed in a variety of hematologic malignancies and solid tumors. Hematologic malignancies with high ROR1 expression include B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and acute myeloid leukemia (AML). In solid tumors, ROR1-expressing tumors include triple-negative breast cancer, colon cancer, lung cancer, pancreatic cancer, and ovarian cancer. Furthermore, ROR1 expression is closely related to disease progression and treatment efficacy. Detection of ROR1 expression in non-advanced and advanced-stage tumor cells from CLL patients revealed that ROR1 expression in advanced-stage cells was significantly higher than in non-advanced-stage cells; moreover, in CLL, higher ROR1 expression levels were associated with shorter treatment-free survival and overall survival (OS). In breast cancer patients, high ROR1 expression corresponds to shorter metastasis-free survival. The above findings indicate that ROR1 expression is closely related to tumor invasion and poor disease prognosis. These studies reveal ROR1 as a potential target for the development of anti-tumor drugs.

[0003] Regarding the mechanism of action of ROR1, current research suggests that ROR1 participates in the non-canonical Wnt signaling pathway mediated by Wnt5a. Wnt5a activates receptor ROR1 or FZD5, leading to Dvl2 / 3 activation and Akt phosphorylation. Akt then promotes IKKα phosphorylation, activating the IKK complex. The IKK complex degrades IκBα and promotes the phosphorylation of the NF-κB subunit p65. Phosphorylated p65 translocates to the nucleus, promoting the transcriptional expression of target genes, including Wnt5a. The secretion of Wnt5a further promotes a new round of autonomous feedback loops. Activation of the ROR1 / Akt / p65 pathway in these autonomous feedback loops further promotes the secretion of pro-inflammatory factors (such as IL-6) and chemokines (such as CCL2). In addition, some studies suggest that ROR1 is associated with EMT in tumor cells or with the activation of YAP / TAZ transcription, thereby enhancing tumorigenesis and chemotactic resistance. Therefore, there is still a need in this field for diverse drug molecules targeting ROR1. Summary of the Invention

[0004] Based on research on anti-ROR1 antibodies, this application yields an anti-ROR1 antibody with strong binding affinity to ROR1.

[0005] In a first aspect, the present invention provides an anti-ROR1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein...

[0006] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 respectively; and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6 or 17, SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0007] In a preferred embodiment, the anti-ROR1 antibody or its antigen-binding fragment as described above, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0008] In a preferred embodiment, the anti-ROR1 antibody or its antigen-binding fragment as described above, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:17, SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0009] In a preferred embodiment, the anti-ROR1 antibody or its antigen-binding fragment is of animal origin, such as rabbit origin, or is a chimeric antibody; more preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:15, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:15; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:16.

[0010] In a preferred embodiment, the anti-ROR1 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, and its heavy chain variable region further includes a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably including a human IgG1 or IgG4 heavy chain FR region.

[0011] In a preferred embodiment, the anti-ROR1 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1 or 11, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1 or 11, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:5, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5.

[0012] In a preferred embodiment, the anti-ROR1 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:5, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5; or

[0013] The heavy chain variable region amino acid sequence is as shown in SEQ ID NO:11, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:11, and / or the light chain variable region amino acid sequence is as shown in SEQ ID NO:5, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5.

[0014] In some embodiments, the anti-ROR1 antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, and / or further comprises a light chain constant region of human κ, λ chains or variants thereof.

[0015] In some embodiments, the heavy chain constant region of the antigen-binding fragment of the anti-ROR1 antibody is the IgG1 heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO:13, and the light chain constant region is the κ light chain constant region, the amino acid sequence of which is shown in SEQ ID NO:14.

[0016] In some embodiments, the heavy chain sequence of the anti-ROR1 antibody is as shown in SEQ ID NO:9 or 12, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9 or 12; and the light chain sequence is as shown in SEQ ID NO:10, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10.

