Antigen-binding proteins and their use

Abstract

The present application relates to antigen-binding proteins and uses thereof, specifically, those having an IC of about 1.0 μg / mL or lower in competitive ELISA detection. 50 The present invention relates to an antigen binding protein capable of competitively inhibiting the binding of IGF-1R to its ligand at a therapeutically effective level.

Description

[Technical Field]

[0001] This application relates to the field of biopharmaceuticals, and more specifically, to antigen-binding proteins and their use. [Background technology]

[0002] The interaction between IGF-1 (insulin-like growth factor I) and IGF-1R (insulin-like growth factor receptor 1) activates cellular signaling pathways by causing autophosphorylation of receptor tyrosine residues. Activation of this receptor can stimulate the growth and survival of tumor cells. Therefore, inhibition of IGF-1R activity is a potential method for treating or preventing tumors. Overexpression of insulin-like growth factor receptor 1 is observed in several cancer cell lines and tumor tissues, and the degree of IGF-1R expression may correlate with disease severity. In this field, there is a growing demand for methods that can effectively inhibit IGF-1R activity, such as antigen-binding proteins or their functionally active fragments that can bind to IGF-1R. [Overview of the Initiative]

[0003] This invention provides an antigen-binding protein that may have properties selected from the following group: (1) inhibiting the binding of IGF-1R to its ligand, (2) binding to IGF-1R, (3) selectively binding to primate (e.g., human or monkey) IGF-1R, (4) inhibiting the proliferation of cells overexpressing IGF-1R, (5) inhibiting the proliferation of human breast cancer cells, such as inhibiting the proliferation of IGF-1-induced human breast cancer cells, and (6) inhibiting IGF-1-induced MEK, Erk1 / 2 and / or Akt phosphorylation.

[0004] In one embodiment, the present invention provides an antigen-binding protein that can inhibit the competitive binding of IGF-1R to its ligand at an IC50 value of approximately 1.0 μg / mL or lower in ELISA detection competition.

[0005] According to one embodiment, the present invention provides an antigen-binding protein that can competitively bind to a reference antibody and IGF-1R, wherein the reference antibody comprises HCDR1-3, and HCDR1-3 each comprises amino acid sequences shown in SEQ ID NO: 85, 100, and 131, respectively.

[0006] In one embodiment, the present application provides an antigen-binding protein comprising HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 217 or 218.

[0007] According to one embodiment, the present application provides a polypeptide comprising the antigen-binding protein of the present application.

[0008] In one embodiment, the present application provides an immune complex comprising an isolated antigen-binding protein and / or polypeptide of the present application.

[0009] In one embodiment, the present application provides nucleic acids encoding isolated antigen-binding proteins and / or polypeptides of the present application.

[0010] According to one embodiment, the present application provides a vector containing the nucleic acid of the present application.

[0011] In one embodiment, the present application provides cells comprising the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application and / or the vector of the present application.

[0012] In one embodiment, the present application provides a method for producing an isolated antigen-binding protein and / or polypeptide of the present application, comprising culturing cells of the present application under conditions for expressing the isolated antigen-binding protein and / or polypeptide.

[0013] In one embodiment, the present application provides a composition comprising the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application and / or the cells of the present application, and optionally a pharmaceutically acceptable adjuvant.

[0014] According to one aspect, the present application provides a reagent kit comprising the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application and / or the composition of the present application.

[0015] According to one aspect, the present application provides a method for inhibiting the proliferation of cells overexpressing IGF-1R or its functionally active fragment, inhibiting the MAPK pathway and / or inhibiting the interaction between IGF-1R or its functionally active fragment and its ligand, the method comprising administering the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, the composition of the present application and / or the reagent kit of the present application.

[0016] According to one aspect, the present application provides a method for detecting the presence and / or content of IGF-1R or its functionally active fragment, the method comprising administering the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, the composition of the present application and / or the reagent kit of the present application.

[0017] According to one aspect, the present application provides the use of the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, the composition of the present application and / or the reagent kit of the present application in the manufacture of a drug, wherein the drug is used for preventing, alleviating and / or treating a disease or disorder.

[0018] Those skilled in the art can recognize other aspects and advantages of the present application from the detailed description below. The following detailed description only shows and describes exemplary embodiments of the present application. As will be recognized by those skilled in the art, based on the content of the present application, those skilled in the art can modify the disclosed specific embodiments without departing from the spirit and scope of the invention related to the present application. Correspondingly, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive. [Brief explanation of the drawing]

[0019] The specific features of the invention relating to this application are described in the attached claims. The features and advantages of the invention relating to this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows.

[0020] [Figure 1] The SDS-PAGE diagram of the nano-antibody of this invention is shown. [Figure 2] The figure shows the results of the binding of the nanoantibody-Fc fusion protein of this application to recombinant human, monkey, and mouse IGF1R protein and human insulin receptor protein. [Figure 3] The figure shows the results of the inhibitory effect of the nanoantibody-Fc fusion protein of this invention on the phosphorylation of IGF-1-induced Erk protein. [Figure 4] The nanoantibody-Fc fusion protein of this invention exhibits inhibitory activity against the phosphorylation of IGF-1-induced MEK, Erk, and Akt proteins (Note: Fc' indicates that the subtype is IgG1 C220S L234A L235A D356E L358M). [Modes for carrying out the invention]

[0021] Embodiments of the present invention will be described below with reference to specific examples, and those skilled in the art will be able to easily understand other advantages and effects of the present invention from the contents disclosed herein.

[0022] Definition of Terms

[0023] In this application, the term “antigen-binding protein” generally refers to a polypeptide molecule capable of specifically recognizing and / or neutralizing a particular antigen. In this application, the term “antigen-binding protein” may also include “antibody” or “antigen-binding fragment.” The term “antibody” may include monoclonal antibodies, antibody fragments, or antibody derivatives, and includes, but is not limited to, camel antibodies, fully human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies (such as scFv), and antibody fragments that bind to antigens (such as Fab, Fab', and (Fab)2 fragments). The term “antibody” may further include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, and any antibody fragments and derivatives thereof that bind to antigens described herein. In this application, the term “antibody” may also include single-domain antibodies.

[0024] In this application, the term “isolated” generally means obtained from its natural state by artificial means. When an “isolated” substance or component exists in nature, it may be that the natural environment in which it exists has been altered, or that the substance has been separated from its natural environment, or both. For example, if a polynucleotide or polypeptide that is not isolated in vivo exists naturally in a living animal, a high-purity version of the same polynucleotide or polypeptide isolated from this natural state is called isolated. The term “isolated” does not exclude mixtures of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0025] In this application, the term "single-domain antibody" generally refers to a class of antibodies in which the light chain is deleted and only the heavy chain variable region exists. Research has revealed that Bactrian camels, dromedary camels, alpacas, and llamas possess heavy chain antibodies (hcAbs) composed only of heavy chains but possessing complete functionality. The molecular weight of the variable domains of the hcAb (VHH) is only 1 / 10 that of conventional antibodies, making them the smallest molecular fragment with complete antibody function currently available, and they are called single-domain antibodies (sdAbs). Compared to other antibodies, single-domain antibodies have advantages such as low immunogenicity, small molecular size, and strong permeability, and therefore have the potential for a wide range of applications in fields such as basic research, drug development, and disease treatment. For example, single-domain antibodies can be derived from alpacas. Single-domain antibodies can be composed of heavy chain variable regions (VH). The term "heavy chain variable region" generally refers to the amino-terminal structural domain of the heavy chain of the antigen-binding fragment. The heavy chain variable region can be further distinguished into a more conserved region called the framework region (FR) and a hypervariable region called the complementarity-determining region (CDR). Each heavy chain variable region can consist of three CDR and four FR regions, which are arranged in the order H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3, and H-FR4 from the amino terminus to the carboxyl terminus. The heavy chain variable region contains a binding domain that interacts with the antigen of the present invention. The appropriate boundaries of the CDRs are defined differently depending on the different system. The system described by Kabat not only provides a clear residue numbering system applicable to any variable region of an antigen-binding protein, but also provides precise residue boundaries that define the CDRs. These CDRs may be called Kabat CDRs. Chothia et al. found that despite great diversity at the amino acid sequence level, certain subparts within Kabat CDRs adopt nearly identical peptide backbone structures.These subparts are named L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light chain region and heavy chain region, respectively. These regions may also be called Chothia CDRs, and the Chothia CDRs have boundaries that overlap with Kabat CDRs. Other boundaries that limit CDRs that overlap with Kabat CDRs are described in Padlan and MacCallum. Other CDR boundary definitions may not strictly follow any of the above systems, but they still overlap with Kabat CDRs and may be shortened or lengthened according to specific residues or groups of residues or even the entire CDR without significantly affecting antigen binding prediction or experimental expression. In this application, CDRs may be defined using the Contact numbering system. In this application, CDRs may be defined using the Abm numbering system. In this application, CDRs may be defined using the Chothia numbering system. In this application, CDRs may be defined using the Kabat numbering system. In this application, CDRs may be defined using the IMGT numbering system. In this application, the term “single-domain antibody” is used interchangeably with “nano antibody” and “VHH.” The scope of protection claimed in this application is the sequence shown according to the definition of the IMGT numbering system, but amino acid sequences corresponding to other CDR definition rules should also be included in the scope of claims. Exemplary definition rules are shown in the table below.

[0026] In this application, the term "monoclonal antibody" generally refers to a group of antibodies that are essentially the same; that is, each antibody in the group is identical except for any naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and directly target a single antigen site. For example, such monoclonal antibodies can be produced by hybridoma technology or by recombinant DNA methods in bacteria, eukaryotic cells, or plant cells. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques known in the art.

[0027] In this application, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is the same as the corresponding amino acid sequence of an antibody derived from a particular species, or which belongs to a particular class, but the remaining segments of the chain are the same as the corresponding sequence of another species. For example, both the variable regions of the light and heavy chains are derived from the antibody variable region of a certain animal species (mouse, rat, etc.), while the constant region is the same as the antibody sequence of another species (human, etc.). For example, to obtain a chimeric antibody, non-human B cells or hybridoma cells are used to generate the variable region, and the constant region combined with them is derived from humans. The variable region has the advantage of being easy to manufacture, and its specificity is not affected by the origin of the constant region combined with it. At the same time, because the constant region of the chimeric antibody can be derived from humans, the likelihood of the antibody inducing an immune response when the chimeric antibody is injected is lower than when the constant region is derived from a non-human antibody.

