Antigen-binding proteins and uses thereof

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] The present application relates to the biopharmaceutical field, specifically to antigen binding proteins and uses thereof. [Background technology]

[0002] Interaction of IGF-1 (insulin-like growth factor I) with IGF-1R (insulin-like growth factor receptor 1) activates cell signaling pathways by causing autophosphorylation of receptor tyrosine residues. Activation of the receptor can stimulate tumor cell growth and survival. Thus, inhibition of IGF-1R activity is a potential method for treating or preventing tumors. Overexpression of insulin-like growth factor receptor 1 is also found in several cancer cell lines and tumor tissues, and the degree of IGF-1R expression may correlate with disease severity. The art is looking for methods that can effectively inhibit IGF-1R activity, such as antigen-binding proteins or functionally active fragments thereof that can bind to IGF-1R. Summary of the Invention

[0003] The present application provides antigen binding proteins, which can have a property selected from the group consisting of: (1) inhibiting 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 proliferation of cells overexpressing IGF-1R, (5) inhibiting human breast cancer cell proliferation, such as inhibiting IGF-1-induced human breast cancer cell proliferation, and (6) inhibiting IGF-1-induced MEK, Erk1 / 2 and / or Akt phosphorylation.

[0004] According to one aspect, the present application provides an antigen binding protein capable of inhibiting competitive binding of IGF-1R to its ligand with an IC50 value of about 1.0 μg / mL or lower in a competitive ELISA detection.

[0005] According to one aspect, the present application provides an antigen-binding protein capable of competitively binding to IGF-1R with a reference antibody, said reference antibody comprising HCDRs 1-3, said HCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 85, 100 and 131, respectively.

[0006] According to one aspect, the present application provides an antigen binding protein comprising an HCDR3, wherein said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:217 or 218.

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

[0008] According to one aspect, the present application provides an immunoconjugate comprising the isolated antigen-binding protein of the present application and / or the polypeptide of the present application.

[0009] According to one aspect, the present application provides an isolated antigen binding protein of the present application and / or a nucleic acid encoding a polypeptide of the present application.

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

[0011] According to one aspect, the present application provides a cell comprising an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, and / or a vector of the present application.

[0012] According to one aspect, the present application provides a method of producing an isolated antigen binding protein of the present application and / or a polypeptide of the present application, the method comprising culturing a cell of the present application under conditions that allow expression of said isolated antigen binding protein and / or said polypeptide.

[0013] According to one aspect, the present application provides a composition comprising an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, a vector of the present application, and / or a cell of the present application, and optionally a pharma- ceutically acceptable adjuvant.

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

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

[0017] According to one aspect, the present application provides the use of an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, a vector of the present application, a cell of the present application, a composition of the present application, and / or a reagent kit of the present application in the manufacture of a medicament, wherein the medicament is used to prevent, alleviate and / or treat a disease or condition.

[0018] Those skilled in the art can recognize other aspects and advantages of the present application from the following detailed description. The following detailed description merely illustrates and describes exemplary embodiments of the present application. As will be recognized by those skilled in the art, the present application may enable those skilled in the art to modify the specific embodiments disclosed without departing from the spirit and scope of the invention related to the present application. Correspondingly, the drawings and description in the present application are illustrative only and not limiting. [Brief description of the drawings]

[0019] Particular features of the invention related to this application are set forth in the appended claims. A better understanding of the features and advantages of the invention related to this application can be obtained by reference to the following detailed description of exemplary embodiments and drawings, the brief description of which is as follows:

[0020] [Figure 1] FIG. 2 shows an SDS-PAGE diagram of the nanobody of the present application. [Diagram 2] FIG. 1 shows the binding results of the nanobody-Fc fusion proteins of the present application to recombinant human, monkey and mouse IGF1R proteins and human insulin receptor protein. [Diagram 3] FIG. 1 shows a graph showing the inhibitory effect of the nanobody-Fc fusion protein of the present application on IGF-1-induced phosphorylation of Erk protein. [Figure 4] Figure 2 shows the inhibitory effect of the nanobody-Fc fusion protein of the present application on IGF-1-induced phosphorylation of MEK, Erk and Akt proteins (Note: Fc' indicates that the subtype is IgG1 C220S L234A L235A D356E L358M). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0022] Definition of Terms