[0017] In some embodiments, the heavy chain sequence of the anti-ROR1 antibody is as shown in SEQ ID NO:9, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9; and the light chain sequence is as shown in SEQ ID NO:10, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10.

[0018] In a preferred embodiment, the antigen-binding fragment is selected from Fab, Fv, scFv, and F(ab'). 2 , dsFv and dAb.

[0019] In the second aspect, a biomaterial is provided, which may be:

[0020] (1) A nucleic acid molecule that encodes the anti-ROR1 antibody or its antigen-binding fragment as described above; preferably, the nucleic acid molecule is a DNA molecule or an RNA molecule; those skilled in the art can deduce the DNA or RNA sequence encoding the amino acid sequence based on the anti-ROR1 antibody or its antigen-binding fragment disclosed in this application, and set appropriate expression elements for it so that the DNA or RNA molecule can express the antibody or its antigen-binding fragment of the present invention.

[0021] (2) A vector containing nucleic acid molecules as described above; preferably, the vector is an expression vector;

[0022] (3) Host cells containing the nucleic acid molecules or carriers mentioned above, or culture media, bacterial suspensions, etc. obtained after culturing the host cells mentioned above.

[0023] In a fourth aspect, a method for preparing the anti-ROR1 antibody or its antigen-binding fragment of the present invention is provided, comprising:

[0024] (1) Chemical synthesis method: prepared by synthesizing the amino acid sequence of the anti-ROR1 antibody or its antigen-binding fragment according to the present invention;

[0025] (2) Bio-preparation method: culturing host cells as described above; further comprising isolating antibodies from the obtained culture; and purifying the antibodies.

[0026] In a fifth aspect, a composition is provided comprising the antibody or antigen-binding fragment thereof of the present invention, or biological material. Preferably, the composition is a pharmaceutical, further comprising a pharmaceutically acceptable carrier or excipient.

[0027] In a sixth aspect, the use of the anti-ROR1 antibody or its antigen-binding fragment, or biological material, or composition of the present invention in the preparation of a medicament for treating tumors or cancer is provided.

[0028] In another aspect, the present invention also provides a method for treating tumors or cancer, the method comprising administering to a subject a preventive or therapeutically effective amount of an anti-ROR1 antibody or its antigen-binding fragment as described in any of the preceding claims, or a biological material, or a composition.

[0029] In another aspect, this disclosure also provides anti-ROR1 antibodies or antigen-binding fragments thereof as described in any of the preceding claims, or biological materials, or compositions, for use as medicaments. In one embodiment, it is used as a medicament for treating tumors or cancer.

[0030] In some embodiments, the tumor or cancer includes solid tumors and hematologic malignancies. In some embodiments, the tumor or cancer is selected from the group consisting of: breast cancer, melanoma, pancreatic cancer, lung cancer, esophageal cancer, non-small cell lung cancer, laryngeal tumors, sarcomas, pharyngeal tumors, oral tumors, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colon cancer, colorectal cancer, lymphoma, and leukemia.

[0031] In some implementations, the breast cancer referred to is triple-negative breast cancer.

[0032] In some implementations, the leukemias mentioned include B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and non-Hodgkin's lymphoma (NHL).

[0033] In some implementations, the tumor or cancer expresses ROR1.

[0034] In a seventh aspect, the invention provides the use of the anti-ROR1 antibody or its antigen-binding fragment, or biological material, or composition thereof in the preparation of a medicament that inhibits binding to ROR1.

[0035] In an eighth aspect, the use of the antibody or antigen-binding fragment thereof, or biological material, or composition of the present invention in the preparation of a medicament for inhibiting the proliferation of ROR1-expressing cells is provided.

[0036] In some embodiments, the ROR1-expressing cells are cancer cells or tumor cells. In some embodiments, the cancer cells are breast cancer cells, lymphoma cells, ovarian cancer cells, or pancreatic cancer cells.

[0037] In some embodiments, the ROR1-expressing cells are MDA-MB-231, Jeko-1, SK-OV-3, or PANC-1.