[0028] In this application, the terms “IGF-1R,” “IGF1R,” “IGF-IR,” or “IGFIR” generally refer to insulin-like growth factor receptor 1. IGF-1R as used herein may include its isoforms, species homologs, functionally active fragments, and analogs having at least one common epitope. For example, the “functionally active fragment” may include a fragment that retains the endogenous function of at least one naturally occurring protein. An exemplary amino acid sequence of IGF-1R can be shown in UniProt registry number P08069. For example, ligands for IGF-1R may include IGF-1 and / or IGF-2.

[0029] In this application, the terms “IGF-1,” “IGF1,” “IGF-I,” or “IGFI” generally refer to insulin-like growth factor 1. IGF-1 as used herein may include its isoforms, species homologs, functionally active fragments, and analogs having at least one common epitope. For example, the “functionally active fragment” may include a fragment that retains the endogenous function of at least one naturally occurring protein. An exemplary amino acid sequence of IGF-1 can be shown in UniProt registry number P05019.

[0030] In this application, the terms “IGF-2,” “IGF2,” “IGF-II,” or “IGFII” generally refer to insulin-like growth factor 2. IGF-2 as used herein may include its isoforms, species homologs, functionally active fragments, and analogs having at least one common epitope. For example, the “functionally active fragment” may include a fragment that retains the endogenous function of at least one naturally occurring protein. An exemplary amino acid sequence of IGF-2 can be shown in UniProt registry number P01344.

[0031] In addition to the specific proteins and nucleotides referred to herein, this application may also include functional variants, derivatives, analogues, homologs and fragments thereof.

[0032] The term "functional variant" refers to a polypeptide having an amino acid sequence that is essentially identical to a naturally occurring sequence, or encoded by an essentially identical nucleotide sequence, and capable of having one or more of the activities of a naturally occurring sequence. In the context of this application, a variant of any given sequence refers to a sequence in which a specific sequence of residues (both amino acid and nucleotide residues) is modified to cause the polypeptide or polynucleotide to retain essentially at least one endogenous function. As long as the original functional activity is preserved, variant sequences can be obtained by adding, deleting, substituting, modifying, replacing and / or muting at least one amino acid residue and / or nucleotide residue present in naturally occurring proteins and / or polynucleotides.

[0033] In this application, the term “derivative” generally refers to any substitution, mutation, modification, exchange, deletion and / or addition of one (or more) amino acid residues of the self / paired sequence, insofar as the resulting polypeptide or polynucleotide retains at least one of its endogenous functions.

[0034] In this application, the term “analog” generally includes any mimicry of a polypeptide or polynucleotide, i.e., a chemical compound having at least one endogenous function of the polypeptide or polynucleotide that the mimicry imitates.

[0035] In general, amino acid substitutions, such as at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, can be made, as long as the modified sequence retains the essential activity or capability. Amino acid substitutions may include the use of unnaturally occurring analogs.

[0036] In this application, the term “homologous” generally refers to an amino acid sequence or nucleotide sequence that has a certain degree of homology to a naturally occurring sequence. The term “homologous” can be equated with sequence “identical.” A homologous sequence may include an amino acid sequence that is at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. Generally, a homologous sequence includes the same active site as the subject amino acid sequence. Homologousity can be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), and homology can also be expressed in terms of sequence identity. In this application, a sequence in which any one of the SEQ ID NO: of the amino acid sequence or nucleotide sequence referred to has percentage identity refers to a sequence in which the SEQ ID NO: has said percentage identity over the entire length of the referred SEQ ID NO: To determine sequence identity, sequence alignment can be performed, using various methods familiar to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithm necessary to achieve optimal alignment across the full-length sequences being compared.

[0037] The proteins or polypeptides used in this invention may have deletions, insertions, or substitutions of amino acid residues that result in silent changes and functionally equivalent proteins. Intentional amino acid substitutions can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as the endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with nonpolar head groups having similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0038] In this application, the term “tumor” generally refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumor-forming factors. For example, the tumor may include solid tumors. For example, the tumor may include hematological tumors. For example, the tumor may include tumors associated with the expression of the antigen of this application. The term “tumor associated with the expression of the antigen of this application” generally refers to a tumor formed by changes in the expression of the antigen of this application resulting in disease progression or evasion of immune surveillance. For example, the “tumor associated with the expression of the antigen of this application” may be a tumor formed by upregulation of the expression level of the antigen of this application resulting in disease progression or evasion of immune surveillance. The tumor associated with the protein expression of the antigen of this application may be a tumor that is positive for the antigen of this application. In a tumor that is positive for the antigen of this application, the expression level of the protein of the antigen of this application on the tumor cell surface or in the tumor microenvironment is approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher compared to normal cells.

[0039] In this application, the term "solid tumor" generally refers to a tangible mass that can be detected clinically (e.g., by X-ray, CT scan, B-ultrasound, or palpation). For example, the solid tumor may be selected from breast cancer.

[0040] In this application, the term "hematological malignancy" generally refers to tumors that are not visible or palpable by X-ray, CT scan, B-ultrasound, and palpation.

[0041] In this application, the term "immune complex" generally refers to a complex formed when the aforementioned other reagents (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents) are combined (e.g., covalently bonded by linking molecules) with the isolated antigen-binding protein, and this complex delivers the aforementioned other reagents to target cells (such as tumor cells) through the specific binding of the isolated antigen-binding protein to the antigen in the target cells. The immune complex then enters the interior of the target cell through internalization (e.g., into vesicles such as lysosomes), in which case the linking molecules in the immune complex are cleaved, releasing the aforementioned other reagents and thereby exerting their cytotoxic effects. The antigen may also be secreted by the target cell and located in the gaps outside the target cell.

[0042] In this application, the term “subject” generally refers to humans or non-human animals, including but not limited to cats, dogs, horses, pigs, cattle, sheep, rabbits, mice, rats, or monkeys.

[0043] In this application, the term "nucleic acid molecule" generally refers to isolated forms of nucleotides, deoxyribonucleotides, or ribonucleotides of any length, isolated from the natural environment or artificially synthesized, or analogs thereof.

[0044] In this application, the term “vector” generally refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. The vector can transport the inserted nucleic acid molecule intracellularly and / or between cells. The vector may include vectors primarily used for the insertion of DNA or RNA into cells, vectors primarily used for the replication of DNA or RNA, and vectors primarily used for the transcription and / or translation of DNA or RNA. The vector may be a polynucleotide that can be transcribed and translated into polypeptides when appropriate cells are introduced. Generally, by culturing appropriate cells containing the vector, the vector can produce a desired expression product. In this application, the vector may include a lentiviral vector.

[0045] In this application, the term “cell” generally refers to individual cells, cell lines, or cell cultures that may include, or already include, plasmids or vectors containing the nucleic acid molecules described herein, or that are capable of expressing the polypeptides or antigen-binding proteins described herein. The cells may include single-cell progeny. Due to natural, accidental, or intentional mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, as long as they are capable of expressing the polypeptides or antigen-binding proteins described herein. The cells can be obtained by transfecting cells in vitro using the vectors described herein. The cells may be prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells, e.g., COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some embodiments, the cells may be immune cells. For example, the immune cells can be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0046] In this application, the term “treatment” generally refers to (i) preventing the onset of a disease, symptom, or condition in a patient who is susceptible to such disease, symptom, or condition but has not yet been diagnosed with the disease; (ii) suppressing the disease, symptom, or condition, i.e., preventing its progression; and (iii) alleviating the disease, symptom, or condition, i.e., regressing the disease, symptom, or condition and / or symptoms associated with the disease, symptom, or condition.

[0047] In this application, the terms “polypeptide,” “peptide,” “protein,” and “protein” are used interchangeably and generally refer to polymers of amino acids having any length. Such polymers may be linear or branched, and may contain modified amino acids or be interrupted by non-amino acids. These terms further encompass modified amino acid polymers. These modifications may include the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other operation (such as binding to a marker component). The term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycy, D and L optical isomers, as well as amino acid analogs and peptide mimics.

[0048] In this application, the terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “nucleotide” are used interchangeably and generally refer to polymeric forms of nucleotides having any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after the assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by compounding with labeling components.

[0049] In the present application, the term "K" D "(similarly, "K" D " or "KD") generally refers to the "affinity constant" or the "equilibrium dissociation constant", and at the equilibrium of titration measurements, or the value obtained by dividing the dissociation rate constant (k d ) by the association rate constant (k a ). The association rate constant (k a ), the dissociation rate constant (k d ), and the equilibrium dissociation constant (K D ) represent the binding affinity of a binding protein (e.g., the isolated antigen-binding protein described in the present application) to an antigen (e.g., the antigen protein of the present application). Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K D value can be measured by an Octet molecular interaction analyzer (Sartorius), and can also be measured by other experimental methods and instruments such as a BIAcore biomolecular interaction analysis system (GE Healthcare Life Sciences). Also, the K D value can be used using the KinExA system (Kinetic Exclusion Assay) of Sapidyne Instruments, or the K D value can be measured using a surface plasmon resonance apparatus (SPR). In the present application, the K D value can be measured by BIAcore.

[0050] In the present application, the term "and / or" should be understood to mean either or both of the options.

[0051] In the present application, the term "comprising" generally means including the explicitly specified features but not excluding other elements. In some cases, "comprising" also includes the case of including only the specified components. In some cases, "comprising" also includes the meaning of "consisting of...".

[0052] In this application, the term "approximately" generally refers to a variation within a range of ±0.5% to 10% of the specified value. For example, a variation within a range of ±0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the specified value.