[0023] In the present application, the term "antigen-binding protein" generally refers to a polypeptide molecule capable of specifically recognizing and / or neutralizing a particular antigen. In the present application, the term "antigen-binding protein" may include "antibody" or "antigen-binding fragment". The term "antibody" may include monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to camelid antibodies, fully human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies (such as scFv), and antibody fragments (such as Fab, Fab' and (Fab)2 fragments) that bind to an antigen. 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 an antigen as described herein. In the present application, the "antibody" may include single domain antibodies.

[0024] In this application, the term "isolated" generally means obtained by artificial means from a natural state. If an "isolated" substance or component occurs in nature, the natural environment in which it occurs may have been altered or the substance may have been separated from the natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in vivo in a living animal, and the same polynucleotide or polypeptide of high purity 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 of the antibody is deleted and only the heavy chain variable region is present. Research has found that heavy chain antibodies (hcAbs) exist in Bactrian camels, dromedaries, alpacas and llamas that are composed only of heavy chains but have full functionality, and the molecular weight of the variable domains of the hcAbs (abbreviated as VHH) is only 1 / 10 of that of conventional antibodies, making them the smallest molecular fragments currently available with full antibody function, and are called single domain antibodies (sdAbs). Compared with other antibodies, single domain antibodies have the advantages of low immunogenicity, small molecule size, and strong penetrating power, and therefore have a wide range of application prospects 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 an antigen-binding fragment. Heavy chain variable regions can be further differentiated into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conservative regions called framework regions (FRs). Each heavy chain variable region can be composed of three CDRs and four FR regions, which are arranged from the amino terminus to the carboxyl terminus in the following order: H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3, and H-FR4. The heavy chain variable region contains a binding domain that interacts with the antigen of the present application. The appropriate boundaries of the CDRs are defined differently according to different systems. The system described by Kabat not only provides a clear residue numbering system that can be applied to any variable region of an antigen binding protein, but also provides precise residue boundaries that define the CDRs. These CDRs may be referred to as Kabat CDRs. Chothia et al. found that, despite the great diversity at the amino acid sequence level, certain subparts within the Kabat CDRs adopt nearly the same peptide backbone structure.These subparts are designated L1, L2 and L3 or H1, H2 and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define the CDRs that overlap with the Kabat CDRs are described in Padlan and MacCallum. Other CDR boundary definitions may not strictly follow any one of the above systems, but still overlap with the Kabat CDRs, and may be shorter or longer without significantly affecting the predicted or experimental expression of antigen binding according to specific residues or groups of residues, or even the CDRs as a whole. In this application, the CDRs may be defined using the Contact numbering system. In this application, the CDRs may be defined using the Abm numbering system. In this application, the CDRs may be defined using the Chothia numbering system. In this application, the CDRs may be defined using the Kabat numbering system. In the present application, CDRs can be defined using the IMGT numbering system. In the present application, the term "single domain antibody" is used interchangeably with "nanobody" and "VHH". The scope of protection claimed in the present 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 the present application. Exemplary definition rules are shown in the table below.

[0026] In this application, the term "monoclonal antibody" generally refers to a population of essentially identical antibodies, i.e., each antibody comprising the population is identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific and directly target a single antigenic site. For example, the monoclonal antibodies can be produced by hybridoma technology or produced in bacteria, eukaryotic animal cells or plant cells using recombinant DNA methods. 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 part 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 belonging 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 one animal species (mouse, rat, etc.), while the constant parts are the same as the antibody sequence derived from another species (human, etc.). For example, to obtain a chimeric antibody, non-human B cells or hybridoma cells are used to generate the variable regions, and the constant regions combined with them are derived from humans. The variable regions have the advantage of being easy to manufacture, and their specificity is not affected by the origin of the constant regions combined with them. At the same time, since the constant regions of the chimeric antibody can be derived from humans, the antibody is less likely to induce an immune response when the chimera is injected than when the constant regions are used as antibodies of non-human origin.

