Igf1r antibodies and uses thereof

Abstract

This disclosure relates to IGF1R antibodies and their applications. Specifically, this disclosure relates to antibodies that bind to IGF1R or antigen-binding fragments thereof, and the use of such antibodies or antigen-binding fragments in the diagnosis, prevention, or treatment of IGF1R-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to antibodies or antigen-binding fragments targeting IGF1R and their applications. Background Technology

[0002] Thyroid ophthalmopathy (TED), also known as Graves' eye disease, is an autoimmune inflammatory disease of the orbit and surrounding tissues characterized by upper eyelid retraction, eyelid lag, swelling, redness, conjunctivitis, and proptosis. It is most common in patients with Graves' disease and less common in patients with thyroiditis or those with normal thyroid function. It is part of a systemic process with varying expression in the eye, thyroid, and skin, caused by autoantibodies that bind to these organs and tissues. These autoantibodies target fibroblasts in the eye muscles, which can differentiate into adipocytes. The adipocytes and muscles swell and become inflamed, veins are compressed, and fluid cannot drain, leading to edema.

[0003] Insulin-like growth factor 1 receptor (IGF1R) belongs to the tyrosine protein kinase receptor family and is a transmembrane protein on the cell surface. It can be activated by IGF-1 and IGF-2 (both insulin-like growth factors), and its overexpression may be associated with the pathogenesis of malignant tumors such as multiple sclerosis, Crohn's disease, and pulmonary fibrosis, as well as autoimmune diseases. Anti-IGF-1R antibodies can be detected in most patients with Graves' disease, but are rarely found in normal individuals. IgG (Graves-associated immunoglobulin G) isolated from the serum of patients with Graves' disease can replace IGF-1R in binding to sites on the surface of orbital fibroblasts. IGF-1R or Graves' disease-associated IgG can activate IGF-1R-positive orbital fibroblasts from patients with thyroid-associated ophthalmopathy, thereby activating Akt / FRAP / mTOR / P70s6k channels to induce the expression of interleukin-16 and factors that regulate the expression and secretion of activated normal T cells on orbital fibroblasts, promote the synthesis of T cell chemokines, and cause inflammatory infiltration of T lymphocytes and the production of hyaluronic acid. All of these suggest that IGF-1R may participate in the development of Graves' disease as a secondary antigen.

[0004] However, there is still a significant unmet need for treatment of TED patients and IGF1R-related symptoms. Summary of the Invention

[0005] To address one of the aforementioned technical problems in the prior art, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to IGF1R. The disclosed antibody or antigen-binding fragment can block the binding of IGF1R to its ligands IGF-1 / IGF-2. This disclosure also provides the antibody or antigen-binding fragment thereof for the treatment or prevention of tumors and thyroid eye diseases.

[0006] According to one aspect of this disclosure, an antibody or antigen-binding fragment thereof that binds to IGF1R is provided. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a complementarity-determining region (VH-CDR) of a heavy chain variable domain, comprising: VH-CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, VH-CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and VH-CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and a complementarity-determining region (VL-CDR) of a light chain variable domain, comprising: VL-CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, VL-CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and VL-CDR3 having an amino acid sequence as shown in SEQ ID NO: 6, wherein any one of the amino acid sequences in the CDR sequence optionally includes an amino acid sequence that has been added, deleted, modified, and / or substituted with 1 to 3 amino acid residues and retains IGF1R binding activity.

[0007] In some embodiments, the antibody or its antigen-binding fragment specifically binds to IGF1R. In some embodiments, the antibody or its antigen-binding fragment specifically binds to IGF1R or fragments thereof derived from humans, mice, and cynomolgus monkeys. In some embodiments, the antibody or its antigen-binding fragment can block the binding of IGF1R to its ligands IGF-1 / IGF-2, thereby inhibiting the activation of downstream signaling pathways.

[0008] In some embodiments, the heavy chain variable domain of the antibody or its antigen-binding fragment may include frame regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4. In some embodiments, the light chain variable domain of the antibody or its antigen-binding fragment may include frame regions VL-FR1, VL-FR2, VL-FR3, and VL-FR4.

[0009] In some embodiments, at least a portion of the VH-FR1~4 and / or VL-FR1~4 are derived from antibodies or mutants of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, and geese. In some embodiments, at least a portion of the VH-FR1~4 and / or VL-FR1~4 are derived from antibodies or mutants of mice, rats, lemurs, macaques, chimpanzees, or humans, preferably from human antibodies or mutants.

[0010] In some embodiments, the VH-FR1 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 9.

[0011] In some embodiments, the VH-FR2 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 10.

[0012] In some embodiments, the VH-FR3 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 11.

[0013] In some embodiments, the VH-FR4 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12.

[0014] In some embodiments, the VL-FR1 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 13.

[0015] In some embodiments, the VL-FR2 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 14.

[0016] In some embodiments, the VL-FR3 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 15.

[0017] In some embodiments, the VL-FR4 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0018] In some embodiments, the VH-FR3 includes one or more mutations of M4L, R6S, T8K, and I10S compared to the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the VH-FR3 includes the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0019] In some embodiments, the VL-FR2 includes one or more mutations of Q4E, K8Q, and L12V compared to the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the VL-FR2 includes the amino acid sequence shown in WYQEKPGQVPKVLIY (SEQ ID NO: 18) or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0020] In some embodiments, the heavy chain variable region may include an amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 19, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0021] In some embodiments, the light chain variable region may include an amino acid sequence as shown in SEQ ID NO: 8 or SEQ ID NO: 20, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0022] In some embodiments, the antibody or its antigen-binding fragment may further include a heavy chain constant region and / or a light chain constant region. In some embodiments, at least a portion of the heavy chain constant region and / or the light chain constant region may be derived from antibodies or mutants of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese. In some embodiments, at least a portion of the heavy chain constant region and / or the light chain constant region is derived from antibodies or mutants of mice, rats, lemurs, macaques, chimpanzees, or humans, preferably from human antibodies or mutants.