[0053] Details of the invention Section 1. It is an antigen-binding protein, In ELISA detection competition, approximately 1.0 μg / mL or lower IC50 is effective. 50 The value can inhibit the competitive binding of IGF-1R to its ligand. Antigen-binding protein. Section 2. The aforementioned IGF-1R includes IGF-1R derived from primates. The antigen-binding protein described in item 1. Section 3. The ligand for IGF-1R includes IGF-1 and / or IGF-2, as well as the functionally active fragment. The antigen-binding protein described in item 1 or 2. Section 4. It is an antigen-binding protein, The antigen-binding protein can competitively bind to a reference antibody and IGF-1R, and the reference antibody comprises HCDR1-3, where HCDR1-3 each comprises the amino acid sequences shown in SEQ ID NO: 85, 100, and 131, respectively. Antigen-binding protein. Section 5. The reference antibody includes a heavy chain variable region VH, and VH includes the amino acid sequence shown in SEQ ID NO:1. The antigen-binding protein described in item 4. Section 6. It is an antigen-binding protein, The antigen-binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 217 or 218. Antigen-binding protein. Section 7. The antigen-binding protein comprises HCDR3, and the HCDR3 comprises an amino acid sequence represented by any one of SEQ ID NO: 130-153. An antigen-binding protein as described in any one of items 1 to 6. Section 8. The antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 215 or 216. An antigen-binding protein as described in any one of items 1 to 7. Section 9. The antigen-binding protein comprises HCDR2, and the HCDR2 comprises an amino acid sequence represented by any one of SEQ ID NO: 96 to 129. An antigen-binding protein as described in any one of items 1 to 8. Section 10. The antigen-binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 213 or 214. An antigen-binding protein as described in any one of items 1 to 9. Section 11. The antigen-binding protein comprises HCDR1, and the HCDR1 comprises an amino acid sequence represented by any one of SEQ ID NO: 81 to 95. An antigen-binding protein as described in any one of items 1 to 10. Section 12. The heavy chain variable region VH is included, and the VH comprises the HCDR1, HCDR2, and HCDR3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 217 or 218, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 215 or 216, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 213 or 214. An antigen-binding protein as described in any one of items 1 to 11. Section 13. The heavy chain variable region VH is included, and the VH comprises the HCDR1, HCDR2, and HCDR3, wherein the HCDR3 comprises an amino acid sequence represented by any one of SEQ ID NO: 130 to 153, the HCDR2 comprises an amino acid sequence represented by any one of SEQ ID NO: 96 to 129, and the HCDR1 comprises an amino acid sequence represented by any one of SEQ ID NO: 81 to 95. An antigen-binding protein as described in any one of items 1 to 12. Section 14. The aforementioned antigen-binding proteins include HCDR1, HCDR2, and HCDR3, where HCDR1, HCDR2, and HCDR3 are selected from the group consisting of the following amino acid sequences. Table 1 Table 2 An antigen-binding protein as described in any one of items 1 to 13. Item 15. A molecule comprising H-FR1, wherein the C-terminus of H-FR1 is directly or indirectly ligated to the N-terminus of HCDR1, and H-FR1 comprises an amino acid sequence represented by any one of SEQ ID NO: 219, 220, and 154-186. An antigen-binding protein as described in any one of items 1 to 14. Section 16. It contains H-FR2, the H-FR2 is located between HCDR1 and HCDR2, and the H-FR2 contains an amino acid sequence represented by any one of SEQ ID NO: 221, 222, and 187-195. An antigen-binding protein as described in any one of items 1 to 15. Section 17. It contains H-FR3, the H-FR3 is located between HCDR2 and HCDR3, and the H-FR3 contains an amino acid sequence represented by either SEQ ID NO: 223 or 196-203. An antigen-binding protein as described in any one of items 1 to 16. Section 18. It contains H-FR4, the N-terminus of H-FR4 is directly or indirectly ligated to the C-terminus of HCDR3, and H-FR4 contains the amino acid sequence shown in either SEQ ID NO: 224 or 204-206. An antigen-binding protein as described in any one of items 1 to 17. Section 19. It comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 219 or 220, H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 221 or 222, H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 223, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 224. An antigen-binding protein as described in any one of items 1 to 18. Section 20. It includes a heavy chain variable region VH, and said VH includes the amino acid sequence shown in SEQ ID NO: 225 or 226. An antigen-binding protein as described in any one of items 1 to 19. Section 21. It includes a heavy chain variable region VH, and said VH includes an amino acid sequence represented by any one of SEQ ID NO: 1 to 80. An antigen-binding protein as described in any one of items 1 to 20. Section 22. Containing an antibody or its antigen-binding fragment, An antigen-binding protein as described in any one of items 1 to 21. Section 23. A single-domain antibody or its antigen-binding fragment, An antigen-binding protein as described in any one of items 1 to 22. Section 24. The antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb. The antigen-binding protein described in item 22. Section 25. The aforementioned antibodies include camel antibodies, chimeric antibodies, humanized antibodies and / or fully human antibodies. The antigen-binding protein described in item 22. Section 26. Contains an amino acid sequence shown in any one of SEQ ID NO:1~80. An antigen-binding protein as described in any one of items 1 to 25. Section 27. It is a polypeptide, A substance comprising an antigen-binding protein described in any one of items 1 to 26, Polypeptide. Section 28. The polypeptide further comprises a structure that can improve the stability of the antigen-binding protein and / or bind to the Fc receptor. Polypeptides as described in item 27. Section 29. The polypeptide further comprises an immunoglobulin Fc region. A polypeptide as described in any one of items 27-28. Section 30. The antigen-binding protein is directly or indirectly linked to the Fc region. Polypeptides as described in item 29. Section 31. The C-terminus of the VH of the antigen-binding protein is directly or indirectly linked to the N-terminus of the Fc region. A polypeptide as described in any one of items 29 to 30. Section 32. The Fc region includes Fc derived from IgG1 and / or Fc derived from IgG4. A polypeptide as described in any one of items 29 to 31. Section 33. The Fc region includes the amino acid sequence shown in SEQ ID NO:227. A polypeptide as described in any one of items 29 to 32. Section 34. The Fc region includes an amino acid sequence represented by any one of SEQ ID NO:209~212. A polypeptide as described in any one of items 29 to 33. Section 35. It is an immune complex, A comprising an isolated antigen-binding protein as described in any one of items 1 to 26 and / or a polypeptide as described in any one of items 27 to 34, immune complex. Section 36. nucleic acids, Encoding an isolated antigen-binding protein and / or a polypeptide as described in any one of items 1 to 26, Nucleic acid. Section 37. It is a vector, Including the nucleic acids described in item 36, vector. Section 38. It is a cell, A comprising an isolated antigen-binding protein as described in any one of items 1 to 26, a polypeptide as described in any one of items 27 to 34, an immune complex as described in item 35, a nucleic acid as described in item 36, and / or a vector as described in item 37. cell. Section 39. A method for producing an isolated antigen-binding protein and / or a polypeptide as described in any one of items 1 to 26 and / or as described in any one of items 27 to 34, The process involves culturing the cells described in item 38 under conditions that express the isolated antigen-binding protein and / or the polypeptide, method. Section 40. A composition, A combination comprising an isolated antigen-binding protein as described in any one of items 1 to 26, a polypeptide as described in any one of items 27 to 34, an immune complex as described in item 35, a nucleic acid as described in item 36, a vector as described in item 37 and / or a cell as described in item 38, and optionally a pharmaceutically acceptable adjuvant. composition. Section 41. It is a reagent kit, A composition comprising an isolated antigen-binding protein as described in any one of items 1 to 26, a polypeptide as described in any one of items 27 to 34, an immune complex as described in item 35, a nucleic acid as described in item 36, a vector as described in item 37, a cell as described in item 38, and / or a composition as described in item 40. Reagent kit. Section 42. A method for inhibiting the proliferation of cells overexpressing IGF-1R or its functionally active fragment, inhibiting the MAPK pathway, and / or inhibiting the interaction between IGF-1R or its functionally active fragment and its ligand, The method includes administering an isolated antigen-binding protein as described in any one of items 1 to 26, a polypeptide as described in any one of items 27 to 34, an immune complex as described in item 35, a nucleic acid as described in item 36, a vector as described in item 37, cells as described in item 38, a composition as described in item 40, and / or a reagent kit as described in item 41. method. Section 43. Inhibiting the aforementioned MAPK pathway includes inhibiting the phosphorylation of MAPK pathway proteins. The method described in item 42. Section 44. The MAPK pathway protein comprises MEK, Erk1 / 2 and Akt, or the above-mentioned functionally active fragment. The method described in any one of items 42 to 43. Section 45. The ligand for IGF-1R includes IGF-1 and / or IGF-2, as well as the functionally active fragment. The method described in any one of items 42 to 44. Section 46. A method for detecting the presence and / or content of IGF-1R or its functionally active fragment, The method includes administering an isolated antigen-binding protein as described in any one of items 1 to 26, a polypeptide as described in any one of items 27 to 34, an immune complex as described in item 35, a nucleic acid as described in item 36, a vector as described in item 37, cells as described in item 38, a composition as described in item 40, and / or a reagent kit as described in item 41. method. Section 47. The use of an isolated antigen-binding protein according to any one of claims 1 to 26, a polypeptide according to any one of claims 27 to 34, an immune complex according to claim 35, a nucleic acid according to claim 36, a vector according to claim 37, a cell according to claim 38, a composition according to claim 40, and / or a reagent kit according to claim 41 in the manufacture of a drug, The aforementioned drug is used to prevent, alleviate and / or treat a disease or illness. use. Section 48. The aforementioned diseases or conditions include tumors. Use as described in item 47. Section 49. The aforementioned disease or condition includes tumors associated with the expression of IGF-1R or its functionally active fragment. Use as described in any one of paragraphs 47-48. Section 50. The aforementioned diseases or conditions include breast cancer. Use as described in any one of paragraphs 47-49.

[0054] This application provides an antigen-binding protein that can inhibit the competitive binding of IGF-1R to its ligand at an IC50 value of approximately 1.0 μg / mL or lower in ELISA detection competition. The isolated antigen-binding protein described herein has an IC50 value of approximately 2 μg / mL or lower. 50 (For example, the EC 50The binding of IGF-1 to IGF-1R can be inhibited at concentrations of approximately 1.9 μg / mL or less, approximately 1.8 μg / mL or less, approximately 1.7 μg / mL or less, approximately 1.5 μg / mL or less, approximately 1.3 μg / mL or less, approximately 1.1 μg / mL or less, approximately 1.0 μg / mL or less, approximately 0.9 μg / mL or less, approximately 0.8 μg / mL or less, approximately 0.7 μg / mL or less, 0.6 μg / mL or less, approximately 0.5 μg / mL or less, approximately 0.4 μg / mL or less, approximately 0.3 μg / mL or less, approximately 0.2 μg / mL or less, or approximately 0.1 μg / mL or less or less.