[0038] The anti-ROR1 antibody of the present invention has significant specific binding to ROR1, high binding affinity, and good prospects for drug development. Attached Figure Description

[0039] Figure 1A The humanized antibody was shown to have binding activity to the ROR1 protein. Figure 1B The humanized antibody was shown to have binding activity with the ROR2 protein.

[0040] Figure 2 The humanized antibody was shown to bind to ROR1 on the MDA-MB-231 cell membrane. Detailed Implementation

[0041] definition

[0042] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields.

[0043] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of…”. For example, for HCDR1 containing the amino acid sequence of SEQ ID NO: 2, it explicitly covers the amino acid sequence of HCDR1 as shown in SEQ ID NO: 2.

[0044] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0045] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be used to provide clear support for both meanings or either meaning.

[0046] As used herein, the term "antibody" or "immunoglobulin" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (knO) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, and ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain consists of a variable region (VH) and a constant region (CH). The constant region consists of three domains (CH1, CH2, and CH3). The light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating 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. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of the heavy / light chain pairs form the antigen-binding sites.

[0047] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the antibody variable region that are primarily responsible for binding to the antigen. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as those defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct16; 9:2278), etc. For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system.

[0048] Unless otherwise stated, the variable region and CDR sequence in the embodiments of the present invention are governed by the "Kabat" numbering rule. Although a numbering system (such as Kabat) is used to define amino acid residues in a specific implementation, the corresponding technical solutions of other numbering systems are considered equivalent.

[0049] As used herein, the term "framework region" or "FR" residue refers to those amino acid residues in the variable region of an antibody, other than the CDR residues as defined above. The term "antibody" is not limited to any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0050] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen; it is also referred to as an “antigen-binding moiety.” See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Exemplary examples include (i) a Fab fragment having VL, CL, VH, and CH1 junctions. (ii) A domain having a disulfide bond between the heavy and light chains; (iii) A Fab' fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iv) An Fd fragment having VH and CH1 domains; (v) An Fd' fragment having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) An Fv fragment having VL and VH domains of one arm of the antibody; (vi) A dAb fragment consisting of a VH domain; (vii) (viii) Hingeless antibodies, which contain at least VL, VH, CL, and CH1 domains but lack a hinge region; (viii) F(ab)2 fragments, which are bivalent fragments containing two Fab' fragments linked by disulfide bridges in the hinge region; (ix) Single-chain antibody molecules (scFv) refer to molecules containing a variable region (VH) of the antibody heavy chain and a variable region (VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH; x) "Diabodies" have two antigen-binding sites, including a heavy chain variable domain (VH) and a light chain variable domain (VL) linked together in the same polypeptide chain; (xi) "Linear antibodies" contain a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions; (xii) dsFv refers to a fragment formed by replacing one amino acid residue in each of VH and VL with a cysteine ​​residue via an SS bond between the cysteine ​​residues.

[0051] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0052] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions.

[0053] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0054] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0055] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al., Nature 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0056] Antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to achieve immunospecific antibody purification. For purification of immunoglobulins, see, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0057] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three (heavy and / or light immunoglobulin chains) of the receptor CDR of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDR is called the "donor," and the immunoglobulin providing the framework is called the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the receptor framework can be a naturally occurring human framework or a sequence having approximately 85%, 90%, 95%, 99%, or higher identity with it. Humanized antibodies typically retain the intended properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc. Donor antibodies can be mouse, rat, rabbit, or non-human primate antibodies with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0058] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids, phage particles, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site. Based on the given nucleic acid sequence, those skilled in the art can easily construct a suitable vector that enables the nucleic acid to be replicated or expressed.

[0059] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0060] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its bound ligand (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while the terms "kdis" or "kd" as used herein are intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the equilibrium dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).