[0023] In some embodiments, at least a portion of the heavy chain constant region comprises a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM immunoglobulins. In some embodiments, at least a portion of the light chain constant region comprises a light chain constant region derived from κ and λ immunoglobulins.

[0024] In some embodiments, the antigen-binding fragment of the antibody may include, but is not limited to, Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, single-chain Fv (scFv), dsFv or Fd fragment.

[0025] In some embodiments, the antibody or its antigen-binding fragment may include a heavy chain and / or a light chain.

[0026] In some embodiments, the heavy chain may include an amino acid sequence as shown in SEQ ID NO: 23 or SEQ ID NO: 28, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0027] In some embodiments, the light chain may include an amino acid sequence as shown in SEQ ID NO: 24 or SEQ ID NO: 29, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0028] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith; and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 24, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith.

[0029] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 28, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith; and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 29, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity therewith.

[0030] According to another aspect of this disclosure, a chimeric antigen receptor is provided, the chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain and an intracellular signal transduction domain, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof that binds to IGF1R according to this disclosure.

[0031] According to another aspect of this disclosure, an isolated nucleic acid molecule is provided that encodes the antibody or antigen-binding fragment thereof that binds to IGF1R as disclosed herein, or the chimeric antigen receptor described above in this disclosure.

[0032] According to another aspect of this disclosure, a carrier is provided that includes the nucleic acid molecules described above.

[0033] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include eukaryotic cell expression vectors and / or prokaryotic cell expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0034] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0035] According to another aspect of this disclosure, a cell is provided, said cell comprising the nucleic acid molecule of this disclosure, the antibody or antigen-binding fragment thereof that binds to IGF1R of this disclosure, the chimeric antigen receptor of this disclosure, and the nucleic acid molecule of this disclosure.

[0036] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the IGF1R-binding antibody or its antigen-binding fragment, chimeric antigen receptor, or multispecific antibody or its antigen-binding fragment as disclosed above. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells.

[0037] In some embodiments, the cells may be immune cells. In some embodiments, the immune cells may include, but are not limited to, monocytes (e.g., T cells) and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptors described above in this disclosure.

[0038] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising the nucleic acid molecule of this disclosure, the antibody or antigen-binding fragment thereof that binds to IGF1R of this disclosure, the chimeric antigen receptor of this disclosure, the multispecific antibody or antigen-binding fragment thereof of this disclosure, and the cell of this disclosure. In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0039] In some embodiments, the pharmaceutical composition can be used to prevent or treat IGF1R-related diseases. In some embodiments, IGF1R-related diseases include thyroid eye disease and tumors. In some embodiments, the tumors may include, but are not limited to, prostate cancer, nervous system tumors (e.g., glioma), malignant melanoma, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, liver cancer, lymphoma, pancreatic cancer, Ewing sarcoma, and cervical cancer.

[0040] According to another aspect of this disclosure, a product is provided comprising the antibody or antigen-binding fragment thereof that binds to IGF1R as described above, wherein the product comprises at least one of reagents, chips, test strips, and detection kits. In some embodiments, the product can be used for: 1) detecting the presence or amount of IGF1R in a sample; 2) diagnosing or prognostically assessing IGF1R-related diseases; or, 3) inhibiting IGF-1 and / or IGF-2-induced cell proliferation.

[0041] In some embodiments, the IGF1R-related diseases include thyroid eye disease and tumors. In some embodiments, the tumors may include, but are not limited to, prostate cancer, nervous system tumors (e.g., glioma), malignant melanoma, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, liver cancer, lymphoma, pancreatic cancer, Ewing sarcoma, and cervical cancer.

[0042] In some embodiments, the antibodies or antigen-binding fragments of the IGF1R described above in this disclosure may be conjugated to detectable markers.

[0043] In some embodiments, the detectable marker may be a fluorescent or luminescent marker. In some embodiments, the detectable marker may be selected from, for example, any one of acridine ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase, and alkaline phosphatase.

[0044] According to another aspect of this disclosure, the use of the antibody or antigen-binding fragment thereof that binds to IGF1R, the nucleic acid molecule, the expression vector, the cell, or the pharmaceutical composition thereof in the preparation of a medicament for the prevention or treatment of IGF1R-related diseases is provided.

[0045] According to another aspect of this disclosure, a method for preventing or treating IGF1R-related diseases is provided, the method comprising administering to a subject in need a therapeutically effective amount of the aforementioned IGF1R-binding antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned cells, or the aforementioned pharmaceutical composition.

[0046] In some embodiments, the IGF1R-related diseases include thyroid eye disease and tumors. In some embodiments, the tumors may include, but are not limited to, prostate cancer, nervous system tumors (e.g., glioma), malignant melanoma, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, liver cancer, lymphoma, pancreatic cancer, Ewing sarcoma, and cervical cancer. Attached Figure Description

[0047] Figure 1 The binding activity of the antibody of this disclosure to human IGF1R is shown.

