Antibody targeting IGF-1r and use thereof

Abstract

Disclosed in the present invention are an antibody targeting IGF-1R and a use thereof. The antibody comprises a light chain variable region and a heavy chain variable region; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and amino acid sequences thereof are shown in SEQ ID NOs: 2, 3 and 4, respectively; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and amino acid sequences thereof are shown in SEQ ID NOs: 6, 7 and 8, respectively. The antibody has high specificity and high affinity, can significantly inhibit the phosphorylation of IGF-1R, blocks the binding of IGF-1 to human IGF-1R protein, exhibits a very low ADCC effect and favorable pharmacokinetic activity, and is suitable for efficient detection of IGF-1R and for the treatment of autoimmune diseases and tumors.

Description

An antibody targeting IGF-1R and its application

[0001] This application claims priority to Chinese Patent Application No. 2024111842702, filed on August 27, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of antibodies, specifically relating to an antibody targeting IGF-1R and its applications. Background Technology

[0003] The insulin-like growth factor 1 receptor (IGF-1R) is located on the surface of human cells. It is a transmembrane receptor activated by the hormone insulin-like growth factor 1 (IGF-1) and another related hormone insulin-like growth factor 2 (IGF-2). It regulates the metabolism, growth and differentiation of various mammalian cells and belongs to the tyrosine kinase (ATK) family.

[0004] After IGF-1R binds to its ligand, it leads to receptor autophosphorylation, which induces cell signal transduction and promotes cell growth and proliferation.

[0005] IGF-1R is expressed in many normal tissues, but it is overexpressed in various tumor cells, such as thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer. It is also overexpressed in orbital fibroblasts in Graves' orbitopathy (GO). TED (thyroid ophthalmopathy) is characterized by the proliferation of fibroblasts expressing IGF-1R and thyroid-stimulating hormone receptor (TSHR) in orbital tissue, which mediate hyaluronic acid deposition, orbital muscle expansion, and increased fat. TED is driven by TSHR-stimulating autoantibodies and the interaction between TSHR and IGF-1R.

[0006] The autoimmune antibody TSI (TSHR-activated form, GD-IgG) binds to IGF-1R (IGF-1 binding site) and activates TSHR in tandem, leading to increased secretion of hyaluronic acid (HA) and glycosaminoglycans (GAG). Anti-IGF-1R antibodies inhibit the activation of other TSHRs by blocking the binding of TSI to IGF-1R, thereby reducing HA and GAG secretion. Therefore, developing an IGF-1R antibody drug is of great significance.

[0007] Tepezza (tetruminumab) is the world's first anti-IGF-1R monoclonal antibody, launched in 2020. It is the first and only biologic drug approved by the US FDA for the treatment of thyroid-associated ophthalmopathy. Initial approval was for patients with disease duration ≤9 months (acute phase) and high disease activity. Currently, the US FDA has approved an update to the indications and usage sections of the tetruminumab product label to approve its use in treating thyroid ophthalmopathy, but its efficacy needs further improvement. Currently, there are no original antibody drugs targeting the same target available in China, indicating significant clinical demand. Summary of the Invention

[0008] This invention addresses the shortcomings of existing anti-IGF-1R monoclonal antibodies by providing an antibody targeting IGF-1R and its application.

[0009] The present invention mainly solves the above-mentioned technical problems through the following technical means.

[0010] A first aspect of the present invention provides an antibody targeting IGF-1R, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:2, the amino acid sequence of LCDR2 is shown in SEQ ID NO:3, the amino acid sequence of LCDR3 is shown in SEQ ID NO:4, the amino acid sequence of HCDR1 is shown in SEQ ID NO:6, the amino acid sequence of HCDR2 is shown in SEQ ID NO:7, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:8.

[0011] In some embodiments of the present invention, the frame region of the light chain variable region and / or heavy chain variable region is a human or mouse-derived frame region.

[0012] In some embodiments of the present invention, the framework region of the light chain variable region and / or heavy chain variable region is a human-derived framework region.

[0013] In some embodiments of the present invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:1; and / or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5.

[0014] In some specific embodiments of the present invention, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5.

[0015] In some embodiments of the present invention, the antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv, wherein the Fv is preferably scFv.

[0016] In some embodiments of the present invention, the antibody is a full-length antibody, wherein the heavy chain constant region and / or light chain constant region are preferably derived from human antibodies.

[0017] In some embodiments of the present invention, the heavy chain constant region is derived from the human heavy chain IgG1 or IgG4 constant region or variants thereof; and / or, the light chain constant region is derived from the human light chain κ chain constant region or variants thereof.

[0018] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region of the antibody is as shown in SEQ ID NO:9 or 10; and / or, the amino acid sequence of the light chain constant region of the antibody is as shown in SEQ ID NO:11.

[0019] In some embodiments of the present invention, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:14 or 16; and / or, the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO:18.

[0020] A second aspect of the present invention provides a chimeric antigen receptor comprising an antibody as described in the first aspect of the present invention.