[0061] The term "antigen" refers to a molecule or molecular moiety that can be selectively recognized by antigen-binding protein molecules (such as antibodies) or bound by a binding agent. An antigen may have one or more epitopes that can interact with different antigen-binding protein molecules (such as antibodies).

[0062] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., the tertiary folding of the antigen as a protein), which allows non-consecutive amino acids to be spatially close. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0063] The terms "specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 The equilibrium dissociation constant (KD) of an antibody to an antigen or epitope (M or less) is used to determine its binding antigen or epitope. In some embodiments, the KD of an antibody to an antigen is 10% or less (e.g., 1%) of the KD of the antibody to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, e.g., cynomolgus macaques (Macaca fascicularis) (cynomolgus, cyno) or chimpanzees (Pan troglodytes) (chimpanzee, chimp) or marmosets (Callithrix jacchus) (commonmarmoset, marmoset).

[0064] The term "non-binding" means that the antibody cannot bind to an antigen or its epitope within that antigen in the manner described above for specific binding. For example, when the antibody binds at approximately 1 × 10⁻⁶... -6 M or a larger equilibrium dissociation constant (KD) binds to the antigen or its epitope within the antigen.

[0065] The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to a target antigen or its epitope. Specific antigen-binding modules include the antigen-binding domain of an antibody, such as those containing heavy chain variable regions and light chain variable regions.

[0066] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0067] The term "sequence identity" refers to the degree (percentage) to which two sequences have the same amino acids / nucleic acids at equivalent positions when optimally aligned. During alignment, gaps may be introduced where necessary to achieve the maximum percentage of sequence identity, but any conserved substitutions are not considered part of sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0068] As an example, "the amino acid sequence of the heavy chain variable region has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1" means that the heavy chain variable region contains HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:4, and that amino acid mutations are allowed to be introduced in the region outside the CDRs, thereby having at least 85% sequence identity with SEQ ID NO:1.

[0069] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding molecules or antibodies described herein, along with other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0070] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0071] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless explicitly stated otherwise, the terms “patient” or “subject” are used interchangeably herein. As used herein, the term “cynomolgus monkey” refers to the cynomolgus monkey (Macacafascicularis). In some embodiments, the individual or subject is a human.

[0072] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0073] "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention intended to be applied to the individual being treated, and can be implemented for preventative purposes or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, the molecules disclosed herein are used to delay the onset of disease or slow its progression.

[0074] Antibody sequences in the examples of this invention

[0075] Table 1. Sequences of ROR1 humanized monoclonal antibodies VH12VL9 and VH13VL9

[0076]

[0077]

[0078]

[0079] The present invention will be further illustrated by specific embodiments below, but the scope of the present invention is not limited thereto.

[0080] Unless otherwise specified, the reagents and instruments used in this invention are all conventional reagents and instruments that can be obtained commercially; the methods used are all conventional techniques, and those skilled in the art can undoubtedly complete the experiments and obtain the corresponding results based on the contents of the specification.

[0081] Example 1: Screening of anti-ROR1 rabbit monoclonal antibodies

[0082] This embodiment describes a method for preparing rabbit anti-human ROR1 monoclonal antibody. First, the extracellular ROR1 protein (UniProt ID: Q01973) was expressed as an immunogen. A 6xHis tag was added to the C-terminus of the extracellular ROR1 protein amino acid sequence (Gln30-Glu403), and then cloned into an expression vector. The expression vector was transiently transfected into 293 cells, and the cell culture medium was collected and purified after 7 days. To prepare the rabbit anti-human ROR1 monoclonal antibody, New Zealand rabbits (purchased from Wuhan Wanqianjiaxing Biotechnology Co., Ltd.) were immunized with ROR1 protein five times. B cells from the immunized New Zealand rabbits were isolated and cultured as monoclonal antibodies. The titer of the monoclonal B cell supernatant was detected using ELISA. ROR1 protein was coated at 1 μg / mL onto microplates and incubated overnight at 4°C, then blocked with blocking buffer at 37°C for 1 h. After washing, the B cell supernatant was added to the plate and incubated at 37°C for 1 h. After washing the plate, HRP-labeled goat anti-rabbit IgG (manufacturer: Jakson) was added, and the reaction was carried out at 37°C for 1 hour. After washing again, TMB solution was added, and the reaction was carried out at room temperature in the dark for 5 minutes, then 2NH₂SO₄ was added to terminate the reaction. The absorbance was measured at 450 nm using a microplate reader. Based on the binding activity with ROR1, monoclonal 34A4 heavy chain variable region and light chain variable region sequences with high binding activity were screened, as shown in SEQ ID NO:15 and SEQ ID NO:16, respectively.