[0055] For example, the IGF-1R may include IGF-1R derived from primates. For example, the ligand for the IGF-1R may include IGF-1 and / or IGF-2, as well as the functionally active fragment.

[0056] This application provides an antigen-binding protein that can competitively bind to a reference antibody and IGF-1R, wherein the reference antibody may contain HCDR1-3, and the HCDR1-3 may contain amino acid sequences shown in SEQ ID NO: 85, 100, and 131, respectively. In this application, CDRs can be defined using the Contact numbering system. In this application, CDRs can be defined using the Abm numbering system. In this application, CDRs can be defined using the Chothia numbering system. In this application, CDRs can be defined using the Kabat numbering system. In this application, CDRs can be defined using the IMGT numbering system. Those skilled in the art can determine the CDR region using different numbering systems depending on the sequence and structure of the antibody. Using different numbering systems may result in different CDR regions. In this application, the CDR encompasses CDR sequences partitioned by any CDR partitioning scheme, and also includes variants thereof, the variants including substitutions, deletions, and / or additions of one or more amino acids to the amino acid sequence of the CDR. For example, substitutions, deletions, and / or insertions of 1 to 30, 1 to 20, or 1 to 10 amino acids, and for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, including homologs thereof, wherein the homologs may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology to the amino acid sequence of the CDR. In some embodiments, the CDR can be determined by a Kabat numbering method.

[0057] For example, the reference antibody may include a heavy chain variable region VH, and VH may include the amino acid sequence shown in SEQ ID NO:1. For example, the reference antibody may be the 5115 antibody of this application.

[0058] In this application, the antigen-binding protein may include a heavy chain variable region VH, and the VH may include at least one, two, or three of HCDR1, HCDR2, and HCDR3. For example, the sequence of HCDR3 of the antigen-binding protein can be determined according to Kabat definition rules.

[0059] For example, this application provides several methods for defining CDR. Taking the 5115 antibody as an example:

[0060] [Table 1]

[0061] This application provides an antigen-binding protein. The antigen-binding protein may contain HCDR3, and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 217 (NPR[RY]AT[PT][DG][FLMY][HIKLMQRST][AQRS]Y[AKL]Y) or 218 (NPRRATPD[LY]TQY[AL]Y). The letters in square brackets indicate the types of amine acids that can be selected at that site.

[0062] For example, the antigen-binding protein may contain HCDR3, and the HCDR3 may contain an amino acid sequence represented by any one of SEQ ID NO: 130 to 153. CDRs can be defined using the Kabat numbering system.

[0063] For example, the antigen-binding protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 215 (AISG[FGHKQRST]G[IKLMQRST][QRV][AFHLMQSTVY][LNRTY]YADSVKG) or 216 (AISGSG[MS]RA[RY]YADSVKG). The letters in square brackets indicate the types of amino acids that can be selected at that site.

[0064] For example, the antigen-binding protein may include HCDR2, and the HCDR2 may include an amino acid sequence represented by any one of SEQ ID NO: 96 to 129. CDRs can be defined using the Kabat numbering system.

[0065] For example, the antigen-binding protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 213 ([GRST][FLY]V[LM][AGMS]) or 214 (S[FL]V[LM][AS]). The letters in square brackets indicate the types of amino acids that can be selected at that site.

[0066] For example, the antigen-binding protein may include HCDR1, and the HCDR1 may include an amino acid sequence represented by any one of SEQ ID NO: 81 to 95. CDRs can be defined using the Kabat numbering system.

[0067] For example, the antigen-binding protein may include a heavy chain variable region VH, and VH may include HCDR1, HCDR2, and HCDR3, HCDR3 may include the amino acid sequence shown in SEQ ID NO: 217 or 218, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 215 or 216, and HCDR1 may include the amino acid sequence shown in SEQ ID NO: 213 or 214.

[0068] For example, the antigen-binding protein may include a heavy chain variable region VH, and VH may include HCDR1, HCDR2, and HCDR3, HCDR3 may include an amino acid sequence shown in any one of SEQ ID NO: 130 to 153, HCDR2 may include an amino acid sequence shown in any one of SEQ ID NO: 96 to 129, and HCDR1 may include an amino acid sequence shown in any one of SEQ ID NO: 81 to 95.

[0069] For example, the antigen-binding protein may include H-FR1, the C-terminus of H-FR1 being directly or indirectly linked to the N-terminus of HCDR1, and H-FR1 may include the amino acid sequence shown in any one of SEQ ID NO: 219 (EVQLVES[GS]GGLVQ[AP]G[DG]SLRLSCA[AV]S[GHLMRST][AGIKPRTV][AEFGPRSTY][FLR][KNPRS]), 220 (EVQLVESGGGLVQPGGSLRLSCAASGRTF[RS]), and 154-186. The letters in square brackets indicate the types of amino acids that can be selected at that site.

[0070] For example, the antigen-binding protein may contain H-FR2, which is located between HCDR1 and HCDR2, and which may contain an amino acid sequence represented by SEQ ID NO: 221 (WFRQAPGK[EG][LR]E[FL]V[AS]), 222 (WFRQAPGK[EG][LR]EFVS), and one of 187-195. The letters in square brackets indicate the types of amino acids that can be selected at that site.

[0071] For example, the antigen-binding protein may contain H-FR3, which is located between HCDR2 and HCDR3, and which may contain the amino acid sequence shown in SEQ ID NO:223(RF[AT][IV]SRDN[AS][KN]NT[LV]YLQM[NS]SL[KR][AP][DE]DT[AG][LV]YYCAA) and any one of 196-203. The letters in square brackets indicate the types of amino acids that can be selected at that site.

[0072] For example, the antigen-binding protein may include H-FR4, the N-terminus of H-FR4 may be directly or indirectly linked to the C-terminus of HCDR3, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 224 (WGQGT[LQ]VTVSS) and any one of 204-206. The letters in square brackets indicate the types of amino acids that can be selected at that site.

[0073] For example, the antigen-binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, where H-FR1 may include the amino acid sequence shown in SEQ ID NO: 219 or 220, H-FR2 may include the amino acid sequence shown in SEQ ID NO: 221 or 222, H-FR3 may include the amino acid sequence shown in SEQ ID NO: 223, and H-FR4 may include the amino acid sequence shown in SEQ ID NO: 224.

[0074] For example, the amino acid information (SEQ ID NO) for each region corresponding to the antigen-binding protein number of this application is shown below.

[0075] [Table 2]

[0076] [Table 3]

[0077] [Table 4]

[0078] [Table 5]

[0079] For example, the antigen-binding protein of the present invention may include an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 as the above-mentioned antigen-binding protein (e.g., having the same HCDR1-3). In some cases, the antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above-mentioned antigen-binding protein.

[0080] For example, the antigen-binding protein may include a heavy chain variable region VH, and the VH is SEQ ID NO: 225(EVQLVES[GS]GGLVQ[AP]G[DG]SLRLSCA[AV]S[GHLMRST][AGIKPRTV][AEFGPRSTY][FLR][KNPRS][GRST][FLY]V[LM][AGMS]WFRQAPGK[EG][LR]E[FL]V[AS]AISG[FGHKQRST]G[IKLMQRST][QRV][AFHLMQSTVY][LNRTY]YADSVKGRF[AT][IV]SRDN[AS][KN]NT[LV]YLQM[NS]SL[KR][AP][DE]DT[AG][LV]YYCAANPR[RY]AT[PT][DG][FLMY][HIKLMQRST][AQRS]Y[AKL]YWGQGT[LQ]VTVSS) or 226(EVQ The amino acid sequence may include the amino acid sequence shown in LVES[GS]GGLVQ[AP]G[DG]SLRLSCA[AV]S[GHLMRST][AGIKPRTV][AEFGPRSTY][FLR][KNPRS][GRST][FLY]V[LM][AGMS]WFRQAPGK[EG][LR]E[FL]V[AS]AISG[FGHKQRST]G[IKLMQRST][QRV][AFHLMQSTVY][LNRTY]YADSVKGRF[AT][IV]SRDN[AS][KN]NT[LV]YLQM[NS]SL[KR][AP][DE]DT[AG][LV]YYCAANPR[RY]AT[PT][DG][FLMY][HIKLMQRST][AQRS]Y[AKL]YWGQGT[LQ]VTVSS). The letters in square brackets indicate the type of amino acid that can be selected at that site.

[0081] For example, the antigen-binding protein may include a heavy chain variable region VH, and VH may include an amino acid sequence shown in any one of SEQ ID NO: 1 to 80.

[0082] For example, the antigen-binding protein may include HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH, and VH may include an amino acid sequence shown in any one of SEQ ID NO: 1 to 80.

[0083] For example, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof.

[0084] For example, the antigen-binding protein may include a single-domain antibody or an antigen-binding fragment thereof.

[0085] For example, the antigen-binding fragment here may be selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0086] For example, the antibody here may include camel antibodies, chimeric antibodies, humanized antibodies, and / or fully human antibodies.

[0087] For example, the antigen-binding protein may contain an amino acid sequence represented by any one of SEQ ID NO: 1 to 80.

[0088] This application provides a polypeptide which may contain the antigen-binding protein of this application.

[0089] For example, the polypeptide may further include a structure that improves the stability of the antigen-binding protein and / or can bind to the Fc receptor.

[0090] For example, the polypeptide may further contain an immunoglobulin Fc region.

[0091] For example, the antigen-binding protein can be directly or indirectly linked to the Fc region.

[0092] For example, the C-terminus of the VH region of the antigen-binding protein can be directly or indirectly linked to the N-terminus of the Fc region.

[0093] For example, the Fc region here may include Fc derived from IgG1 and / or Fc derived from IgG4.

[0094] For example, the Fc region here may include the amino acid sequence shown in SEQ ID NO:227(EPKS[ACS]DKTHTCPPCPAPE[AL][AL]GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR[DE]E[LM]TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK). The letters in square brackets indicate the types of amine acids that can be selected at that site.

[0095] For example, the Fc region here may include an amino acid sequence represented by any one of SEQ ID NO:209~212.

[0096] This application provides an immune complex which may contain the isolated antigen-binding protein and / or polypeptide of the present application.

[0097] This application provides nucleic acids capable of encoding the isolated antigen-binding protein and / or polypeptide of the present application.