[0021] A third aspect of the present invention provides an isolated nucleic acid that encodes an antibody as described in the first aspect of the present invention or a chimeric antigen receptor as described in the second aspect of the present invention.

[0022] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain of the antibody is as shown in SEQ ID NO:15 or 17; and / or, the nucleotide sequence encoding the light chain of the antibody is as shown in SEQ ID NO:19.

[0023] A fourth aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acids as described in the third aspect of the present invention.

[0024] In some embodiments of the present invention, the recombinant expression vector is a plasmid vector, a granular vector, a bacteriophage vector, or a viral vector, and the viral vector is preferably a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0025] In some specific embodiments of the present invention, the backbone of the plasmid vector is pCDNA3.4.

[0026] A fifth aspect of the present invention provides a transformant comprising the isolated nucleic acid as described in the third aspect of the present invention or the recombinant expression vector as described in the fourth aspect of the present invention, wherein the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0027] In some embodiments of the present invention, the eukaryotic cells are yeast cells or mammalian cells.

[0028] In some embodiments of the present invention, the eukaryotic cells are mammalian cells, such as HEK293 cells or CHO cells.

[0029] A sixth aspect of the present invention provides a genetically modified cell comprising a chimeric antigen receptor as described in a second aspect of the present invention.

[0030] In some embodiments of the present invention, the genetically modified cells are eukaryotic cells, preferably isolated human cells, and more preferably immune cells such as T cells or NK cells.

[0031] A seventh aspect of the present invention provides a method for preparing an antibody targeting IGF-1R, the method comprising the following steps:

[0032] The transformant as described in the fifth aspect of the present invention is cultured to obtain the antibody targeting IGF-1R from the culture.

[0033] An eighth aspect of the present invention provides an antibody-drug conjugate comprising a cytotoxic agent or tag, and an antibody as described in the first aspect of the present invention.

[0034] In some embodiments of the present invention, the cytotoxic agent is MMAF or MMAE, and the tag is a fluorescent agent.

[0035] A ninth aspect of the present invention provides a pharmaceutical composition comprising one or more of the group consisting of an antibody as described in the first aspect of the present invention, a genetically modified cell as described in the sixth aspect of the present invention, and an antibody-drug conjugate as described in the eighth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0036] In some embodiments of the present invention, the pharmaceutical composition further comprises one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0037] The tenth aspect of the present invention provides the use of one or more of the group consisting of antibodies as described in the first aspect of the present invention, chimeric antigen receptors as described in the second aspect of the present invention, isolated nucleic acids as described in the third aspect of the present invention, recombinant expression vectors as described in the fourth aspect of the present invention, transformants as described in the fifth aspect of the present invention, genetically modified cells as described in the sixth aspect of the present invention, antibody-drug conjugates as described in the eighth aspect of the present invention, and pharmaceutical compositions as described in the ninth aspect of the present invention in the preparation of medicaments for diagnosing, preventing, and / or treating IGF-1R-related diseases, or in the preparation of reagents for inhibiting IGF-1R phosphorylation or blocking the binding of IGF-1 to human IGF-1R protein.

[0038] In some embodiments of the present invention, the IGF-1R-related diseases are selected from autoimmune diseases and tumors.

[0039] In some embodiments of the present invention, the autoimmune disease described is a thyroid eye disease, such as Graves' ophthalmopathy; the tumor is selected from one or more of thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer, preferably breast cancer or colon cancer.

[0040] The eleventh aspect of the present invention provides a kit comprising one or more of the group consisting of antibodies as described in the first aspect of the present invention, chimeric antigen receptors as described in the second aspect of the present invention, genetically modified cells as described in the sixth aspect of the present invention, antibody-drug conjugates as described in the eighth aspect of the present invention, and pharmaceutical compositions as described in the ninth aspect of the present invention.

[0041] In some embodiments of the present invention, the kit further includes (i) means for administering one or more of the group consisting of the antibody, the chimeric antigen receptor, the genetically modified cell, the antibody-drug conjugate, and the pharmaceutical composition; and / or (ii) instructions for use.

[0042] The twelfth aspect of the present invention provides a pillbox kit comprising pillbox A and pillbox B, wherein:

[0043] The kit A contains one or more of the following: antibodies as described in the first aspect of the present invention, chimeric antigen receptors as described in the second aspect of the present invention, genetically modified cells as described in the sixth aspect of the present invention, antibody-drug conjugates as described in the eighth aspect of the present invention, and pharmaceutical compositions as described in the ninth aspect of the present invention.

[0044] The kit B contains other antibodies for treating IGF-1R-related diseases or pharmaceutical compositions containing said other antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines. In this invention, the other antibodies do not contain antibodies targeting IGF-1R.

[0045] In some embodiments of the present invention, the IGF-1R-related diseases are selected from autoimmune diseases and tumors.

[0046] In some embodiments of the present invention, the autoimmune disease is a thyroid eye disease, such as Graves' ophthalmopathy; the tumor is selected from one or more of thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer, preferably breast cancer or colon cancer.