[0028] In the present application, the term "IGF-1R", "IGF1R", "IGF-IR" or "IGFIR" generally refers to insulin-like growth factor receptor 1. The IGF-1R of the present application may include its isoforms, species homologues, functionally active fragments, and analogs having at least one common epitope therewith. For example, the "functionally active fragments" may include fragments that retain at least one endogenous function of the naturally occurring protein. An exemplary IGF-1R amino acid sequence can be shown in UniProt Accession No. P08069. For example, ligands of IGF-1R may include IGF-1 and / or IGF-2.

[0029] As used herein, 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 homologues, functionally active fragments, and analogs having at least one common epitope therewith. For example, the "functionally active fragments" may include fragments that retain at least one endogenous function of the naturally occurring protein. An exemplary amino acid sequence of IGF-1 can be found in UniProt Accession No. P05019.

[0030] As used herein, 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 homologues, functionally active fragments, and analogs having at least one common epitope therewith. For example, the "functionally active fragments" may include fragments that retain at least one endogenous function of the naturally occurring protein. An exemplary amino acid sequence of IGF-2 can be shown in UniProt Accession No. P01344.

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

[0032] The term "functional variant" refers to a polypeptide that has essentially the same amino acid sequence as a naturally occurring sequence or is encoded by essentially the same nucleotide sequence and can have one or more activities of the 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 or nucleotide residues) has been modified, thereby causing said polypeptide or polynucleotide to essentially retain at least one intrinsic function. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one amino acid residue and / or nucleotide residue present in the naturally occurring protein and / or polynucleotide, so long as the original functional activity is retained.

[0033] As used herein, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application that includes any substitution, mutation, modification, replacement, deletion and / or addition of one or more amino acid residues of the self / paired sequence, so long as the resulting polypeptide or polynucleotide essentially retains at least one of its endogenous functions.

[0034] As used herein, the term "analog" generally refers to a polypeptide or polynucleotide and includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that has at least one endogenous function of the polypeptide or polynucleotide that it mimics.

[0035] Generally, amino acid substitutions can be made, such as at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, so long as the modified sequence retains essentially the requisite activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0036] In the present application, the term "homolog" generally refers to an amino acid sequence or a nucleotide sequence that has a certain homology with a naturally occurring sequence. The term "homology" can be equated to sequence "identity". A homologous sequence may include an amino acid sequence that may be 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. In general, a homolog contains an active site, etc., that is identical to the subject amino acid sequence. Homology can be considered in terms of similarity (i.e., amino acid residues that have similar chemical properties / functions) or homology can be expressed in terms of sequence identity. In the present application, a sequence with a percentage identity to any one of the SEQ ID NOs: of a referenced amino acid sequence or nucleotide sequence refers to a sequence with said percentage identity over the entire length of the referenced SEQ ID NO:. To determine sequence identity, sequence alignment can be performed and can be done in a variety of ways as known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art will be able to determine appropriate parameters for the alignment, including any algorithms needed to achieve optimal alignment over the full length of the sequences being compared.

[0037] Proteins or polypeptides used herein may have deletions, insertions, or substitutions of amino acid residues which produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, so long as the intrinsic function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids containing non-polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0038] In the present application, the term "tumor" generally refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors. For example, the tumor may include a solid tumor. For example, the tumor may include a blood tumor. For example, the tumor may include a tumor associated with the expression of the antigen of the present application. The term "tumor associated with the expression of the antigen of the present application" generally refers to a tumor formed due to changes in the expression of the antigen of the present application, which leads to disease progression or evasion of immune surveillance. For example, the "tumor associated with the expression of the antigen of the present application" may be a tumor formed due to upregulation of the expression level of the antigen of the present application, which leads to disease progression or evasion of immune surveillance. The tumor associated with the protein expression of the antigen of the present application may be a tumor that is positive for the antigen of the present application. In a tumor that is positive for the antigen of the present application, the protein expression level of the antigen of the present application on the tumor cell surface or tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than that of 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 can be selected from breast cancer.