[0098] The nucleic acid molecules described herein may be isolated. For example, they may be produced or synthesized by (i) in vitro amplification such as polymerase chain reaction (PCR) amplification, (ii) clonal recombination, (iii) purification by enzymatic digestion and gel electrophoresis fractionation, or (iv) chemical synthesis. For example, the isolated nucleic acid may be a nucleic acid molecule produced by recombinant DNA technology. In this application, nucleic acids encoding the isolated antigen-binding protein may be produced by various methods known in the art. These methods include, but are not limited to, obtaining the nucleic acid molecule of the isolated antigen-binding protein described herein using reverse transcription PCR and PCR.

[0099] This application provides an expression vector which may contain the nucleic acid of the present application. Each vector may contain one or more of the nucleic acid molecules. The vector may also contain other genes, such as marker genes, that enable selection of the vector in a suitable host cell and under suitable conditions. The vector may further contain an expression regulatory element that enables precise expression of the coding region in a suitable host. Such regulatory elements are well known to those skilled in the art and include, for example, promoters, ribosome binding sites, enhancers, and other regulatory elements that regulate gene transcription or mRNA translation. In some embodiments, the expression regulatory sequence is a tunable element. The specific structure of the expression regulatory sequence may vary depending on the function of the species or cell type, but generally includes a 5' untranscribed sequence and 5' and 3' untranslated sequences, such as a TATA cut, cap sequence, and CAAT sequence, which are involved in the initiation of transcription and translation, respectively. For example, the 5' untranscribed expression regulatory sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptionally regulating functionally linked nucleic acids. The expression regulatory sequence may further include an enhancer sequence or an upstream activator sequence. In this application, suitable promoters may include, for example, promoters for use with SP6, T3, and T7 polymerases, human U6RNA promoters, CMV promoters, and their artificial hybrid promoters (such as CMV), wherein a portion of the promoter may be fused with a portion of the promoter of another cellular protein (such as human GAPDH or glyceraldehyde-3-phosphate dehydrogenase) gene, and may or may not include additional introns. One or more nucleic acid molecules described in this application are operably linked to the expression regulatory element.

[0100] The vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. For example, the vector may be a viral vector. The viral vector may be administered directly to the patient (in vivo) or indirectly, for example, by treating cells with the virus in vitro and then administering the treated cells to the patient (ex vivo). Viral vector technology is well known in the art and is described, for example, in manuals on virology and molecular biology. Conventional virus-based systems may include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene transfer. In some cases, genes can be introduced and incorporated into the host genome using retroviral, lentiviral, and adeno-associated virus methods, and the inserted gene can be expressed for a long period of time. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce relatively high viral titers. The lentiviral vector may include a long terminal repeat sequence (5'LTR) and a truncated 3'LTR, RRE, a rev response element (cPPT), a central termination sequence (CTS), and / or a post-translational regulatory element (WPRE). The vectors described herein can be introduced into cells.

[0101] This application provides a host cell which may contain the isolated antigen-binding protein, the polypeptide, the immune complex, the nucleic acid, and / or the vector of the Application. The cell may contain the isolated antigen-binding protein, the polypeptide, the immune complex, one or more nucleic acid molecules, and / or one or more vectors. For example, each type of cell or each cell may contain one or one nucleic acid molecule or vector of the Application. For example, each type of cell or each cell may contain multiple (e.g., two or more) or multiple types (e.g., two or more) nucleic acid molecules or vectors of the Application. For example, the vector of the Application can be introduced into the host cell, for example, a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / O cell, a HEK293T cell, a 293F cell, or a HEK293A cell, or other eukaryotic cells such as plant-derived cells, fungal or yeast cells. The vector described herein can be introduced into host cells by methods known in the art, such as electroporation, lipofectine transfection, or lipofectamin transfection. For example, the cells may include yeast cells. For example, the cells may include Escherichia coli cells. For example, the cells may include mammalian cells. For example, the cells may include immune cells.

[0102] A method for producing an isolated antigen-binding protein and / or polypeptide of the present application, the method comprising culturing cells of the present application under conditions for expressing the isolated antigen-binding protein and / or polypeptide.

[0103] The composition may comprise the isolated antigen-binding protein of the Application, the polypeptide of the Application, the immune complex of the Application, the nucleic acid of the Application, the vector of the Application, and / or the cells of the Application, and optionally a pharmaceutically acceptable adjuvant. In the Application, the pharmaceutically acceptable adjuvant may comprise buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants. Any conventional medium or reagent is intended for use in the pharmaceutical compositions herein, unless it is compatible with the cells described herein. In the Application, the pharmaceutically acceptable excipients may comprise additives other than the main active ingredient in the drug formulation, also known as adjuvants.

[0104] This application provides a reagent kit which may contain the isolated antigen-binding protein, the polypeptide, the immune complex, the nucleic acid, the vector, the cells, and / or the composition of the application.

[0105] The present application provides a method for inhibiting the proliferation of cells overexpressing IGF-1R or a functionally active fragment thereof, which may include administering the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, the composition of the present application, and / or the reagent kit of the present application. The present application also provides a method for inhibiting the MAPK pathway, which may include administering the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, the composition of the present application, and / or the reagent kit of the present application. For example, inhibiting the MAPK pathway may include inhibiting the phosphorylation of MAPK pathway proteins. For example, the MAPK pathway proteins may include MEK, Erk1 / 2 and Akt, or the functionally active fragments described above. In the present application, the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.

[0106] The present application provides a method for inhibiting the interaction between IGF-1R or a functionally active fragment thereof and its ligand, which may include administering the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, the composition of the present application, and / or the reagent kit of the present application. For example, the ligand may include IGF-1 or a functionally active fragment thereof. In the present application, the method may include an in vitro method, an ex vivo method, a method for non-diagnostic or non-therapeutic purposes.

[0107] The present application provides a method for detecting the presence and / or content of IGF-1R or its functionally active fragment, which may include administering the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immune complex of the present application, the nucleic acid of the present application, the vector of the present application, the cells of the present application, the composition of the present application, and / or the reagent kit of the present application. In the present application, the method may include an in vitro method, an ex vivo method, a method for non-diagnostic or non-therapeutic purposes. For example, the presence and / or expression level of IGF-1R in a sample obtained from a subject may be determined by contacting a sample with the antigen-binding protein of the present application and detecting the presence and / or content of the antigen-binding protein bound to the sample.

[0108] This application provides the use of the isolated antigen-binding protein, polypeptide, immune complex, nucleic acid, vector, cell, composition, and / or reagent kit of the Application in the manufacture of a drug, wherein the drug is used to prevent, alleviate, and / or treat a disease or condition. For example, the disease or condition may include a tumor. For example, the disease or condition may include a tumor associated with the expression of IGF-1R or a functionally active fragment thereof. For example, the disease or condition may include breast cancer.

[0109] This application provides isolated antigen-binding proteins, polypeptides, immune complexes, nucleic acids, vectors, cells, compositions, and / or reagent kits for preventing, mitigating, and / or treating diseases or conditions. For example, the disease or condition may include tumors. For example, the disease or condition may include tumors associated with the expression of IGF-1R or its functionally active fragments. For example, the disease or condition may include breast cancer.

[0110] The present invention provides a method for preventing, mitigating and / or treating a disease or condition, comprising administering an effective amount of the isolated antigen-binding protein, polypeptide, immune complex, nucleic acid, vector, cell, composition, and / or reagent kit of the present invention to a target subject of interest. For example, the disease or condition may include a tumor. For example, the disease or condition may include a tumor associated with the expression of IGF-1R or a functionally active fragment thereof. For example, the disease or condition may include breast cancer.

[0111] While not intended to be limited by any theory, the following examples are merely illustrative of the protein, preparation method and use of the present invention and do not limit the scope of the present invention.

[0112] Examples

[0113] Example 1: Immunity of Alpaca

[0114] Healthy adult alpacas were immunized with recombinant human IGF-1R protein (his tag). In the initial immunization, 0.5 mg of recombinant human IGF-1R protein was emulsified with an equal volume of Freund's complete adjuvant and then subcutaneously injected at multiple sites. In the booster immunization, 0.25 mg of recombinant human IGF-1R protein was emulsified with an equal volume of Freund's incomplete adjuvant and then subcutaneously injected at multiple sites. A total of three booster immunizations were performed, and antiserum titers were monitored by collecting blood before the initial immunization and one week after each booster immunization.

[0115] Example 2: Construction of an Alpaca Immunology Library

[0116] After immunization is complete, peripheral blood is collected from alpacas, and lymphocytes are separated using lymphocyte separatory solution, and TRIzol TM Total RNA was extracted using Reagent, and cDNA was obtained by reverse transcription using SuperScript® III First-Strand Synthesis System for RT-PCR. The VHH gene was amplified by nested PCR. After recovering the VHH gene fragment using a gel purification kit, it was enzymatically digested with restriction endonuclease sfiI, and then cloned into the phagemide vector pComb 3XSS. The constructed clone product was transformed into E. coli TG1 electroporated competent cells to construct a VHH gene library, and the library volume was measured using plate gradient dilution to obtain a volume of 1.3 × 10⁶. 9 The cells were measured to be cfu, and colony PCR results showed a 100% library insertion rate. After inoculating the gene library into logarithmic culture, the M13K07 phage was used for rescue. After rescue culture, the phages were collected by centrifugation, purified with PEG-NaCl to obtain a phage display library, which was then used for subsequent screening.