[0083] Rabbit source 34A4 VH (SEQ ID NO:15):

[0084] QSLEESGGRLVTPGGSLTLTCTVSGIDLSSYAMIWVRQAPGEGLEWIGIIYASGGTWYASW AKGRFTFSRTSTTVDLKMTSLTTEDTATYFCARYIDGAGNIWGPGTLVTVSS;

[0085] Rabbit source 34A4 VL (SEQ ID NO:16):

[0086] QVLTQTPSPVSAAVGGTVTINCQASQSVYNNRLAWYQQKPGQPPKLLIYWASTLTSGVPS RFKGSGSGTQFTLTISEVQCDDAATYHCQGGYSGNIAAFGGGTEVVVK;

[0087] The amino acid sequences of the three CDR regions of rabbit-derived 34A4 VH are shown in SEQ ID NO:2, 3, and 4, respectively, and the amino acid sequences of the three CDR regions of VL are shown in SEQ ID NO:17, 7, and 8, respectively. The amino acid sequence of SEQ ID NO:17 is: QASQSVYNNRLA.

[0088] The aforementioned heavy chain variable region and light chain variable region were combined with the human heavy chain constant region SEQ ID NO:13 and light chain constant region SEQ ID NO:14, respectively, to obtain the chimeric antibody Ch34A4. The affinity of Ch34A4 for ROR1 was detected using the method described in Example 5. The results showed that the chimeric antibody had extremely high affinity for ROR1, with an affinity constant of 2.95 × 10⁻⁶. -9 M.

[0089] Example 2: Humanization and Expression of Anti-ROR1 Rabbit Monoclonal Antibody

[0090] 34A4 was humanized using the CDR transplantation method, and the N at position 8 of LCDR1 (QASQSVYNNRLA, SEQ ID NO:17) was mutated to Q, resulting in the sequence QASQSVYQNRLA (SEQ ID NO:6). This yielded the humanized variable region. The humanized variable region was then combined with a heavy chain constant region (e.g., IgG1, sequence see SEQ ID NO:13) and a light chain constant region (e.g., the kappa light chain constant region, sequence see SEQ ID NO:14) to obtain the humanized antibodies VH12VL9 and VH13VL9. VH12VL9 contains the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:5 (Table 1). The heavy chain variable region contains CDR1 of SEQ ID NO:2, CDR2 of SEQ ID NO:3, and CDR3 of SEQ ID NO:4, while the light chain variable region contains CDR1 of SEQ ID NO:6, CDR2 of SEQ ID NO:7, and CDR3 of SEQ ID NO:8. VH13VL9 contains the heavy chain variable region of SEQ ID NO:11 and the light chain variable region of SEQ ID NO:5, and its CDR sequence is identical to that of VH12VL9. The heavy and light chain sequences of the VH12VL9 and VH13VL9 antibodies are shown in Table 1.

[0091] The antibody of this invention was expressed and purified in CHO-K1 cells. First, a vector containing the antibody heavy and light chain coding sequences was transferred into CHO-K1 cells by electroporation. After culturing at 37°C and 5% CO2 for 4 days, the cell culture supernatant was centrifuged at 3000 rpm for 10 min. The supernatant was collected and purified using protein A to achieve an antibody purity >95%.