[0047] The thirteenth aspect of the present invention provides a method for diagnosing, treating, and / or preventing IGF-1R-related diseases, the method comprising administering to a patient in need a therapeutically effective amount of an antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the second aspect of the present invention, a genetically modified cell as described in the sixth aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, or a pharmaceutical composition as described in the ninth aspect of the present invention, or treating the patient in need using a kit as described in the eleventh aspect of the present invention or a kit as described in the twelfth aspect of the present invention.

[0048] In some embodiments of the present invention, the IGF-1R-related diseases are selected from autoimmune diseases and tumors.

[0049] In some embodiments of the present invention, the autoimmune disease is a thyroid eye disease, such as Graves' ophthalmopathy; the tumor is selected from one or more of thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer, more preferably breast cancer or colon cancer.

[0050] The fourteenth aspect of the present invention provides a method for detecting IGF-1R, the method comprising using an antibody as described in the first aspect of the present invention or a kit as described in the eleventh aspect of the present invention.

[0051] In some embodiments of the present invention, the detection is a non-diagnostic detection.

[0052] In some embodiments of the present invention, the method is based on Western Blot, ELISA or flow cytometry techniques for detection.

[0053] The fifteenth aspect of the present invention provides a combination therapy comprising administering to a patient in need an antibody as described in the first aspect of the present invention, a chimeric antigen receptor as described in the second aspect of the present invention, a genetically modified cell as described in the sixth aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, or a pharmaceutical composition as described in the ninth aspect of the present invention, and a second therapeutic agent; the second therapeutic agent preferably comprises other antibodies for treating IGF-1R-related diseases or a pharmaceutical composition comprising said other antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0054] In some embodiments of the present invention, the IGF-1R-related diseases are selected from autoimmune diseases and tumors.

[0055] In some embodiments of the present invention, the autoimmune disease is a thyroid eye disease, such as Graves' ophthalmopathy; the tumor is selected from one or more of thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer, more preferably breast cancer or colon cancer.

[0056] The sixteenth aspect of the present invention provides one or more of the following groups: antibodies as described in the first aspect of the present invention, chimeric antigen receptors as described in the second aspect of the present invention, isolated nucleic acids as described in the third aspect of the present invention, recombinant expression vectors as described in the fourth aspect of the present invention, transformants as described in the fifth aspect of the present invention, genetically modified cells as described in the sixth aspect of the present invention, antibody-drug conjugates as described in the eighth aspect of the present invention, and pharmaceutical compositions as described in the ninth aspect of the present invention, for the preparation of medicaments for the diagnosis, prevention, and / or treatment of IGF-1R-related diseases, or for the preparation of reagents for inhibiting IGF-1R phosphorylation or blocking the binding of IGF-1 to human IGF-1R protein.

[0057] In some embodiments of the present invention, the IGF-1R-related diseases are selected from autoimmune diseases and tumors.

[0058] In some embodiments of the present invention, the autoimmune disease is a thyroid eye disease, such as Graves' ophthalmopathy; the tumor is selected from one or more of thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer, preferably breast cancer or colon cancer.

[0059] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0060] The reagents and raw materials used in this invention are all commercially available.

[0061] The positive and progressive effects of this invention are as follows:

[0062] This invention provides an antibody targeting IGF-1R obtained by constructing a phage library from fully human transgenic mice. This antibody exhibits high specificity and high affinity, significantly inhibits IGF-1R phosphorylation, blocks the binding of IGF-1 to human IGF-1R protein, has a very low ADCC effect, and good pharmacokinetic activity. It is suitable for efficient detection of IGF-1R, treatment of autoimmune diseases and tumors, etc. Attached Figure Description

[0063] Figure 1 shows the serum titers of hIGF-1R-immunized mice.

[0064] Figure 2 shows the curve of antibody binding to IGF-1R.

[0065] Figure 3 shows that the CA379 antibody blocked the binding of IGF-1R and human IGF-1 protein.

[0066] Figure 4 shows the flow cytometry binding results of the anti-IGF-1R antibody on MCF-7 cells.

[0067] Figure 5 shows the inhibitory effect of IGF-1R antibody on the proliferation of MCF-7 cells.

[0068] Figure 6 shows the results of phosphorylation experiments of anti-hIGF-1R antibody on MCF-7 cells.

[0069] Figure 7 shows the binding results of the anti-IGF-1R antibody with INSR.

[0070] Figure 8 shows the ADCC effect of IGF-1R antibody on cells.

[0071] Figure 9 shows the in vivo pharmacokinetic results of the anti-hIGF-1R antibody.

[0072] Figure 10 shows the efficacy of the anti-hIGF-1R antibody against the Colo205 model.