[0117] Example 3 Screening and identification of nanoantibodies

[0118] The nanoantibody phage display library constructed in Example 2 was subjected to immunoassay in a test tube blocked with 1% casein (Nunc-lmmuno TMThe phages were placed in an immunotubule (IVT) and incubated upside down at room temperature for 90 minutes to bind, then allowed to stand for 30 minutes to remove nonspecifically binding phages. Subsequently, the library was transferred to immunotubules coated with IGF-1R (hIGF-1R-his) antigen, incubated upside down at room temperature for 90 minutes to bind, then allowed to stand for 30 minutes. The immunotubules were washed 10 times with PBST to remove phages that did not bind or had weak binding affinity. 1 mL of freshly prepared 100 mM triethylamine was added to the immunotubules, and 500 μL of 1 M Tris-HCl, pH 6.4 was added to neutralize and dissociate specific phages. After gentle shaking, the eluate was collected and placed on ice. The eluted material was collected from E. coli TG1 during logarithmic growth, followed by gradient dilution of the post-infection E. coli TG1, and then spread onto LB solid medium (plate dilution method). After 12-14 hours, the plates were counted, the amount of phage eluted was calculated, monoclonal samples were randomly selected, amplified to obtain phages, and identified using enzyme-linked immunosorbent assay (ELISA). The method involved coating 96-well Greiner ELISA plates with 50 nM recombinant human IGF-1R (hIGF1R-his) protein, coating overnight at 4°C, and then blocking with 1% casein. The phages awaiting identification were blocked with 0.1% casein for 1 hour, then placed in the ELISA plates and incubated for 2 hours, washing with PBST between different blocks / incubations. After washing the plate, add anti-M13-HRP (Sinobio, 11973-MM05), bind at 37°C for 1 hour, wash, add TMB substrate solution (Thermo, 34029) to develop color, stop the reaction, and then use a microplate reader to obtain OD 450The following was observed: Positive control wells (enriched libraries for each round), negative control (helper phage), and blank wells (PBS) were set up. A well was considered positive if its OD value was more than three times greater than that of the negative control. Clones corresponding to the positive wells were cultured, plasmids were extracted, sequenced, and the DNA sequences were translated into amino acid sequences and then aligned. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same antibody strain, while clones with different CDR sequences were considered different antibody strains, resulting in a total of 43 unique sequences.

[0119] Using 1 μM IPTG identified by the above ELISA binding experiment, TG1 with a specific sequence was induced overnight, and after repeated freeze-thaw cycles, the supernatant was collected by centrifugation. After blocking with 0.1% BSA for 1 hour, the supernatant sample was placed in a microplate reader coated with 5 μg / mL antigen protein along with 1.25 μg / mL IGF1-Fc and blocked with 1% casein, and incubated at room temperature for 1 hour. The plate was washed with PBST between different incubation steps. Anti-human IgG-HRP (Abcam ab6759, 1:5000 dilution in 1% casein) was added and incubated for 1 hour, then washed three times with PBST, and TMB substrate solution (Thermo, 34029) was added to develop color. After the reaction was complete, OD was performed using a microplate reader. 450 The results were read. Based on the results, the blocking activity of each clone was sorted, and a subset of sequences with the highest blocking function were selected for subsequent purification and expression.

[0120] Example 4 Expression and purification of nanoantibody VHH

[0121] Positive clones with a specific sequence and high inhibitory activity, as screened in Example 3, were transformed into Top10F' cells, and the expression of the nanoantibody VHH was induced using IPTG. Cell precipitates were collected by centrifugation, disrupted by sonication, and the supernatant was collected. The supernatant was purified using TALON® Metal Affinity Resins (Takara, 635502), and the buffer was replaced with phosphate buffer. The purified VHH was OD 280 The substance was quantified by measurement and stored at 4°C for use.

[0122] The SDS-PAGE results of the purified nanoantibodies are shown in Figure 1.

[0123] Example 5: ELISA experiment on competitive binding of nanoantibodies to antigen proteins and their ligands.

[0124] Recombinant human IGF-1R-his antigen (5 μg / mL) was added to a 96-well plate and coated overnight at 4°C. After washing the plate with PBS, it was blocked at room temperature for 1 hour with 1% casein. The VHH sample was gradient diluted 2-fold with 0.1% BSA / PBS to a starting concentration of 20 μg / mL. Different concentrations of VHH and 1.25 μg / mL of IGF-1-Fc were added to the antigen-coated 96-well plate and incubated at room temperature for 1 hour. After washing the well plate three times with 0.1% PBST, anti-human Fc tag antibody (anti-human IgG-HRP, Abcam ab6759, 1:5000, containing 1% casein) labeled with horseradish peroxidase was added and incubated at room temperature for 30 minutes. After washing three times with 0.1% PBST, TMB substrate solution (Thermo, 34028) was added to develop color, and OD was performed using a microplate reader. 450 The data is read, and the curve is fitted using Graphpad Prism software, and the EC 50 The values ​​were calculated. The results showed that multiple nanoantibodies can effectively and competitively bind to human IGF-1R, and here the IC20 of two nanoantibodies 50 The values ​​are shown in Table 1.

[0125] [Table 6]

[0126] Example 6: Expression and purification of nanoantibody-Fc fusion protein

[0127] A pTT5 vector containing a nanoantibody and a nucleic acid sequence encoding a human Fc segment (the C-terminus of VHH linked to the N-terminus of human Fc) was synthesized, and a liposome-DNA complex was prepared using the following steps: 15 μg of plasmid DNA was diluted to a total volume of 0.5 mL with Opti-MEM® I (GIBCO 31985), and 30 μL of 293fectin was added. TM The reagent was diluted to a total volume of 0.5 mL with Opti-MEM(registered trademark) I, gently mixed, and incubated at room temperature for 5 minutes. The diluted DNA was then mixed with diluted 293fectin. TM (Added to the (Invitrogen 12347) reagent, gently mixed uniformly, and incubated at room temperature for 30 minutes. The above mixture was divided into 1 × 10⁻⁶ 6 cells / mL cells (FreeStyle TM 293F cells (Invitrogen R790-07) were placed in a flask and incubated in a cell incubator containing 8% CO2 at 37°C in an orbital oscillator rotating at 125 rpm. On day 4 after transfection, the supernatant was collected and Pierce TM The protein was purified using a Protein A agarose gel, and the buffer was replaced with phosphate buffer.

[0128] Example 7: ELISA experiment on competitive binding of nanoantibody-Fc fusion protein to antigen protein and its ligand.

[0129] Recombinant human IGF1R-his antigen (5 μg / mL) was added to a 96-well plate and coated overnight at 4°C. After washing the plate four times with 1×TBST, it was blocked at room temperature for 1.5 hours with 2% BSA. After washing the plate four times with 1×TBST, 50 μL of nanoantibody-Fc fusion protein at different concentrations (starting concentration 10 μg / mL, 2-fold gradient dilution) and 50 μL of biotinylated IGF-1 (2.5 μg / mL) were added to the antigen-coated 96-well plate, and incubated at 37°C for 1 hour. After washing the well plate four times with 1×TBST, add streptavidin (Thermo, 21126) labeled with horseradish peroxidase, incubate at 37°C for 1 hour, wash four times with 1×TBST, add 200 μL of TMB substrate solution and allow to develop in the dark for 20 minutes, stop the reaction by adding 50 μL of 1M H2SO4, and then use a microplate reader to obtain OD 450 The data is read, and the curve is fitted using Graphpad Prism software, and the IC 50 The values ​​were calculated. As shown in Table 2, the results indicate that IGF1R-5115 Fc can effectively and competitively bind to IGF-1R, and its inhibitory activity is superior to that of the positive control antibody Teprotumumab.

[0130] [Table 7]

[0131] Example 8: Humanization of nanoantibodies and detection of the binding activity of humanized nanoantibody-Fc fusion proteins to antigen proteins.

[0132] The above nano-antibody 5115 was selected and humanized. By sequence analysis of the sequence database, the IGHV3-23 germline gene Germline was selected as a template, and CDR transplantation (retaining some susceptible camel amino acids) was performed to obtain 5115-H0. Based on 5115-H0, further combination reverse mutation design was performed empirically to obtain a total of six humanized sequences: 5115-H1, 5115-H2, 5115-H3, 5115-H4, 5115-H5, and 5115-H6. The humanized VHH gene sequence and Fc sequence were synthesized into full-length sequences, the pcDNA3.4 plasmid was enzymatically digested with NotI / XbaI (NEB, Cat.R0189L, R0145L), and then recombinant with the above full-length sequences. The recombinant plasmid was transformed into TOP10 competent cells, the bacterial suspension was spread, and the cells were cultured at 37°C for 16-20 hours. A single colony was selected from the plate and subjected to colony PCR. Positive clones were identified by electrophoresis, selected, and sequenced. The correctly sequenced clones were expanded and cultured, and the plasmid was extracted. Using the ExpiCHO-S expression system, the plasmid was expressed in ExpiCHO-S cells for 7 days as needed. Finally, the VHH-Fc fusion protein was obtained by one-step purification using Protein A affinity chromatography.

[0133] Recombinant human IGF-1R-his antigen (2 μg / mL) was added to a 96-well plate and coated overnight at 4°C. After washing the plate three times with PBST, it was blocked at room temperature for 2 hours using PBST containing 5% skim milk powder. The blocking solution was discarded, the well plate was washed three times with PBST, and then 30 μL of nanoantibody-Fc fusion protein of different concentrations was added and incubated at room temperature for 1 hour. After washing the well plate three times with PBST, 30 μL / well of horseradish peroxidase-labeled anti-human Fc antibody (abcam, ab97225) was added and incubated at room temperature for 1 hour, then washed three times with PBST, 30 μL of TMB substrate solution was added and allowed to develop in the dark for 5-30 minutes, and the reaction was stopped by adding 30 μL of 2 M H2SO4. The OD was then measured using a microplate reader. 450The data is read, and the curve is fitted using Graphpad Prism software, and the EC 50 The values ​​were calculated. The results showed that all of the above nanoantibody-Fc fusion proteins could effectively bind to the antigen protein, and that the binding activity of some humanized nanoantibodies was equivalent to or better than that of the positive control. EC of each sample 50 The values ​​are shown in Table 3.

[0134] [Table 8]

[0135] Example 9: Species cross-validation and selective binding validation between nanoantibody-Fc fusion protein and antigen protein.

[0136] Recombinant human, monkey, and mouse IGF-1R-his antigens and human insulin receptor protein (5 μg / mL) were added to a 96-well plate and coated overnight at 4°C. After washing the plate with PBS, it was blocked at room temperature for 1 hour with 1% casein. The nanoantibody-Fc fusion protein was diluted to 2 μg / mL in 0.1% BSA / PBS, and the above nanoantibody-Fc fusion protein was added to the antigen-coated 96-well plate and incubated at room temperature for 1 hour. After washing the well plate three times with 0.1% PBST, anti-human Fc-tagged antibody (anti-human IgG-HRP, Abcam ab6759, 1:5000, containing 1% casein) labeled with horseradish peroxidase was added and incubated at room temperature for 30 minutes. After washing three times with 0.1% PBST, TMB substrate solution (Thermo, 34028) was added to develop color, and OD was performed using a microplate reader. 450 I read it.