[0092] Example 3: Specific binding of humanized antibody to ROR1

[0093] The binding of humanized antibodies to human ROR1 protein was detected using an ELISA method. hROR1 (manufacturer: Kaika Biotechnology, catalog number: ROR-HM401) was diluted to 0.5 μg / mL in PBS and coated onto microplates, then incubated at 37°C for 1 hour. The plates were then blocked with 5% BSA-PBS blocking buffer at 37°C for 1 hour. After washing with PBST, the antibody was diluted to different concentrations (starting at 66.67 nM, 6-fold dilution, for a total of 8 concentrations), added to the plates, and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG (manufacturer: Sigma-Aldrich, catalog number A0170, 1:10000 dilution) was added, and the reaction was incubated at 37°C for 1 hour. After washing, TMB solution was added, and the reaction was incubated at room temperature in the dark for 15 minutes. The reaction was then terminated by adding ELISA stop solution. The absorbance was measured at 450 nm using a microplate reader. The results are as follows: Figure 1A As shown, VH12VL9 and VH13VL9 exhibit good binding activity to human ROR1 protein, with EC50 values ​​of 0.10 nM and 0.09 nM, respectively.

[0094] The binding activity between the humanized antibody and the ROR1 homologous protein ROR2 was detected using the ELISA method described in this embodiment. The ROR2 protein (manufacturer: Kaika Biotechnology, catalog number: ROR-HM402) coating concentration was 1 μg / mL, and the antibody concentration started at 133.33 nM, diluted 4-fold, for a total of 8 concentrations. An anti-ROR2 antibody (manufacturer: Abcam, catalog number: ab190145) was used as a positive control. Results are as follows: Figure 1B As shown, VH12VL9 and VH13VL9 do not bind to human ROR2 protein, indicating that VH12VL9 and VH13VL9 specifically recognize human ROR1 protein.

[0095] Example 4: Binding of humanized antibodies to MDA-MB-231 cells

[0096] Flow cytometry (FACS) was used to detect the binding of humanized antibodies to the human breast cancer cell line MDA-MB-231 (source: Shanghai Cell Bank) endogenously expressing ROR1. MDA-MB-231 cells were incubated with different concentrations of antibody (starting at 66.67 nM, 6-fold dilution, for a total of 8 concentrations) at 4°C for 30 minutes. After washing the cells twice with 2% BSA-PBS, PE-labeled goat anti-human IgG Fc (Invitrogen, catalog number: 12-4998-82, 1:100 dilution) was added, and the reaction was carried out at 4°C in the dark for 30 minutes. After washing the cells twice with 2% BSA-PBS, the median fluorescence intensity was detected by flow cytometry using a BD C6 plus instrument. The results are as follows: Figure 2 As shown, VH12VL9 and VH13VL9 can bind to ROR1 expressed on MDA-MB-231 cells, with EC50 values ​​of 0.44 nM and 0.41 nM, respectively.

[0097] Example 5: Affinity of humanized antibody to ROR1

[0098] The affinity of humanized antibodies for human ROR1 was measured using an OctetR8 (Sartorius) instrument and an AHC (Anti-hIgG Fc Capture) biosensor (Sartorius) as the sensor. The antibody was captured, and the sensor was then immersed in the ROR1 antigen analyte. The experiment consisted of five steps: 1. Baseline (60s), 2. Loading (antibody capture) (150s, 1.5nm), 3. Baseline (100s), 4. Association (binding antigen ROR1, 60s), 5. Dissociation (dissociating antigen ROR1, 60s). After the test, the sensor was regenerated by alternating immersion in regeneration buffer (glycine, pH 1.5) and neutralization buffer (PBS) for 5 seconds each, for a total of three cycles. PBS was used as the running buffer in this experiment.