[0048] Figure 2 The binding activity of the antibody disclosed herein with monkey IGF1R is demonstrated.

[0049] Figure 3 The binding activity of the antibody disclosed herein with human IGF1R overexpressing cells is demonstrated.

[0050] Figure 4 The binding activity of the antibody disclosed herein with monkey IGF1R overexpressing cells is demonstrated.

[0051] Figure 5 The antibody disclosed herein demonstrates its blocking activity against ligand IGF1 and human IGF1R overexpressing cells.

[0052] Figure 6 The inhibitory effect of the antibodies disclosed herein on the proliferation of IGF-1 (A) and IGF-2 (B) induced by MCF-7 cells is shown.

[0053] Figure 7 The antibody-mediated endocytosis activity of this disclosure is demonstrated. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0055] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0056] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0057] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0058] As used in this article, "antibody" refers to a globulin produced by plasma cells, which are formed from the proliferation and differentiation of B lymphocytes in response to antigen stimulation. Antibodies specifically bind to the corresponding antigens and mediate immune effects. They are mainly found in serum and body fluids and are important immune molecules mediating humoral immunity. Antibodies can encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, single-chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulin (Ig).

[0059] As used herein, the term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that, apart from possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies within the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0060] The terms “full-length antibody” and “intact antibody” as used herein are used interchangeably to refer to antibodies that are structurally similar to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant regions). The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: k-light chains and λ-light chains.

[0061] Within the light and heavy chains, variable and constant regions are linked by a "J" region containing approximately 12 or more amino acid residues, and the heavy chain also contains a "D" region containing approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates 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.

[0062] As used herein, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR1-4) and three hypervariable regions (HVR1-3), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain is sufficient to confer antigen-binding specificity. The three HVRs within the VH and VL domains together constitute the antigen-binding site of Ig, which can bind complementary to the corresponding antigenic epitope; therefore, the HVRs are also called complementarity-determining regions (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The VH or VL chain of the antibody may further contain all or part of the constant regions of the heavy or light chain.

[0063] As used herein, the term "variable" refers to the fact that certain segments of the variable domain are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable domain, but is concentrated in three segments called hypervariable regions (HVRs) within the variable domains of the light and heavy chains. The relatively highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are tightly held together by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. The constant domain does not directly participate in antibody-antigen binding but has other effector functions, such as participating in antibody-dependent cytotoxicity.

[0064] As used herein, the term "framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences typically appear in the VH (or VL) in the following sequence: VH(VL)FR1-VH(VL)CDR1-VH(VL)FR2-VH(VL)CDR2-VH(VL)FR3-VH(VL)CDR3-VH(VL)FR4.

[0065] In a specific embodiment, the antibody or antigen-binding fragment of the present disclosure that specifically binds to IGF1R includes heavy chain CDR1-3 and light chain CDR1-3 as shown in Table # below, wherein any one or more of heavy chain CDR1-3 and light chain CDR1-3 includes an amino acid sequence having 1, 2 or 3 conserved amino acid substitutions compared to the amino acid sequence shown in Table 1.

[0066] Table 1. Amino acid sequence of the CDR region of anti-IGF1R antibody (defined according to the Kabat numbering system).

[0067] In specific embodiments, the antibody or antigen-binding fragment of the present disclosure that specifically binds to IGF1R may be a humanized antibody or antigen-binding fragment of the present disclosure. In such embodiments, the antibody or antigen-binding fragment of the present disclosure that specifically binds to IGF1R includes: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0068] Table 2. Amino acid sequence of the heavy chain variable region of B145 CDR transplantation antibody.

[0069] In specific embodiments, the antibodies or antigen-binding fragments of the present disclosure that specifically bind to IGF1R include heavy chains VH-FR1, VH-FR2, VH-FR3 and / or VH-FR4, each comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with VH-FR1, VH-FR2, VH-FR3 and / or VH-FR4 as shown in Table 3 below.

[0070] In specific embodiments, the antibodies or antigen-binding fragments of the present disclosure that specifically bind to IGF1R include heavy chains VL-FR1, VL-FR2, VL-FR3 and / or VL-FR4, each comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with VL-FR1, VL-FR2, VL-FR3 and / or VL-FR as shown in Table 3 below.

[0071] Table 3. FR region of antibody B145.

[0072] In a preferred embodiment, one or more amino acid residues are selected and reverse-mutated in the framework region of the heavy chain variable region and / or the light chain variable region to mutate human amino acid residues into mouse amino acid residues in order to maintain the affinity and biological activity of the humanized antibody.

[0073] In such an embodiment, the VH-FR3 includes one or more mutations of M4L, R6S, T8K, and I10S compared to the amino acid sequence shown in SEQ ID NO: 11. In such an embodiment, the heavy chain VH-FR3 includes the amino acid sequence shown in SEQ ID NO: 17.

[0074] In one embodiment, the VL-FR2 includes one or more mutations of Q4E, K8Q, and L12V compared to the amino acid sequence shown in SEQ ID NO: 14. In another embodiment, the VL-FR2 includes the amino acid sequence shown in WYQEKPGQVPKVLIY (SEQ ID NO: 18).

[0075] Antibody "classes" refer to the types of constant structural domains or constant regions possessed by the antibody's heavy chain. Based on differences in heavy chain structure and antigenicity, they can be classified into five classes: μ chain, γ chain, α chain, δ chain, and ε chain. Immunoglobulins composed of different heavy and light chains are respectively called IgA, IgD, IgE, IgG, and IgM. Even within the same class of Ig, the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain differ, thus further subdividing the same class of Ig into different subclasses. For example, human IgG can be divided into IgG1–IgG4; IgA can be divided into IgA1 and IgA2. Based on differences in light chain structure and antigenicity, immunoglobulin (Ig) light chains are divided into κ (kappa) chains and λ (lambda) chains, thus classifying Ig into two types: κ type and λ type.