[0137] As shown in Figure 2, the 5115-Fc fusion protein and the humanized nanoantibody-Fc fusion protein (5115-H0 Fc) exhibited binding activity to both recombinant human and cynomolgus monkey IGF-1R, but not to mouse IGF-1R. Furthermore, the two nanoantibody-Fc fusion proteins tested showed good selectivity for the IGF-1R antigen because they did not exhibit binding activity to the human insulin receptor protein.

[0138] Example 10: Construction of mutant humanized nanoantibody-Fc fusion proteins and detection of their binding activity to antigen proteins

[0139] First round of mutant construction: 5115-H4 was selected as the maternal lineage, and its VHH gene was ligated to a dAb phage display vector to obtain the maternal plasmid. A series of primers were designed, and single-point or double-point mutations were introduced into the maternal lineage to construct four affinity-mature phage display libraries. Using immunotubule screening (i.e., solid-phase screening), immunotubules were coated with recombinant human IGF-1R-His antigen protein, incubated with the affinity-mature phage display libraries, washed, eluted, and enriched with high-affinity specific monoclonal antibodies through three rounds of panning. Based on the above panning process and VHH ELISA detection, 15 candidate molecules were selected based on VHH-level affinity and sequence similarity, and nano-antibody-Fc fusion proteins were expressed and detected by ELISA.

[0140] Second round of mutant construction: 5115-H4-M34 and 5115-H1 were selected as maternal lines, and their VHH genes were ligated to dAb phage display vectors to obtain maternal plasmids. A series of primers were designed, and single-point or double-point mutations were introduced into each maternal line to construct a total of eight affinity-mature phage display libraries. Using immunotubule screening (i.e., solid-phase screening), immunotubules were coated with the antigen protein hIGF1R-His, the affinity-mature phage display libraries were added and incubated, washed, eluted, and high-affinity specific monoclonal antibodies were enriched through three rounds of panning. Based on the above panning process and VHH ELISA detection, 16 candidate molecules were selected from the 5115-H4-M34 maternal mutants and expressed as nanoantibody-Fc fusion proteins, and 40 candidate molecules were selected from the 5115-H1 maternal mutants and expressed as nanoantibody-Fc fusion proteins and detected by ELISA.

[0141] The binding activity of nanoantibody-Fc fusion proteins obtained by screening after constructing two rounds of mutants using the ELISA method to the antigen protein (recombinant human IGF-1R-His) was detected. 2 μg / mL of recombinant human IGF1R-His antigen protein was added to each 96-well microplate reader at 30 μL / well and coated overnight at 4°C. The plates were washed three times with PBST. PBS containing 5% skim milk powder was added and blocked at room temperature for 2 hours. The plates were washed three times with PBST. The nanoantibody-Fc fusion proteins were gradient diluted 3-fold, and 30 μL / well of different concentrations of nanoantibody-Fc fusion protein were added to each well and incubated at room temperature for 1 hour. After washing the well plates three times with PBST, 30 μL / well of anti-human Fc antibody labeled with horseradish peroxidase was added and incubated at room temperature for 50 minutes. The plates were washed three times with PBST. 30 μL of TMB substrate solution was added and allowed to develop color in the dark for 1 to 5 minutes. After stopping the reaction by adding 30 μL of 2M hydrochloric acid, the OD was measured using a microplate reader.450 The data is read, and the curve is fitted using Graphpad Prism software, and the EC 50 The values ​​were calculated. As shown in Tables 4 and 5, all of the nanoantibody-Fc fusion proteins obtained after mutant construction had relatively high affinity for the antigen, and the antigen-binding activity of some of the nanoantibody fusion proteins was equivalent to or better than that of the positive control Teprotumumab.

[0142] [Table 9]

[0143] [Table 10]

[0144] Example 11: Detection of affinity of nanoantibody-Fc fusion protein for recombinant human IGF-1R

[0145] The affinity of nanoantibody fusion proteins to recombinant human IGF-1R was detected using a BIAcore instrument. The antigen was diluted to 12.5 μg / mL in NaAc buffer at pH 4.75, the flow rate was set to 10 μL / min, the default activation tip time for the EDC and NHS mixture was 420 s, and recombinant human IGF-1R-his was bound to the 800 RU level on the CM5 tip using the preset binding volume mode. Unbound activating groups were blocked with ethanolamine. Nanoantibody-Fc fusion proteins (5 concentrations detected for each fusion protein) diluted 2-fold in series were injected sequentially at a flow rate of 50 μL / min at 25°C, with a binding time of 120 s and a dissociation time of 600 s. Using BIAcore T200 analysis software, the curve was fitted using a 1:1 Langmuir binding model to determine the dynamical constants such as the binding rate constant, dissociation rate constant, and binding-dissociation constant.

[0146] The results showed that all of the tested nanoantibody-Fc fusion proteins had relatively high affinity for recombinant human IGF-1R, and that the affinity of some nanoantibody-Fc fusion proteins to the antigen was equivalent to or better than that of the positive control. The dynamical constants of the binding of nanoantibody-Fc fusion proteins to recombinant human IGF-1R-his are shown in Table 6.

[0147] [Table 11]

[0148] Example 12: ELISA experiment on competitive binding of nanoantibody-Fc fusion protein to antigen protein and its ligand.

[0149] From the construction of humanized and mutant libraries, nanoantibody-Fc fusion proteins with relatively high antigen binding activity were selected, and their competitive binding activity to antigen proteins and their ligands was verified.

[0150] Recombinant human IGF-1R-his antigen (5 μg / mL) was added to a 96-well plate and coated overnight at 4°C. After washing the plate four times with 1×TBST, it was blocked with 2% BSA at 37°C for 1.5 hours. After washing the plate four times with 1×TBST, 50 μL of biotinylated IGF-1-Fc (2.5 μg / mL) or IGF-2 (N-Avi&N-Fc, 4 μg / mL) and 50 μL of nanoantibody-Fc fusion protein at different concentrations (starting concentration 10 μg / mL, 2-fold gradient dilution) were added to the 96-well plate, and incubated at 37°C for 1 hour. After washing the well plate four times with 1×TBST, add streptavidin (Thermo, 21126) labeled with horseradish peroxidase, incubate at 37°C for 1 hour, wash four times with 1×TBST, add 200 μL of TMB substrate solution and allow to develop in the dark for 20 minutes, stop the reaction by adding 50 μL of 1M H2SO4, and then use a microplate reader to obtain OD 450 The data is read, and the curve is fitted using Graphpad Prism software, and the IC50 The values were calculated. As shown in Table 7, the above nanobody-Fc fusion protein could effectively competitively bind to IGF-1R, and its inhibitory activity was superior to that of the positive control antibody Teprotumumab.

[0151]

Table 12

[0152] Detection of the inhibitory activity of the nanobody-Fc fusion protein in Example 13 on IGF-1-induced BaF3 cell proliferation overexpressing human IGF-1R

[0153] The VHH gene sequence of 5115-H4-M34 and the Fc sequence (IgG1 C220S L234A L235A D356E L358M or IgG1 C220A L234A L235A D356E L358M) were synthesized into the full-length sequence. The pcDNA3.4 plasmid was digested with NotI / XbaI (NEB, Cat.R0189L, R0145L), and then recombined with the above full-length sequence. The recombinant plasmid was transformed into TOP10 competent cells, the bacterial solution was spread, and cultured at 37 °C for 16 - 20 h. Single colonies on the plate were selected for colony PCR, positive clones were identified by electrophoresis, positive clones were selected for sequencing, correctly sequenced clones were expanded and cultured, and the plasmid was extracted. HEK293 cells were transiently transfected using a liposome transfection reagent, placed in a cell incubator containing 5% CO2 at 37 °C, and incubated on an orbital shaker rotating at 125 rpm. On the 5th day, the supernatant was collected by centrifugation, purified by a Protein A affinity chromatography column, and the buffer was exchanged with phosphate buffer by dialysis to obtain the nanobody-Fc fusion proteins 5115-H4-M34-Fc’ and 5115-H4-M34-Fc” respectively.

[0154] BaF3 cells overexpressing human IGF-1R can detach from their dependence on IL-3 of wild-type BaF3 cells and instead grow depending on the downstream signaling pathway induced by the binding of IGF-1 and IGF-1R. Therefore, it can be used as a cytological model to detect the blocking activity of anti-IGF-1R antibody against the binding of IGF-1 and IGF-1R. Take a cell line stably transfected with stable human IGF-1R BaF3 in the logarithmic growth phase, resuspend the cells using fresh RPMI1640 culture medium, inoculate the cell suspension into a 96-well cell culture plate at 90 μL / well (3000 cells / well), and place it in a carbon dioxide incubator (37 °C, 5% CO2). Prepare a 10×nanobody-Fc fusion protein solution or a control antibody solution, add it to the 96-well plate inoculated with cells at 10 μL / well, and set the final concentration of the protein to 20 μg / mL, dilute it 3.16-fold, and there were 9 concentrations. The final concentration of IGF-1 in the incubation system was 100 ng / mL. After continuously culturing the cell culture plate in a carbon dioxide incubator (37 °C, 5% CO2) for 72 hours, equilibrate the cell culture plate to room temperature and detect cell viability using CellTiter-Glo reagent (Promega, G7573). Calculate the cell survival rate according to the following formula. Cell survival rate (%) = (Lum 測定される薬物 -Lum 培養液対照 ) / (Lum 細胞対照 -Lum 培養液対照 ) × 100%. Analyze the data using Graphpad Prism software, fit the data using non-linear S-curve regression to obtain a dose-effect curve, thereby calculate the IC 50 value, and the fitting results are shown in Table 8. The results showed that both of the two nanobody-Fc fusion proteins tested had a relatively strong inhibitory effect on the proliferation of BaF3 cells overexpressing human IGF-1R, and the inhibitory effect was significantly better than that of the positive control antibody.

[0155]

Table 13

[0156] Note: Fc' indicates that the subtype is IgG1 C220S L234A L235A, and Fc'' indicates that the subtype is IgG1 C220A L234A L235A.

[0157] Example 14: Detection of the inhibitory activity of nanoantibody-Fc fusion protein against IGF-1-induced MCF-7 cell proliferation.