[0073] Figure 11 shows the semi-quantitative expression level of IGF-1R protein in Colo205 tumor samples by Western blot. Detailed Implementation

[0074] 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 operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below:

[0075] In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0076] In this invention, the amino acid sequences of the listed complementarity determining regions (CDRs) are all as defined by the Kabat numbering rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), Kabat et al. (1987) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT, imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0077] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of protection claimed by this invention is based on the sequence defined according to the Kabat numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0078] In this invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the second and third constant regions of the two heavy chains (and, for IgE and IgM, a fourth constant region) linked together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0079] In this invention, a "Fab fragment" consists of a light chain and a heavy chain, comprising the CH1 domain and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. A "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form the F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0080] In this invention, scFv refers to a single-chain antibody fragment, which includes a heavy chain variable region, a light chain variable region, and a linker peptide of 15-20 amino acids. The VL and VH domains enable the linker peptides to pair and form monovalent molecules as single polypeptide chains [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker peptide-VH-COOH or NH2-VH-linker peptide-VL-COOH.

[0081] In this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0082] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0083] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0084] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan.

[0085] In this invention, the pharmaceutical composition may comprise a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the invention can be prepared by mixing antibodies of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Preferably, the composition is in the form of a lyophilized formulation or an aqueous solution.

[0086] The pharmaceutical compositions of the present invention may also comprise more than one active ingredient required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release formulations can be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody of the present invention, said matrix being a shaped article, such as a film or microcapsule.

[0087] In this invention, the chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of an antigen-specific antibody with the function of a T-cell receptor. Generally, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In an exemplary aspect, the extracellular domain of the CAR contains an antigen recognition region, which may be a scFV of an antigen-specific antibody.

[0088] In this invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker unit. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, radioactive isotopes, toxic drugs, chemotherapeutic agents, antibiotics, or ribolysins, or derivatives thereof.

[0089] In this invention, the application scenarios for "non-diagnostic purposes" include, but are not limited to: for example, detecting the presence or absence of antigen IGF-1R in vitro in the laboratory; or using it as a positive antibody to screen other antibodies targeting IGF-1R; or competing with other antibodies targeting IGF-1R to detect whether there is competition between the antibodies, i.e., whether the antigen epitopes are the same or similar.

[0090] In this invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0091] It is known in the art that IGF-1R is overexpressed in various tumor cells and orbital tissues, promoting tumorigenicity or malignant transformation of cells, or driving the development of thyroid eye diseases. Therefore, abnormal expression of IGF-1R can lead to a variety of diseases. In this invention, the term "IGF-1R-related disease" refers to diseases caused by IGF-1R overexpression, including but not limited to: autoimmune diseases, such as thyroid eye diseases, specifically Graves' ophthalmopathy in one specific embodiment of this invention; tumors, such as thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer.

[0092] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0093] Example 1: Production of hIGF-1R monoclonal antibody

[0094] 1.1 Mouse immunization method

[0095] The mice used in the immunization experiments were fully human antibody transgenic mice independently developed by Shandong Boan Biotechnology Co., Ltd. (A total of 5 mice were immunized.) Immunization was performed using the hIGF-1R (10164-H08H) antigen protein produced by Beijing Yiqiao Shenzhou Technology Co., Ltd. Mice with higher serum titers were selected for booster immunization. Three days later, the mice were sacrificed, and their spleens were harvested for subsequent library construction experiments.

[0096] Serum titers were primarily determined using the ELISA method. hIGF-1R-his protein at a concentration of 1 μg / mL was coated with CBS coating buffer; the protein was blocked with 3% skim milk powder at 37°C for 1 h; serum diluted with PBST was added and incubated at 37°C for 1 h; then HRP-goat anti-human H+L was added, and incubation was continued at 37°C for 1 h. After 10 min of color development, the reaction was stopped with 2M concentrated sulfuric acid, and the OD450 was read using a microplate reader. The serum titers are shown in Figure 1.

[0097] 1.2 Establishment of the phage library

[0098] Mice were euthanized, and their spleens were dissected and removed. The spleens were then crushed using a syringe stopper and filtered through a sieve. The filtered spleen cells were frozen, and RNA was extracted to obtain cDNA. The phage library was constructed using standard methods. The library size data are shown in Table 1.

[0099] Table 1. Library size of phage libraries constructed from each immunized mouse group.

[0100] 1.3 Screening using two methods

[0101] 1.3.1 Plate screening: Plates were coated with IGF-1R (Beijing Yiqiao Shenzhou, 10164-H08H). The next day, phage libraries were added and incubated for 2 hours. After washing 4-10 times, the specifically bound phages were eluted with elution buffer.

[0102] 1.3.2 Magnetic bead screening: IGF-1R-biotin (IGR-H82E3) purchased from Bipsys Biotechnology Co., Ltd. was blocked with BSA and incubated with a phage library for 2 hours. After washing 4-10 times, the specifically bound phages were eluted with elution buffer. The antibody clones obtained and their sources are shown in Table 3.

[0103] Table 2. Sources of anti-IGF-1R antibodies obtained through screening.

[0104] Example 2: Molecular Construction and Production of Complete Antibodies

[0105] Sixty-three positive IgG1 clones were constructed and sequenced. The amino acid sequence of the variable region of the lead antibody CA379 is shown in Table 3 below (the CDR region is underlined and the system is defined as the Kabat system).

[0106] Table 3. Amino acid sequence of the variable region of CA379 antibody.