[0076] "Humanized antibodies" comprise amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0077] "Humanized antibodies" have an amino acid sequence that corresponds to that of antibodies produced by humans or human cells, or derived from non-human antibodies using sequences encoded by human antibody libraries or other human antibodies. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0078] As used herein, the term "Fc domain" or "Fc region" is used to define a C-terminal region of an antibody heavy chain containing at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. An IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The CH2 domain in this document may be a native sequence CH2 domain or a variant CH2 domain. The CH3 region in this document may be a native sequence CH3 domain or a variant CH3 domain. The CH2 domain may contain one or more mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIII) and / or complement.

[0079] "Regions equivalent to the Fc region of immunoglobulins" include variants of the naturally occurring alleles of the Fc region of immunoglobulins, as well as modified variants that have the ability to produce substitutions, additions, or deletions that substantially do not diminish the function of immunoglobulins-mediated effectors, such as antibody-dependent cytotoxicity. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially losing its biological function. Such variants can be selected according to general rules known in the art to minimize the impact on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0080] As used in this article, the term "effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0081] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0082] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody that retains the antibody's antigen-binding activity. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0083] As used herein, the terms "antigen-binding domain" or "antigen-binding site" refer to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0084] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformation composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used as antigens in this invention can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Antigens can also be human proteins, or antigens can be "full-length," unprocessed proteins, and any form of protein produced by intracellular processing, or naturally occurring protein variants, such as splice variants or allelic variants.

[0085] The specific “binding strength” or “affinity” of an antibody or its antigen-binding fragment to an antigen refers to the strength of the non-covalent interaction between a single binding site and its binding ligand (e.g., antigen), and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. Binding affinity is typically expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (Kd and Ka, respectively). In some embodiments, the dissociation constant (Kd) of the molecule binding to the antigen is ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10).-7 M or lower, such as 10 -7 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0086] As used herein, the term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its natural environment. In this invention, the recombinant polynucleotide encoding a polypeptide contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids of this disclosure further include synthetically generated molecules of this type. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0087] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably to refer to a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes vectors that function as self-replicating nucleic acid structures and vectors incorporated into the genome of the host cell into which they have been introduced. The expression vectors of the present invention comprise expression cassettes. Expression vectors can be transcribed into large quantities of stable mRNA. Once the expression vector is within the target cell, cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding a fragment of the bispecific antigen-binding molecule or thereof of the present invention. The term "expression cassette" of the present invention refers to a recombinant or synthetically generated polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a bispecific antigen-binding molecule or thereof of the present invention.

[0088] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the bispecific antigen-binding molecule of this invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0089] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a receptor having desired antigen specificity and signal transduction domains to propagate intracellular signals upon antigen binding. For example, T lymphocytes recognize specific antigens via the interaction of T cell receptors (TCRs) with short peptides presented by class I or II major histocompatibility complex (MHC) molecules. For initial activation and clonal expansion, naïve T cells depend on antigen-presenting cells (APCs) that provide additional co-stimulatory signals. In some embodiments, monocytes and macrophages can be engineered to express, for example, chimeric antigen receptors (CARs). Modified cells can be recruited to the tumor microenvironment, where they act as potent immune effectors by infiltrating the tumor and killing target cancer cells. CARs may include antigen-binding domains, transmembrane domains, and intracellular domains. The antigen-binding domain binds to the antigen on the target cell. Examples of cell surface markers that can be used as antigens binding to the antigen-binding domain of a CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (e.g., tumor antigens).

[0090] As used herein, the term "modified immune cell" refers to an immune cell that has been genetically modified to express a CAR. In some embodiments, the immune cell is a T cell or a cell derived therefrom. In some embodiments, the immune cell is a natural killer (NK) cell or a cell derived therefrom. In some embodiments, the immune cell is a B cell or a cell derived therefrom. In some embodiments, the immune cell is a B cell or a cell derived therefrom. In some embodiments, the immune cell is a monocyte or macrophage, or a cell derived therefrom.

[0091] An "effective amount" of a drug is the amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes the amount sufficient to improve or prevent the symptoms or signs of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of any side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0092] The "therapeutic effective amount" of a drug (such as a pharmaceutical composition) refers to the amount necessary to effectively achieve the desired therapeutic or preventive effect in terms of dosage, dosing intervals, and time. For example, a therapeutically effective amount of a drug eliminates, mitigates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0093] As used herein, the term “individual” or “subject” refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human being.

[0094] As used herein, the term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments of the present disclosure, along with other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0095] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0096] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of this disclosure or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, to a patient having one or more IGF1R-related diseases or symptoms, the therapeutic agent having a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms, or to inhibit the development of such symptoms to any clinically measurable extent.

[0097] As used herein, the term "prevention" refers to delaying, suppressing, or preventing the onset of IGF1R-related diseases in mammals where the initiation of cancer or tumorigenesis has not been confirmed, but a susceptibility to cancer has been identified, for example, through genetic screening or other methods. The term also includes treating mammals with precancerous lesions to halt the progression of the precancerous lesions to malignancy or to induce their regression.