[0099] The control antibody UC961 was expressed according to patent CN111587124A (SEQ ID NO:3 and SEQ ID NO:4). Antibodies VH12VL9, VH13VL9, and UC961 were diluted to a working concentration of 10 μg / mL using running buffer. The antigen ROR1 protein (manufacturer: Kaika Biotechnology, catalog number: ROR-HM401) was serially diluted to five working concentrations: 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. Data analysis was performed using Octet Analysis Studio CFR (version 12.2.2.26) to calculate response signal values ​​(signal of the coupled analyte sample minus the signal of the blank analyte sample), and a 1:1 binding model was used to fit the data. The results showed that the affinity constants of VH12VL9, VH13VL9, and UC961 were 4.63 × 10⁻⁶. -9 M, 3.90×10 -9 M and 2.64×10 -8 M indicates that VH12VL9 and VH13VL9 have a higher affinity for the ROR1 protein compared to UC961.

[0100] Table 2 Sequence Comparison Table

[0101]

Claims

1. An anti-ROR1 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 respectively; and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6 or 17, SEQ ID NO:7 and SEQ ID NO:8 respectively.

2. The anti-ROR1 antibody or its antigen-binding fragment according to claim 1, wherein, The anti-ROR1 antibody or its antigen-binding fragment is a rabbit-derived antibody or a chimeric antibody.

3. The anti-ROR1 antibody or its antigen-binding fragment according to claim 2, wherein, The amino acid sequence of the heavy chain variable region of the anti-ROR1 antibody or its antigen-binding fragment has at least 95% sequence identity with SEQ ID NO:15; the amino acid sequence of the light chain variable region has at least 95% sequence identity with SEQ ID NO:

16.

4. The anti-ROR1 antibody or its antigen-binding fragment according to claim 2, wherein, The amino acid sequence of the heavy chain variable region of the anti-ROR1 antibody or its antigen-binding fragment has at least 96% sequence identity with SEQ ID NO:15; the amino acid sequence of the light chain variable region has at least 96% sequence identity with SEQ ID NO:

16.

5. The anti-ROR1 antibody or its antigen-binding fragment according to claim 2, wherein, The amino acid sequence of the heavy chain variable region of the anti-ROR1 antibody or its antigen-binding fragment has at least 97% sequence identity with SEQ ID NO:15; the amino acid sequence of the light chain variable region has at least 97% sequence identity with SEQ ID NO:

16.

6. The anti-ROR1 antibody or its antigen-binding fragment according to claim 2, wherein, The amino acid sequence of the heavy chain variable region of the anti-ROR1 antibody or its antigen-binding fragment has at least 98% sequence identity with SEQ ID NO:15; the amino acid sequence of the light chain variable region has at least 98% sequence identity with SEQ ID NO:

16.

7. The anti-ROR1 antibody or its antigen-binding fragment according to claim 2, wherein, The amino acid sequence of the heavy chain variable region of the anti-ROR1 antibody or its antigen-binding fragment has at least 99% sequence identity with SEQ ID NO:15; the amino acid sequence of the light chain variable region has at least 99% sequence identity with SEQ ID NO:

16.

8. The anti-ROR1 antibody or its antigen-binding fragment according to claim 2, wherein, The amino acid sequence of the heavy chain variable region of the anti-ROR1 antibody or its antigen-binding fragment is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

9. The anti-ROR1 antibody or its antigen-binding fragment according to claim 1, wherein, The anti-ROR1 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

10. The anti-ROR1 antibody or its antigen-binding fragment according to claim 9, wherein, The amino acid sequence of the heavy chain variable region has at least 95% sequence identity with SEQ ID NO:1 or 11, and the amino acid sequence of the light chain variable region has at least 95% sequence identity with SEQ ID NO:

5.

11. The anti-ROR1 antibody or its antigen-binding fragment according to claim 9, wherein, The amino acid sequence of the heavy chain variable region has at least 96% sequence identity with SEQ ID NO:1 or 11, and the amino acid sequence of the light chain variable region has at least 96% sequence identity with SEQ ID NO:

5.