[0107] The antibody variable region gene was amplified using conventional molecular biology techniques, PCR (2×Phanta Max Master Mix, manufacturer: Vazyme, catalog number: P515-P1-AA, batch number: 7E512E1). The antibody heavy chain variable region gene was then ligated into the vector pCDNA3.4 (Life Technology) containing the antibody heavy chain constant region sequence via homologous recombination. Similarly, the antibody light chain variable region gene was ligated into the vector pCDNA3.4 containing the antibody light chain constant region sequence. The sequence information for the heavy and light chains is shown in Table 4.

[0108] Table 4. Sequence information of the heavy and light chains of the antibodies.

[0109] After sequencing, the positive clones were extracted and the plasmids were co-transfected into HEK293 cells and cultured in a shaker at 37°C, 8% CO2, and 125 rpm. After transient expression for 7 days, the supernatant was purified by Protein A affinity chromatography to obtain antibodies CA379-IgG1 and CA379-IgG4. The antibody concentration was determined by the theoretical extinction coefficient of MV280 binding.

[0110] Example 3: Detection of the binding between anti-hIGF-1R antibody and human IGF-1R protein

[0111] Dilute hIGF-1R-His protein (Beptase, IGR-H5229) to 2 μg / mL with PBS buffer. Add 100 μL of the diluted antigen to each well of a 96-well ELISA plate and incubate overnight at 4°C. Wash the plate three times with washing buffer (200 μL / well). Add 200 μL of blocking buffer to each well and incubate at room temperature for 1 hour. Dilute IGF-1R monoclonal antibody with antibody dilution buffer, starting from 15 μg / mL, with 4-fold serial dilutions in 8 concentration gradients. Discard the blocking buffer, add the diluted sample (100 μL / well), cover with sealing film, and incubate at room temperature for 2 hours. After washing, dilute anti-human IgG H&L (HRP) (Bethyl, A80-319P) (1:40000) with dilution buffer. Add 100 μL / well to each well of a 96-well plate, cover with a new sealing film, and incubate at room temperature for 1 hour. After washing, add 100 μL of mixed TMB solution to each well. Incubate at room temperature for 5-10 min, avoiding light. Add 50 μL of stop solution to each well, gently tap the plate to ensure thorough mixing, and immediately read the optical density (OD) value (450 nm) of each well using a TECAN Spark microplate reader. Results showed that CA379-IgG1 and CA379-IgG4 bound to IGF-1R protein with high affinity (Figure 2). In this experiment and the following experiments, the antibody against Horizon (Tepezza, sequence below), expressed and purified in our laboratory, was used as the control antibody. Table 5 shows the EC50 of the antibody calculated based on Figure 2. 50 The values ​​show that CA379 antibody can bind to IGF-1R at the ELISA level, and its binding activity is better than that of Tepezza, with CA379-IgG4 showing the best binding activity.

[0112] Tepezza antibody heavy chain sequence: QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGR RYFDLWGRGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:12)

[0113] Tepezza antibody light chain sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKWPPWTFGQG TKVESKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:13)

[0114] Table 5. Binding values ​​of anti-IGF-1R antibodies to IGF-1R protein.

[0115] Example 4: Detection of the effect of anti-hIGF-1R antibody on blocking the binding of IGF-1 to human IGF-1R protein

[0116] hIGF-1 protein (ACRO, IG1-H4269) was diluted to 1 μg / mL with PBS buffer (pH 9.6) and coated onto a microplate at 100 μL / well. The plate was incubated overnight at 4°C. After washing, the plate was blocked with skim milk powder. 15 μg / mL antibody was serially diluted 4-fold with PBS to seven different concentrations. After washing, 50 μL of the diluted antibody was added to each well, followed by 50 μL of biotin-labeled IGF-1R-Protein (Bepsys, IG1-H82Q6) at 37°C for 1 h. After washing, 100 μL of STREP / HRP (Abcam, Ab7403) diluted in PBST was added to each well, and the plate was incubated at 37°C for 1 h. After washing, 100 μL of TMB was added to each well for color development. After 10 min, 50 μL of 2M H2SO4 was added to each well to stop the color development. The OD450 was read using a microplate reader. The results showed that CA379-IgG1 and CA379-IgG4 antibodies could block the binding of IGF-1R protein to IGF-1, as shown in Figure 3.

[0117] Table 6 shows the EC calculated based on Figure 3. 50 The values ​​show that CA379 can block the binding of IGF-1 to IGF-1R at the ELISA level, and its blocking effect is better than that of Tepezza, with CA379-IgG4 showing the strongest blocking activity.

[0118] Table 6. Values ​​of anti-IGF-1R antibody blocking the binding of IGF-1R protein to IGF-1.