[0098] As used herein, the term "detectable marker" encompasses a marker that can be detected directly or indirectly, either attached to the antibody or the bispecific binding protein, or present independently in the kit. Suitable markers include, but are not limited to, molecules detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Suitable markers include, but are not limited to, fluorescent dyes (e.g., GFT and its variants, FITC, TRITC, fluorescein, and rhodamine, etc.), electron-dense reagents (e.g., gold), enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucosyl amylase), molecules containing radionuclides (i.e., radioisotopes), chemiluminescent molecules, electrochemiluminescent molecules, biotin, digoxin / digoxigenin, or haptens, and other entities that are or can be detectable. Antibodies or their antigen-binding fragments or bispecific binding proteins in this disclosure are attached to a "detectable marker," thus being detectably labeled.

[0099] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0100] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0101] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0102] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0103] Example Example 1: Animal Immunization Purchased Human IGF1R ECD-His protein (ACRO, IGR-H5229) was mixed with an equal volume of Freund's complete adjuvant. Ten Balb / c mice were subcutaneously injected with 50 μg of protein per mouse for primary immunization. Booster immunizations were then performed every two weeks by subcutaneous injection of 25 μg of human IGF1R protein mixed with an equal volume of Freund's incomplete adjuvant. Seven to ten days after the third booster immunization, the mice were bled and serum was collected. The titer of human IGF1R protein bound to the immune serum was measured using ELISA. One or more mice with the highest titer were selected and intraperitoneally injected with 50 μg of human IGF1R protein (mixed with an equal volume of Freund's complete adjuvant) for pulse immunization.

[0104] Example 2: Screening for anti-IGF1R antibodies Mice that underwent shock immunization in Example 1 were sacrificed, and their spleens were harvested. RNA was extracted from the ground spleen, and then analyzed using SMARTScribe. TM Reverse transcriptase (Takara) was used to reverse transcribe the DNA into cDNA, which was then used to construct an antibody display library using phage display technology. IGF1R was coated onto a solid-phase vector, and the obtained antibody display library was subjected to positive panning. Positive clones were sequenced and validated by recombinant expression, and the murine antibody mB145 was screened out.

[0105] Example 3: Preparation of humanized antibodies 3.1 Humanized Antibody Design The CDR region of the murine antibody mB145 screened in Example 2 was transplanted onto the template sequence to form a CDR transplanted antibody, labeled as B145-H0L0. Its amino acid sequence is as follows, where B145-H0 is the heavy chain variable region sequence and B145-L0 is the light chain variable region sequence.

[0106] >The amino acid sequence of the heavy chain variable region of the B145 CDR transplanted antibody (B145-H0) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGFFTPYNDGTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTVVYYCARGKRGNYGSSWYFDVWGQGTTVTVSS (SEQ ID NO: 7) >Amino acid sequence of the light chain variable region of the B145 CDR transplanted antibody (B145-L0) DIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKVPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYNSYPWTFGGGTKVEIK (SEQ ID NO: 8) To maintain the affinity and biological activity of the humanized antibody, reverse mutation sequences were designed, resulting in the heavy chain variable region and light chain variable region shown in Table 4 below.

[0107] Table 4. Amino acid sequence of the variable region of humanized anti-IGF1R antibody B145.

[0108] Then, it is combined with the human IgG constant region to obtain a full-length humanized antibody. The human IgG heavy chain constant region can be selected from IgG1, IgG2, or IgG4 subtypes, and the light chain constant region can be selected from Kappa or lambda subtypes, or it can be replaced with other constant regions known in the art. In this embodiment, the heavy chain constant region is selected from IgG1 YTE, and the light chain constant region is selected from Kappa chain constant region to obtain a full-length humanized antibody named hB145. The constant region sequence is as follows.

[0109] >Amino acid sequence of the YTE heavy chain constant region of human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21) >Amino acid sequence of the light chain constant region of human Kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22) 3.2 Expression and purification of humanized antibodies The heavy chain variable region and light chain variable region of the obtained CDR transplantation antibody B145-H0L0 were ligated to the constant region to obtain the full-length heavy chain and light chain sequences (i.e., hB145), with amino acid sequences shown in SEQ ID NO: 23 and 24, respectively. The nucleic acid sequences encoding the full-length heavy chain and light chain sequences of this antibody were cloned into the pTT5 vector to construct the heavy chain and light chain expression plasmids pTT5-HC and pTT5-LC, respectively. Expi293F cells with a viability of over 95% were seeded at a density of 0.8E6 / mL in CD 293 TGE medium (ACROBiosystems) and cultured for 24 hours at 6% CO2, 130 rpm, and 37°C. The mass ratio of 1 mg / mL PEI transfection reagent to expression plasmid pTT5-HC+pTT5-LC (mixed in equal volumes) is 2:1. Taking the transfection of 200 mL Expi293F cells as an example, mix 10 mL of Opti-MEM with 200 μg of plasmid and let stand for 5 min; separately mix 10 mL of Opti-MEM with 400 μl of PEI and let stand for 5 min. Mix the plasmid mixture and PEI mixture and let stand at room temperature for 15 min. Slowly add the plasmid and PEI mixture to 200 mL of Expi293F cells and incubate in a shaker at 6% CO2, 130 rpm, and 37°C. 18-24 h after transfection, add 0.8% of the transfection volume of 250 mM sodium valproate solution. On days 1 and 3 after transfection, supplement with 5% of the volume of OptiVitro® 293 serum-free fed medium HA02 (ExCell Bio). On day 5 after transfection, cell supernatant was collected, and the supernatant was centrifuged at high speed and filtered to remove impurities for purification.