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

[0041] In this application, the term "immunoconjugate" generally refers to a complex formed by conjugating (e.g., covalently linking via a linking molecule) the isolated antigen-binding protein with the other reagent (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents), which delivers the other reagent to a target cell (e.g., a tumor cell) via specific binding of the isolated antigen-binding protein to an antigen in the target cell. The immune complex then enters the target cell via the internalization, eventually entering the interior of the target cell (e.g., into a vesicle such as a lysosome), where the linking molecule in the immune complex is cleaved to release the other reagent, thereby exerting its cytotoxic effect. The antigen can also be secreted by the target cell and located in a gap outside the target cell.

[0042] As used herein, the term "subject" refers generally to a human or a non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0043] As used herein, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, either isolated from a natural environment or artificially synthesized.

[0044] In this application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The vector can transport the inserted nucleic acid molecule into and / or between cells. The vector may include vectors that are primarily used to insert DNA or RNA into cells, vectors that are primarily used to replicate DNA or RNA, and vectors that are primarily used to express DNA or RNA transcriptionally and / or translationally. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide upon introduction into an appropriate cell. Generally, the vector can produce a desired expression product by culturing an appropriate cell containing the vector. In this application, the vector may include a lentiviral vector.

[0045] In the present application, the term "cell" generally refers to an individual cell, cell line, or cell culture that may contain or has contained a plasmid or vector containing a nucleic acid molecule described herein, or that is capable of expressing a polypeptide described herein or an antigen binding protein described herein. The cell may include the progeny of a single cell. Due to natural, accidental, or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, so long as they are capable of expressing a polypeptide or antigen binding protein described herein. The cell may be obtained by transfecting a cell in vitro with a vector described herein. The cell may be a prokaryotic cell (such as E. coli) or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell, or a myeloma cell). In some embodiments, the cell may be an immune cell. 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] As used herein, the term "treatment" generally refers to: (i) preventing the onset of a disease, disease, or condition in a patient who is susceptible to the disease, disease, or condition but who has not yet been diagnosed with the disease; (ii) inhibiting the disease, disease, or condition, i.e., preventing its progression; and (iii) alleviating the disease, disease, or condition, i.e., causing regression of the disease, disease, or condition and / or symptoms associated with the disease, disease, or condition.

[0047] As used herein, the terms "polypeptide", "peptide", "protein" and "proteinaceous" are used interchangeably and generally refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids or it may be interrupted by non-amino acids. These terms also include modified amino acid polymers. These modifications may include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation to a marker moiety). The term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycines, D and L optical isomers, and amino acid analogs and peptidomimetics.

[0048] In this application, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, multiple loci (single locus) defined by linkage 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 contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications of the nucleotide structure can be made before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation to a labeling component.

[0049] In this application, "K D " (Similarly, "K D The term "affinity constant" or "equilibrium dissociation constant" generally refers to the equilibrium dissociation constant (KD) or the dissociation rate constant (k d ) to the binding rate constant (k a The binding rate constant (k a ), dissociation rate constant (k d ) and the equilibrium dissociation constant (K D ) represents the binding affinity of a binding protein (e.g., an isolated antigen binding protein described herein) to an antigen (e.g., an antigen protein of the present application). Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to test samples at equilibrium in physiological buffers. For example, the K can be determined by the Octet Molecular Interaction Analyzer (Sartorius). D The K value can be measured by other experimental methods and instruments such as the BIAcore Biomolecular Interaction Analysis System (GE Healthcare Life Sciences). The K value can also be measured by using the KinExA system (Kinetic Exclusion Assay) from Sapidyne Instruments. D Alternatively, the K can be measured using a surface plasmon resonance (SPR) device. D In the present application, the K value is measured by BIAcore. D The value can be measured.