[0158] Logarithmically growing human breast cancer MCF-7 cells were digested with trypsin, resuspended in RPMI1640 culture medium containing 10% FBS, and inoculated into 96-well cell culture plates at a density of 4000 cells / well. These plates were incubated overnight in a carbon dioxide incubator (37°C, 5% CO2) with the cells attached to the walls. The culture medium was then replaced with 50 μL of serum-free RPMI1640 culture medium, and the cells were incubated in the carbon dioxide incubator for 5 hours. Using RPMI1640 culture medium containing 2% FBS and 20 ng / mL of IGF-1, 2× nano-antibody-Fc fusion protein solutions or control antibody solutions were prepared. The maximum concentration was 20 μg / mL, and these solutions were diluted 5-fold to a total of 9 concentrations. 50 μL of each solution was added to the corresponding wells of the 96-well plates inoculated with the above cells. Cell culture plates were incubated in a carbon dioxide incubator (37°C, 5% CO2) for 6 days, and then cell viability was detected using the CyQUANT® reagent kit. The data was analyzed using Graphpad Prism software, and the data was fitted using nonlinear S-curve regression to obtain a dose-effect curve, thereby determining IC. 50 The values ​​were calculated, and the fitting results are shown in Table 9. The results showed that both of the tested nanoantibody-Fc fusion proteins had a relatively strong inhibitory effect on IGF-1-induced MCF-7 cell proliferation, and that this inhibitory effect was significantly superior to that of the positive control antibody Teprotumumab.

[0159] [Table 14]

[0160] Note: Fc' indicates that the subtype is IgG1 C220S L234A L235A D356E L358M, and Fc'' indicates that the subtype is IgG1 C220A L234A L235A D356E L358M.

[0161] Example 15: Detection of the inhibitory activity of nanoantibody-Fc fusion protein on the IGF-1-induced MCF-7 cell signaling pathway.

[0162] Logarithmic growth phase MCF-7 cells were taken and inoculated into a 6-well plate (approximately 4.5 * 10 e5 cells), and cultured overnight in a 37°C, 5% CO2 cell incubator. The culture medium was discarded, the cells were washed with 2 mL of DPBS, and fresh cell culture medium without FBS was added. The cells were cultured overnight in a 37°C, 5% CO2 cell incubator. Nano antibody-Fc fusion protein, positive or negative control antibody (final concentration 2 μg / mL) was added to each well, gently mixed uniformly, and incubated at 37°C for 10 minutes. IGF-1 (final concentration 50 ng / mL) was added, and incubated at 37°C for 20 minutes. The culture medium was discarded, the cells were washed with 2 mL of DPBS, the washing solution was discarded, and 150 μL of cell lysate containing a protease inhibitor was added to each well. The cells were lysed on ice for 30 minutes, collected, and transferred to a 1.5 mL centrifuge tube. The cells were then centrifuged at 4°C and 12000 rpm for 20 minutes. The concentration of the supernatant protein was measured using a BCA reagent kit. After adjusting the protein concentration, 4× injection buffer containing DTT was added, the mixture was thoroughly combined, and the mixture was heated at 100°C for 5 minutes. The Erk1 / 2 phosphorylation level and total Erk1 / 2 protein level of each sample were detected using the Western Blot method. The primary antibodies were Phospho-p44 / 42 MAPK(Erk1 / 2)(Thr202 / Tyr204)(D13.14.4E)XP(registered trademark) Rabbit mAb (cell signaling, 8544S) and p44 / 42 MAPK(Erk1 / 2)(137F5) Rabbit mAb (cell signaling, 4695T), and the secondary antibody was anti-rabbit IgG-HRP second antibody (cell signaling, 7074P2). The detection results are shown in Figure 3. Compared to blank cell controls, IGF-1 stimulation effectively increased the phosphorylation level of Erk1 / 2, and both nanoantibody-Fc fusion protein 5115-Fc and 5115-H0 pretreatment effectively inhibited IGF-1-induced Erk1 / 2 phosphorylation, with superior inhibitory effects compared to the positive control Teprotumumab.

[0163] Furthermore, logarithmic growth phase MCF-7 cells were taken, inoculated into a 6-well plate (approximately 4.5*10e5 cells), and cultured overnight in a 37°C, 5% CO2 cell incubator. The culture medium was discarded, the cells were washed with 2 mL of DPBS, fresh cell culture medium without FBS was added, and the cells were cultured overnight in a 37°C, 5% CO2 cell incubator. Nano antibody-Fc fusion protein, positive control or negative control antibody was added to each well to a final concentration of 5 μg / mL or 0.5 μg / mL, lightly mixed uniformly, and incubated at 37°C for 30 minutes. IGF-1 (final concentration 300 ng / mL or 30 ng / mL) was added and incubated at 37°C for 20 minutes. The culture medium was discarded, the cells were washed with 2 mL of DPBS, the washing solution was discarded, and 150 μL of cell lysate containing a protease inhibitor was added to each well. The cells were lysed on ice for 30 minutes, collected, and transferred to a 1.5 mL centrifuge tube. The cells were then centrifuged at 4°C and 12000 rpm for 20 minutes. The concentration of the supernatant protein was measured using a BCA reagent kit. After adjusting the protein concentration, 4× injection buffer containing DTT was added, the mixture was thoroughly combined, and the mixture was heated at 100°C for 5 minutes.Using the Western Blot method, MEK phosphorylation and total MEK protein levels, Erk1 / 2 phosphorylation and total Erk1 / 2 protein levels, Akt phosphorylation and total Akt protein levels were detected for each sample. The primary antibodies used were Phospho-p44 / 42 MAPK(Erk1 / 2)(Thr202 / Tyr204)(D13.14.4E)XP(registered trademark) Rabbit mAb(CST, #8544), p44 / 42 MAPK(Erk1 / 2)(137F5)Rabbit mAb(CST, #4695), Phospho-Akt(Ser473)(D9E)XP(registered trademark) Rabbit mAb(CST, #4060), and Akt(pan)(40D4)Mouse The reagents used were mAb (CST, #2920), Phospho-MEK1 / 2 (Ser217 / 221) (41G9) Rabbit mAb (CST, #9154), and MEK1 / 2 Antibody (CST, 9122). The secondary antibody was either anti-rabbit IgG-HRP second antibody (CST, #7074) or anti-Mouse IgG-HRP (CST, #7076). The detection results are shown in Figure 4. Compared to blank cell controls, IGF-1 stimulation effectively increased the phosphorylation levels of MEK, Erk1 / 2, and Akt in MCF-7 cells, and the phosphorylation levels of these proteins increased with increasing IGF-1 concentration. There were no significant differences in the total protein levels of MEK, Erk1 / 2, and Akt among the treatment groups. When the nanoantibody-Fc fusion protein 5115-H4-M34 Fc' was pretreated with 5 μg / mL and 0.5 μg / mL, both effectively inhibited IGF-1-induced MEK, Erk1 / 2, and Akt phosphorylation. The inhibitory effect on protein phosphorylation under 30 ng / mL IGF-1 stimulation was equivalent to that of the positive control, and the inhibitory effect on protein phosphorylation under 300 ng / mL IGF-1 stimulation was superior to that of the positive control Teprotumumab.

Claims

1. An antigen-binding protein capable of binding to IGF-1R, The antigen-binding protein comprises HCDR3, HCDR2, and HCDR1, where HCDR3, HCDR2, and HCDR1 comprises amino acid sequences selected from the following group; Table 1-1 Table 1-2 Furthermore, the antigen-binding protein includes the variable region (VHH) of the heavy chain antibody. Antigen-binding protein.

2. It includes a heavy chain variable region VH, and the VH includes an amino acid sequence represented by any one of SEQ ID NO: 1 to 80. The antigen-binding protein according to claim 1.

3. It is a single-domain antibody. The antigen-binding protein according to claim 1.

4. Containing an amino acid sequence shown in any one of SEQ ID NO: 1 to 80, The antigen-binding protein according to claim 1.

5. It is a polypeptide, A protein comprising the antigen-binding protein according to any one of claims 1 to 4, Polypeptide.

6. The polypeptide further comprises an immunoglobulin Fc region. The polypeptide according to claim 5.

7. The C-terminus of the VH of the antigen-binding protein is directly or indirectly linked to the N-terminus of the Fc region. The polypeptide according to claim 6.

8. The polypeptide according to claim 6, wherein the Fc region comprises Fc derived from IgG1 and / or Fc derived from IgG4.

9. The Fc region includes an amino acid sequence represented by any one of SEQ ID NO: 209 to 212. The polypeptide according to claim 6.

10. nucleic acids, A nucleic acid encoding an isolated antigen-binding protein according to any one of claims 1 to 4.

11. nucleic acids, Encoding the polypeptide described in claim 5, Nucleic acid.

12. A composition, A method comprising an isolated antigen-binding protein according to any one of claims 1 to 4 and an optionally pharmaceutically acceptable adjuvant, composition.

13. A composition, A polypeptide according to claim 5 and an optionally pharmaceutically acceptable adjuvant, composition.

14. Used to inhibit the proliferation of cells overexpressing IGF-1R or its functionally active fragment, to inhibit the MAPK pathway, and / or to inhibit the interaction between IGF-1R or its functionally active fragment and its ligand. A compound comprising an isolated antigen-binding protein according to any one of claims 1 to 4, Pharmaceutical composition.

15. Used to inhibit the proliferation of cells overexpressing IGF-1R or its functionally active fragment, to inhibit the MAPK pathway, and / or to inhibit the interaction between IGF-1R or its functionally active fragment and its ligand. A polypeptide comprising the polypeptide described in claim 5, Pharmaceutical composition.

16. Used to detect the presence and / or content of IGF-1R or its functionally active fragment, A compound comprising an isolated antigen-binding protein according to any one of claims 1 to 4, Pharmaceutical composition.

17. Used to detect the presence and / or content of IGF-1R or its functionally active fragment, A polypeptide comprising the polypeptide described in claim 5, Pharmaceutical composition.

18. The use of an isolated antigen-binding protein according to any one of claims 1 to 4 in the manufacture of a drug, The aforementioned drug is used to prevent, alleviate and / or treat a disease or illness. use.

19. The use of the polypeptide according to claim 5 in the manufacture of a drug, The aforementioned drug is used to prevent, alleviate and / or treat a disease or illness. use.

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