[0110] The supernatant was used to purify the recombinant antibody using a Protein A column. The column was washed with PBS buffer until the A280 reading dropped to baseline, and then the sample was loaded. The target protein was eluted with 100 mM glycine (pH 3.0), and 1 M Tris-HCl (pH 8.0) was added immediately for neutralization. The collected purified antibody was replaced with PBS, and the absorbance at 280 nm was measured to calculate the antibody concentration.

[0111] Example 4: Construction of a stable cell line overexpressing IGF1R receptor To evaluate the affinity of the antibody for the antigen IGF1R, HEK293 cell lines overexpressing human IGF1R (SEQ ID NO: 25), monkey IGF1R (SEQ ID NO: 26), and mouse IGF1R (SEQ ID NO: 27) were constructed.

[0112] In simple terms, the nucleic acid sequence encoding human IGF1R was cloned into the pCDNA3.1 (V79520, Invirogen) vector using a non-liposome transfection method, resulting in the pCDNA3.1(+)-CMV-MCS-NEO-huIGF1R plasmid. Then, using FuGENE® 6 transfection reagent (Promega, Madison, WI, USA), the expression plasmid was transfected into HEK293 cells according to the manufacturer's instructions. After two weeks of selection under a 1 mg / mL genimycin selection pressure, a monoclonal cell line highly expressing human IGF1R was selected by flow cytometry and named HEK293-huIGF1R cells. HEK293 cell lines overexpressing monkey IGF1R and mouse IGF1R were constructed using a similar method and named HEK293 cynoIGF1R cells and HEK293mIGF1R cells, respectively.

[0113] Example 5: Determination of anti-IGF1R antibody affinity 5.1 Assay of human IGF1R / monkey IGF1R binding activity based on ELISA The binding activity of the purified humanized antibody hB145 from Example 3 to the antigen protein human IGF1R / monkey IGF1R was detected by ELISA.

[0114] The specific steps are as follows: Recombinant human IGF1R (Sino Biological, 10164-H08H) or monkey IGF1R (ACROBiosystems, IGR-C5225) was diluted to 1 μg / mL using PBS buffer at pH 7.4. 100 μL of each solution was used to coat a 96-well microplate (Costar, 42592) and incubated overnight at 4 °C. The next day, the liquid in the wells was discarded, and the plate was washed three times with PBST (300 μL / well). Then, blocking buffer (PBST containing 3% BSA) was added to each well, and the plate was blocked at 37 °C for 2 h. After blocking, discard the liquid in the wells, wash the plate three times with PBST (300 μL / well), then add 100 μL of serially diluted antibody sample (initial concentration 10 μg / mL), incubate at 37 ℃ for 1 h, wash three times with PBST (300 μL / well), add 1:5000 diluted HRP-labeled goat anti-human IgG (H+L) secondary antibody (Proteintech), incubate at 37 ℃ for 1 h, wash four times with PBST (300 μL / well), and develop the color. Read the absorbance at 450 nm on a microplate reader, calculate the EC50 value of IGF1R antibody binding to human IGF1R / monkey IGF1R protein, and the results are as follows: Figures 1-2 The results are shown in Table 5 below. The results indicate that the antibodies disclosed herein exhibit excellent binding activity to both human IGF1R and monkey IGF1R. Teprotumumab (Teprotumumab Biosimilar) was used as a control antibody (abinvivo, B975501).

[0115] Table 5. EC50 values ​​of IGF1R antibody binding to human IGF1R and monkey IGF1R proteins.

[0116] 5.2 Determination of antibody affinity constant The affinity constants of the humanized antibody hB145 and the control antibody Teprotumumab for IGF1R were determined by surface plasmon resonance (SPR). Detection was performed using a Protein A chip (Cytiva, Cat. No.: 29127555) with a Biacore 8K instrument. The antibody was injected at a flow rate of 10 μL / min for 12 s. Different concentrations of human IGF1R protein (Sino Biological, 10164-H08H) were injected at a flow rate of 30 μL / min for 120 s, followed by monitoring for dissociation for 360 s. Finally, the chip was regenerated by flowing glycine (pH 1.7) at a flow rate of 30 μL / min for 30 s. Kinetic parameters were calculated using the 1:1 Binding model in the Bia-evaluation analysis software. The affinity constant, expressed as KD, was calculated from the kd / ka (dissociation rate / binding rate) ratio. The affinity assay results for the two antibodies are as follows: hB145 has an affinity approximately 10 times stronger than the control antibody.

[0117] Table 6. Affinity of IGF1R antibody to IGF1R protein.

[0118] Example 6: Detection of antibody binding activity to overexpressing cells The activity of the antibody in binding to HEK293 cells overexpressing IGF1R was evaluated using FACS. HEK293-huIGF1R cells or HEK293 cynoIGF1R cells constructed in Example 4 were collected by trypsin digestion and resuspended in PBS + 2% FBS wash buffer. The cell density was adjusted to 2E6 / mL, approximately 1E5 cells per well. The antibody sample was diluted to 60 μg / mL (final concentration 30 μg / mL) with wash buffer, followed by a 3-fold serial dilution. 50 μL of cells and 50 μL of antibody sample were added to each well sequentially, and the cells were incubated at 4°C for 30 min. The cells were centrifuged at 1000 rpm for 5 min in a 96-well plate, washed once with PBS + 2% FBS, and 100 μL of 1:100 diluted PE-Anti-His flow cytometry antibody (Biolegend, 362603) was added to each well. The cells were resuspended and mixed, and incubated at 4°C for 30 min. After incubation, the cells were centrifuged at 1000 rpm for 5 min in a 96-well plate, washed once with PBS + 2% FBS, and resuspended in 100 μL of PBS + 2% FBS wash buffer in each well. The signal values ​​were then measured using flow cytometry, and the EC50 values ​​were calculated. The results are as follows: Figures 3-4 As shown in Table 7 below. The results show that the antibody disclosed herein has good binding activity with both human and monkey IGF1R overexpressing cells.