12. The anti-ROR1 antibody or its antigen-binding fragment according to claim 9, wherein, The amino acid sequence of the heavy chain variable region has at least 97% sequence identity with SEQ ID NO:1 or 11, and the amino acid sequence of the light chain variable region has at least 97% sequence identity with SEQ ID NO:

5.

13. The anti-ROR1 antibody or its antigen-binding fragment according to claim 9, wherein, The amino acid sequence of the heavy chain variable region has at least 98% sequence identity with SEQ ID NO:1 or 11, and the amino acid sequence of the light chain variable region has at least 98% sequence identity with SEQ ID NO:

5.

14. The anti-ROR1 antibody or its antigen-binding fragment according to claim 9, wherein, The amino acid sequence of the heavy chain variable region has at least 99% sequence identity with SEQ ID NO:1 or 11, and the amino acid sequence of the light chain variable region has at least 99% sequence identity with SEQ ID NO:

5.

15. The anti-ROR1 antibody or its antigen-binding fragment according to claim 9, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1 or 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

16. The anti-ROR1 antibody or its antigen-binding fragment according to any one of claims 2 to 15, wherein, The anti-ROR1 antibody or its antigen-binding fragment contains a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and / or contains a light chain constant region of human κ or λ chain.

17. The anti-ROR1 antibody or its antigen-binding fragment according to claim 16, wherein, The heavy chain constant region is the IgG1 heavy chain constant region, and its amino acid sequence is shown in SEQ ID NO:

13. The light chain constant region is the κ light chain constant region, and its amino acid sequence is shown in SEQ ID NO:

14.

18. The anti-ROR1 antibody or its antigen-binding fragment according to claim 17, wherein, The sequence of the heavy chain has at least 95% sequence identity with SEQ ID NO: 9 or 12; and the sequence of the light chain has at least 95% sequence identity with SEQ ID NO:

10.

19. The anti-ROR1 antibody or its antigen-binding fragment according to claim 17, wherein, The sequence of the heavy chain has at least 96% sequence identity with SEQ ID NO: 9 or 12; and the sequence of the light chain has at least 96% sequence identity with SEQ ID NO:

10.

20. The anti-ROR1 antibody or its antigen-binding fragment according to claim 17, wherein, The sequence of the heavy chain has at least 97% sequence identity with SEQ ID NO: 9 or 12; and the sequence of the light chain has at least 97% sequence identity with SEQ ID NO:

10.

21. The anti-ROR1 antibody or its antigen-binding fragment according to claim 17, wherein, The sequence of the heavy chain has at least 98% sequence identity with SEQ ID NO: 9 or 12; and the sequence of the light chain has at least 98% sequence identity with SEQ ID NO:

10.

22. The anti-ROR1 antibody or its antigen-binding fragment according to claim 17, wherein, The sequence of the heavy chain has at least 99% sequence identity with SEQ ID NO: 9 or 12; and the sequence of the light chain has at least 99% sequence identity with SEQ ID NO:

10.

23. The anti-ROR1 antibody or its antigen-binding fragment according to claim 17, wherein, The sequence of the heavy chain is shown in SEQ ID NO:9 or 12, and the sequence of the light chain is shown in SEQ ID NO:

10.

24. The anti-ROR1 antibody or its antigen-binding fragment according to any one of claims 1-15 or 17-23, wherein, The antigen-binding fragments are selected from Fab, Fv, scFv, F(ab')2 and dsFv.

25. A biomaterial selected from any one of (1)-(3): (1) A nucleic acid molecule encoding an anti-ROR1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 24; (2) A carrier containing the nucleic acid molecule described in (1); (3) A host cell containing the nucleic acid molecule described in (1) or the carrier described in (2).

26. The biomaterial according to claim 25, wherein, The carrier is an expression carrier.

27. A composition comprising an anti-ROR1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 24, or a biological material as described in claim 25 or 26.

28. The composition according to claim 27, wherein, The composition is a drug and further comprises a pharmaceutically acceptable carrier or excipient.

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