[0119] Example 5: Detection of the affinity between anti-hIGF-1R antibody and human IGF-1R protein

[0120] The binding kinetics of the antibody to human IGF-1R protein were measured using a BIAcore 8K instrument based on surface plasmon resonance (SRP) technology. 2 μg / mL of IGF-1R antibody was captured using a ProA chip, and the binding activity of the anti-IGF-1R antibody to human IGF-1R was analyzed. Human IGF-1R protein was serially diluted 2-fold with HBS-EP+ buffer to five concentrations, starting at 50 nM. IGF-1R protein binding kinetics were analyzed using Biacore, and the affinity activity KD value was calculated. Table 7 shows that both the CA379 antibody and Tepezza exhibited high affinity for the human protein.

[0121] Table 7. Biacore detection of IGF-1R antibody binding to human protein

[0122] Example 6: Detection of flow cytometry binding of anti-hIGF-1R antibody on MCF-7 cells

[0123] Antibody binding to cells by flow cytometry: 200 nM antibody was serially diluted 4-fold with PBS to obtain 6 concentrations, and then 50 μL of 2 × 10⁻⁶ cells was added. 6 MCF-7 cells (ATCC, HTB-22) at a concentration of [number] cells / mL and 50 μL of serially diluted antibodies were added to 96-well U-shaped plates and incubated at 4°C for 1 h. After washing twice with PBS, secondary antibody Goat anti-human IgG Fc (Jackson, 109-545-008) diluted in PBS was added and incubated at 4°C for 30 min. After washing twice with PBS, the cells were resuspended in 100 μL of PBS and then subjected to flow cytometry. The fluorescence value (MFI) of the FITC channel was read. The results are shown in Figure 4. EC50 of each antibody... 50 The values ​​are shown in Table 8. The results show that the flow cytometry binding ability of the CA379 antibody is superior to that of Tepezza, with CA379-IgG4 exhibiting the best flow cytometry binding.

[0124] Table 8. Flow cytometry binding values ​​of anti-IGF-1R antibody on MCF-7 cells

[0125] Example 7: Detection of the inhibitory effect of anti-hIGF-1R antibody on the proliferation of MCF-7 cells

[0126] MCF-7 cells were cultured in serum-free MEM medium for 4 hours, and then the cell density was adjusted to 1.6 × 10⁶ cells / year using serum-free MEM medium. 5 cells / mL; 200 nM antibody was diluted 4-fold in 8 gradients with serum-free MEM medium for later use; 50 μL of cells and 25 μL of antibody were added to 96-well cell culture plates and incubated at 37°C for 1 h; 25 μL of hIGF-I-His (ACRO, IG1-H5245) with an initial concentration of 60 nM was added to the cell-antibody mixture to a final concentration of 15 nM, and the plates were sealed and incubated at 37°C for 5 days; the number of viable cells was detected by ATP signal using a microplate reader. 2.0, Novizan, DD1101-01). The results show that the CA379 IC... 50 The smaller size of the control Tepezza indicates that CA379 has a better inhibitory effect on MCF-7 cells compared to the control (see Figure 5 and Table 9).

[0127] Table 9. Relevant values ​​for IGF-1R antibody inhibition of MCF-7 cell proliferation.

[0128] Example 8: Detection of phosphorylation of anti-hIGF-1R antibody on MCF-7

[0129] MCF-7 cells were digested, washed twice with complete culture medium, and then the cell density was adjusted to 5 × 10⁶ cells / year using complete culture medium. 6 cells / mL; 1000 nM antibody was diluted 3-fold in complete culture medium in 8 gradients for later use; 20 μL of cells and 10 μL of serially diluted antibody were added to 96-well plates, sealed and incubated at 37°C for 1 h; 80 nM IGF-I-Fc (ACRO, IG1-H4269) was diluted 4-fold in complete culture medium, and 10 μL was added to the cell-antibody mixture to make a final concentration of 20 nM, sealed and incubated at 37°C for 15 min; 10 μL of lysis buffer was added to lyse the cells and release intracellular phosphorylated AKT protein; the phosphorylation signal of intracellular AKT (S473) in MCF-7 cells was detected by HTRF method using a microplate reader (pAKTs473 kit, Bioauxilium, KIT-AKTS473P-500). As shown in Figure 6 and Table 10, compared with the control antibody, the CA379 antibody has a better ability to inhibit receptor phosphorylation, and the inhibitory activity of CA379-IgG4 is significantly better than that of the control antibody Tepezza.

[0130] Table 10. Relevant values ​​for the inhibition of receptor phosphorylation on MCF-7 cells by anti-hIGF-1R antibody.

[0131] Example 9: Binding experiment of anti-hIGF-1R antibody molecule with INSR

[0132] Dilute insulin receptor (INSR) (11081-H08H) to 10 μg / mL with CBS buffer (pH 9.6), coat ELISA plates (100 μL / well), and incubate overnight at 4°C. After washing, block with skim milk powder. After washing, add 100 μL of antibody diluted in PBST to each well (to final concentrations of 0.625 μg / mL, 2.5 μg / mL, and 10 μg / mL). Incubate at 37°C for 1 h, and wash twice. Then add goat anti-human H+L secondary antibody (KPL, 474-1006) (100 μL / well), and incubate at 37°C for 1 h. After washing, add 100 μL of TMB to each well for color development. After 10 min, add 50 μL of 2M H2SO4 to each well to stop the color development. Read the OD using an ELISA reader. 450 As can be seen from the results (Figure 7), the binding of CA379-IgG1 and CA379-IgG4 antibodies to INSR was very low, which was significantly better than that of the control antibody Tepezza.