[0119] Table 7. Binding activity of the antibodies disclosed herein with human or monkey IGF1R overexpressing cells.

[0120] Example 7: Assay of antibody activity in blocking the binding of IGF1 and IGF1R The activity of the anti-IGF1R antibody in blocking the binding of recombinant IGF-1 protein to HEK293 cells overexpressing IGF1R was further detected using FACS. HEK293-huIGF1R cells were trypsinized and collected, resuspended in PBS + 2% FBS, and the cell density was adjusted to 2E6 / mL, approximately 1E5 cells per well. The antibody sample was serially diluted 3-fold with wash buffer to 400 nM; the ligand huIGF1-His (Acrobiosystem, IG1-H5245) was diluted to 12 μg / mL (final concentration 3 μg / mL). A negative control (Neg) was used without the ligand huIGF1-His. Add 50 μL of cells, 25 μL of antibody sample, and 25 μL of ligand to each well sequentially, and incubate at 4 °C for 30 min. Centrifuge the 96-well plate at 1000 rpm for 5 min, wash once with PBS + 2% FBS, add 100 μL of 1:100 diluted PE-Anti-His flow cytometry antibody to each well, resuspend and mix the cells, and incubate at 4 °C for 30 min. After incubation, centrifuge the 96-well plate at 1000 rpm for 5 min, wash once with PBS + 2% FBS, and resuspend the cells in 100 μL of PBS + 2% FBS wash buffer per well. Detect the signal value of the PE channel using flow cytometry and calculate the IC50 value. The results are shown in Table 8 below. Figure 5 As shown in the figure. The results show that the antibodies disclosed in this invention have good blocking activity in human overexpression cells.

[0121] Table 8. Blocking activity of antibodies against IGF1 and human IGF1R overexpressing cells.

[0122] Example 8: Detection of the inhibitory activity of antibody against IGF-1 / IGF-2 induced MCF-7 cell proliferation Logarithmically growing human breast cancer MCF-7 cells were digested with trypsin, resuspended in RPMI 1640 medium (Gibco, C11875500BT) containing 10% fetal bovine serum, and seeded at a density of 4000 cells per well in 96-well cell culture plates. After overnight incubation in a CO2 incubator (37°C, 5% CO2), the medium was replaced with 50 μL of serum-free RPMI 1640 medium, and the cells were further incubated in a CO2 incubator for 5 hours. Five-fold serial dilutions of humanized antibody or control antibody solutions were prepared in RPMI 1640 medium containing 2% FBS and 20 ng / mL IGF-1 / IGF-2, with a maximum concentration of 133.33 nM (9 concentrations in total). 50 μL of each solution was added to the corresponding well of the 96-well plate. The cell culture plates were incubated in a CO2 incubator (37°C, 5% CO2) for 6 days, and cell viability was assessed using CyQUANT©. The data were analyzed using Graphpad Prism software, and the dose-response curve was obtained by nonlinear s-curve regression fitting. The IC50 value was then calculated, and the results are shown in Table 9 below. Figure 6 As shown in A~6B. The fitting results indicate that the antibodies disclosed in this paper have a strong inhibitory effect on the proliferation of MCF-7 cells induced by IGF-1 / IGF-2, and the inhibitory effect is higher than that of the positive control antibody.

[0123] Table 9. Inhibitory effect of antibodies on IGF-1 / IGF-2 induced proliferation of MCF-7 cells.

[0124] Example 9: Detection of endocytic activity in antibody-mediated IGF1R overexpressing cells HEK293-huIGF1R cells were digested with trypsin and collected. The cells were resuspended in PBS + 2% FBS wash buffer and the cell density was adjusted to 1E5 / mL. Approximately 5K cells per well were seeded into 96-well plates. Humanized antibody or control antibody solutions were prepared in DMEM medium (Gibco, C11995500BT) containing 10 μg / mL DT3C (CUSABIO, CSB-EP360556CQR1), with a maximum concentration of 33.33 nM (9 concentrations in total). 50 μL of each solution was added to the corresponding well of the 96-well plate. Cell culture plates were placed in a CO2 incubator (37℃, 5% CO2) for 72 h. Cell viability was detected using the Cell Counting Lite (Novazia, DD1101-02) assay. Data were analyzed using Graphpad Prism software, and a dose-response curve was obtained by nonlinear s-curve regression fitting. The IC50 value was then calculated. The results are shown in Table 10 below. Figure 7As shown in the figure. The fitting results indicate that the antibody disclosed herein mediates strong endocytic activity, which is significantly stronger than that of the control antibody.

[0125] Table 10. Antibody-mediated endocytosis activity.