[0133] Example 10 Antibody-dependent cell-mediated cytotoxicity (luciferase reporter gene effector cells)

[0134] Prepare ADCC reaction buffer: RPMI 1640 medium containing 1% FBS; collect ADCC effector cells—Jurkat cells (Promega, G7011)—and adjust the cell density to 2.4 × 10⁻⁶ cells using ADCC reaction buffer. 6 Cells / mL; Collect target cells CHO-K1-hIGF-1R (Kangyuan Bochuang, KC-2990), and adjust the cell density to 4.0 × 10⁶ cells / mL using ADCC reaction buffer. 5 The cells / mL were used to dilute the test sample with ADCC reaction buffer, starting from 4 μg / mL and diluting 4-fold to 8 concentrations. 25 μL each of effector cells, target cells, and test sample were added to a reaction plate (Costar, 3917), for a total reaction volume of 75 μL. The reaction system was incubated at 37℃ for 6 h. 75 μL of Bio-Glo Luciferase System (Promega, G7940) was added to each well, and the reaction was allowed to proceed for 15 min. The chemiluminescence value was then read using a microplate reader (BioTek, Synergy Neo2). Figure 8 shows that CA379-IgG1, CA379-IgG4, and Tepezza all exhibited very weak ADCC effects.

[0135] Example 11: In vivo pharmacokinetic experiment of anti-hIGF-1R antibody in mice

[0136] Three Balb / c mice (Jinan Pengyue Experimental Animal Breeding Co., Ltd., female, 18-22g) were selected for each antibody and administered the drug subcutaneously at a dose of 10mg / kg. Serum samples were collected at 0h before administration and at 10min, 1h, 6h, 1d, 3d, 5d, 7d, 10d, and 14d after administration to detect antibody concentration. The serum detection method was ELISA. The specific results are shown in Figure 9 below. It can be seen that, compared with Tepezza, both CA379-IgG1 and CA379-IgG4 have better pharmacokinetic levels.

[0137] Example 12: In vivo efficacy study of anti-IGF-1R monoclonal antibody against a Colo205 subcutaneous xenograft model of colon cancer.

[0138] Balb / c-nude immunodeficient mice, female, 8 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After arrival, the animals were housed in the experimental environment for 3-7 days before experiments could begin. Colo205 cells (Nanjing Kebai, CBP60026) were introduced at a rate of 1×10⁻⁶. 6 / cells per mouse were inoculated subcutaneously into the dorsal region of the right forelimb of mice. The skin of each mouse was cleaned before inoculation to minimize ulceration. The tumors were inoculated when their average volume reached approximately 60-120 mm. 3The mice were administered the drug in groups at the designated time. The candidate antibody CA379 was injected intraperitoneally into mice at a dose of 10 mg / kg, twice weekly. Mouse weight and tumor volume were measured before each administration. Tumor volume (mm²) 3 = Major diameter × Minor diameter × Minor diameter ÷ 2.

[0139] Table 11 and Figure 10 show the efficacy data of each monoclonal antibody in the Colo205 colon cancer model, i.e., the changes in tumor volume in mice after administration. It can be seen that the tumor volume decreased after administration of all three antibodies. The tumor growth inhibition rates (TGI) of CA379-IgG4, CA379-IgG1, and Tepezza groups were 41.9%, 16.2%, and 18.3%, respectively; the tumor weight inhibition rates (IR) were 40%, 12.1%, and 19.2%, respectively. The results indicate that CA379-IgG4 has good efficacy in the Colo205 model.

[0140] Table 11. Pharmacodynamic values ​​of anti-hIGF-1R antibody against the Colo205 model.

[0141] Three tumor tissue samples were prepared from each of the model group, positive control group, and CA379 administration group. The IGF-1R protein content in the samples was semi-quantitatively determined using Western blot. Figure 11 shows that the IGF-1R protein expression level was high in the model group, while the IGF-1R protein expression level was significantly decreased in the CA379 administration group, consistent with the results of the positive control group. This indicates that CA379 injection can downregulate the IGF-1R protein expression level, thereby inhibiting tumor growth.

[0142] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antibody targeting IGF-1R, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, characterized in that, The amino acid sequence of LCDR1 is shown in SEQ ID NO:2, the amino acid sequence of LCDR2 is shown in SEQ ID NO:3, the amino acid sequence of LCDR3 is shown in SEQ ID NO:4, the amino acid sequence of HCDR1 is shown in SEQ ID NO:6, the amino acid sequence of HCDR2 is shown in SEQ ID NO:7, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

8.

2. The antibody as described in claim 1, characterized in that, The framework regions of the light chain variable region and / or heavy chain variable region are human or mouse-derived framework regions. Preferably, the framework region of the light chain variable region and / or heavy chain variable region is a human-derived framework region; More preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:1; and / or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

5.