[0126] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds IGF1R, characterized in that, The antibody or its antigen-binding fragment includes: The complementarity-determining region (VH-CDR) of the heavy chain variable domain includes: VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and The complementarity-determining region (VL-CDR) of the light chain variable domain includes: VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 having the amino acid sequence shown in SEQ ID NO:

6. The above CDR sequence may optionally include an amino acid sequence that has been added, deleted, modified and / or substituted with 1 to 3 amino acid residues and retains IGF1R binding activity.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment specifically binds to IGF1R, preferably to IGF1R or fragments derived from human, mouse, and cynomolgus monkeys.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The heavy chain variable domain of the antibody or its antigen-binding fragment includes frame regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4, and / or the light chain variable domain of the antibody or its antigen-binding fragment includes frame regions VL-FR1, VL-FR2, VL-FR3, and VL-FR4. Preferably, at least a portion of the frame region of the heavy chain variable structural domain and / or the frame region of the light chain variable structural domain are derived from antibodies or mutants of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, and geese, more preferably from antibodies or mutants of mice, rats, lemurs, macaques, chimpanzees, or humans, and more preferably from antibodies or mutants of humans. Preferably, the VH-FR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

9. Preferably, the VH-FR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

10. Preferably, the VH-FR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

11. Preferably, the VH-FR4 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

12. Preferably, the VL-FR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

13. Preferably, the VL-FR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

14. Preferably, the VL-FR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

15. Preferably, the VL-FR4 may include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

16.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, Compared to the amino acid sequence shown in SEQ ID NO: 11, the VH-FR3 includes one or more mutations of M4L, R6S, T8K, and I10S. Preferably, the VH-FR3 includes the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or Compared with the amino acid sequence shown in SEQ ID NO: 14, the VL-FR2 includes one or more mutations of Q4E, K8Q and L12V. Preferably, the VL-FR2 includes the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with it.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 19, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or The light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 8 or SEQ ID NO: 20, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it. Preferably, the antibody or its antigen-binding fragment comprises: 1) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, or 2) Heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 19, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 20, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region. Preferably, at least a portion of the heavy chain constant region and / or the light chain constant region is derived from antibodies or mutants of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, and geese; more preferably, from antibodies or mutants of mice, rats, lemurs, macaques, chimpanzees, or humans; and even more preferably, from antibodies or mutants of humans. Preferably, at least a portion of the heavy chain constant region includes a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM immunoglobulin. Preferably, at least a portion of the light chain constant region comprises a light chain constant region derived from κ and λ immunoglobulins. Preferably, the antigen-binding fragment of the antibody includes a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a single-chain Fv (scFv), a dsFv, or an Fd fragment.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain and / or a light chain, wherein, The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 23 or SEQ ID NO: 28, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or The light chain comprises an amino acid sequence as shown in SEQ ID NO: 24 or SEQ ID NO: 29, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it. Preferably, the antibody or its antigen-binding fragment comprises: 1) A heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 24, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, or 2) a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 28, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 29, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

8. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor includes an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain includes the antibody or antigen-binding fragment thereof that binds to IGF1R as described in any one of claims 1 to 7.

9. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof that binds to IGF1R as described in any one of claims 1 to 7, or the chimeric antigen receptor as described in claim 8.

10. A carrier, characterized in that, The carrier comprises the nucleic acid molecule of claim 9. Preferably, the carrier is an expression carrier.

11. A cell, characterized in that, The cell comprises an antibody or antigen-binding fragment thereof that binds to IGF1R as described in any one of claims 1 to 7, a chimeric antigen receptor as described in claim 8, or a nucleic acid molecule as described in claim 9.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that binds to IGF1R as described in any one of claims 1 to 7, a chimeric antigen receptor as described in claim 8, a nucleic acid molecule as described in claim 9, or a cell as described in claim 11.

13. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is used for the prevention or treatment of IGF1R-related diseases. More preferably, the IGF1R-related diseases include thyroid eye disease and tumors. More preferably, the tumors include prostate cancer, nervous system tumors, malignant melanoma, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, liver cancer, and lymphoma. Pancreatic cancer, Ewing sarcoma, and cervical cancer.

14. A product characterized in that, The product comprises an antibody or antigen-binding fragment thereof that binds to IGF1R as described in any one of claims 1 to 7, and the product comprises at least one of reagents, chips, test strips, and detection kits.

15. The product according to claim 14, characterized in that, The product can be used for: 1) Detect the presence or content of IGF1R in the sample; 2) Diagnostic or prognostic assessment of IGF1R-related diseases; or 3) Inhibits IGF-1 and / or IGF-2-induced cell proliferation. Preferably, the IGF1R-related diseases include thyroid eye disease and tumors. Preferably, the tumors include prostate cancer, nervous system tumors, malignant melanoma, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, liver cancer, lymphoma, pancreatic cancer, Ewing sarcoma, and cervical cancer. Preferably, the antibody or its antigen-binding fragment is conjugated to a detectable marker. Preferably, the detectable marker is a fluorescent or luminescent marker, more preferably selected from any one of acridine ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase, and alkaline phosphatase.

16. Use of any one of claims 1 to 7, the antibody or antigen-binding fragment thereof that binds to IGF1R, the chimeric antigen receptor of claim 8, the nucleic acid molecule of claim 9, the cell of claim 11, or the pharmaceutical composition of claim 12 or 13, in the preparation of a medicament for the prevention or treatment of IGF1R-related diseases.

17. The use according to claim 16, characterized in that, The diseases associated with IGF1R include thyroid eye disease and tumors. Preferably, the tumor includes prostate cancer, nervous system tumors, malignant melanoma, breast cancer, colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, ovarian cancer, liver cancer, lymphoma, pancreatic cancer, Ewing sarcoma, and cervical cancer.

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