3. The antibody as described in claim 1 or 2, characterized in that, The antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv, and the Fv is preferably scFv; Preferably, the antibody is a full-length antibody, and its heavy chain constant region and / or light chain constant region are preferably derived from human antibodies; Further preferably, the heavy chain constant region is derived from the human heavy chain IgG1 or IgG4 constant region or a variant thereof; and / or, the light chain constant region is derived from the human light chain κ chain constant region or a variant thereof; More preferably, the amino acid sequence of the heavy chain constant region of the antibody is as shown in SEQ ID NO:9 or 10; and / or, the amino acid sequence of the light chain constant region of the antibody is as shown in SEQ ID NO:11; Further preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:14 or 16; and / or, the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO:

18.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises the antibody as described in any one of claims 1 to 3.

5. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the antibody as described in any one of claims 1 to 3 or the chimeric antigen receptor as described in claim 4; Preferably, the nucleotide sequence encoding the heavy chain of the antibody is as shown in SEQ ID NO:15 or 17; and / or, the nucleotide sequence encoding the light chain of the antibody is as shown in SEQ ID NO:

19.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 5; Preferably, the recombinant expression vector is a plasmid vector, a granular vector, a bacteriophage vector, or a viral vector, and the viral vector is preferably a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector. More preferably, the backbone of the plasmid vector is pCDNA3.

4.

7. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 5 or the recombinant expression vector as described in claim 6, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; Preferably, the eukaryotic cell is a yeast cell or a mammalian cell; More preferably, the eukaryotic cells are mammalian cells, such as HEK293 cells or CHO cells.

8. A genetically modified cell, characterized in that, The genetically modified cell contains the chimeric antigen receptor as described in claim 4; preferably, the genetically modified cell is a eukaryotic cell, more preferably an isolated human cell, and more preferably an immune cell such as a T cell or NK cell.

9. A method for preparing an antibody targeting IGF-1R, characterized in that, The preparation method includes the following steps: Cultivate the transformant as described in claim 7 to obtain the antibody targeting IGF-1R from the culture.

10. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent or a tag, and an antibody as described in any one of claims 1 to 3; preferably, the cytotoxic agent is MMAF or MMAE, and the tag is a fluorescent agent.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises one or more of the group consisting of the antibody as described in any one of claims 1 to 3, the genetically modified cell as described in claim 8, and the antibody-drug conjugate as described in claim 10, and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition further comprises one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

12. The use of one or more of the group consisting of the antibody as described in any one of claims 1 to 3, the chimeric antigen receptor as described in claim 4, the isolated nucleic acid as described in claim 5, the recombinant expression vector as described in claim 6, the transformant as described in claim 7, the gene-modified cell as described in claim 8, the antibody-drug conjugate as described in claim 10, and the pharmaceutical composition as described in claim 11 in the preparation of a medicament for the diagnosis, prevention, and / or treatment of IGF-1R-related diseases, or in the preparation of a reagent for inhibiting IGF-1R phosphorylation or blocking the binding of IGF-1 to human IGF-1R protein; Preferably, the IGF-1R-related diseases are selected from autoimmune diseases and tumors; More preferably, the autoimmune disease is a thyroid eye disease, such as Graves' ophthalmopathy; the tumor is selected from one or more of thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer, preferably breast cancer or colon cancer.

13. A reagent kit, characterized in that, The kit comprises one or more of the group consisting of the antibody as described in any one of claims 1 to 3, the chimeric antigen receptor as described in claim 4, the genetically modified cell as described in claim 8, the antibody-drug conjugate as described in claim 10, and the pharmaceutical composition as described in claim 11; Preferably, the kit further includes (i) a means for administering one or more of the group consisting of the antibody, the chimeric antigen receptor, the genetically modified cell, the antibody-drug conjugate, and the pharmaceutical composition; and / or (ii) instructions for use.

14. A medicine box set, characterized in that, The pillbox set includes pillbox A and pillbox B, wherein: The kit A contains one or more of the group consisting of the antibody as described in any one of claims 1 to 3, the chimeric antigen receptor as described in claim 4, the genetically modified cell as described in claim 8, the antibody-drug conjugate as described in claim 10, and the pharmaceutical composition as described in claim 11; The kit B contains other antibodies for treating IGF-1R-related diseases or a pharmaceutical composition containing said other antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines. Preferably, the IGF-1R-related diseases are selected from autoimmune diseases and tumors; More preferably, the autoimmune disease is a thyroid eye disease, such as Graves' ophthalmopathy; the tumor is selected from one or more of thyroid cancer, colorectal cancer, gastric cancer, liver cancer, and breast cancer, preferably breast cancer or colon cancer.

15. A method for detecting IGF-1R, characterized in that, The method includes using the antibody as described in any one of claims 1 to 3 or the kit as described in claim 13; Preferably, the detection is for non-diagnostic purposes; and / or the method is based on Western Blot, ELISA, or flow cytometry techniques.

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