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

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

[0052] In this application, the term "about" generally refers to a variation within 0.5% to 10% above and below a specified numerical value, for example, a variation 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% above and below a specified numerical value.

[0053] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0054] The present application provides antigen binding proteins capable of inhibiting competitive binding of IGF-1R to its ligand with an IC50 value of about 1.0 μg / mL or lower in competitive ELISA detection. The isolated antigen binding proteins described herein have an EC50 value of about 2 μg / mL or lower. 50 (For example, the EC 50 can inhibit binding of IGF-1 to IGF-1R at a concentration of about 1.9 μg / mL or less, about 1.8 μg / mL or less, about 1.7 μg / mL or less, about 1.5 μg / mL or less, about 1.3 μg / mL or less, about 1.1 μg / mL or less, about 1.0 μg / mL or less, about 0.9 μg / mL or less, 0.8 μg / mL or less, about 0.7 μg / mL or less, 0.6 μg / mL or less, about 0.5 μg / mL or less, about 0.4 μg / mL or less, about 0.3 μg / mL or less, about 0.2 μg / mL or less, or about 0.1 μg / mL or less or less.

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

[0056] The present application provides an antigen-binding protein capable of competitively binding to IGF-1R with a reference antibody, the reference antibody may comprise HCDRs 1-3, the HCDRs 1-3 may comprise the amino acid sequences shown in SEQ ID NOs: 85, 100 and 131, respectively. In the present application, the CDRs may be defined using the Contact numbering system. In the present application, the CDRs may be defined using the Abm numbering system. In the present application, the CDRs may be defined using the Chothia numbering system. In the present application, the CDRs may be defined using the Kabat numbering system. In the present application, the CDRs may be defined using the IMGT numbering system. Those skilled in the art may determine the CDR regions using different numbering systems depending on the sequence and structure of the antibody. The CDR regions may differ by using different numbering systems. In the present application, the CDRs include CDR sequences divided by any CDR division method, and also include variants thereof, the variants including substitution, deletion and / or addition of one or more amino acids to the amino acid sequence of the CDR. For example, 1 to 30, 1 to 20, or 1 to 10, and for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions, including homologues thereof, which 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 more) sequence homology with the amino acid sequence of the CDR. In some embodiments, the CDRs can be determined by the Kabat numbering method.

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

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

[0059] For example, the present application provides multiple CDR definition methods. Taking the 5115 antibody as an example:

[0060] [Table 1]

[0061] The present application provides an antigen binding protein, which may comprise an HCDR3, and the HCDR3 may comprise 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 brackets indicate the types of amino acids that can be selected at that site.

[0062] For example, the antigen binding protein may comprise an HCDR3, which may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 130 to 153. CDRs may be defined using the Kabat numbering system.

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

[0064] For example, the antigen binding protein may comprise an HCDR2, which may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 96 to 129. CDRs may be defined using the Kabat numbering system.

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

[0066] For example, the antigen binding protein may comprise an HCDR1, which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 81 to 95. CDRs may be defined using the Kabat numbering system.

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

[0068] For example, the antigen binding protein may comprise a heavy chain variable region VH, wherein the VH may comprise the HCDR1, HCDR2 and HCDR3, wherein the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 130-153, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 96-129, and the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 81-95.

[0069] For example, the antigen binding protein may comprise an H-FR1, the C-terminus of the H-FR1 being linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 may comprise any one of the amino acid sequences set forth in SEQ ID NOs: 219 (EVQLVES[GS]GGLVQ[AP]G[DG]SLRLSCA[AV]S[GHLMRST][AGIKPRTV][AEFGPRSTY][FLR][KNPRS]), 220 (EVQLVESGGGLVQPGGSLRLSCAASGRTF[RS]), and 154 to 186. The letters in square brackets indicate the type of amino acid selectable at that site.

[0070] For example, the antigen binding protein may comprise an H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise any one of the amino acid sequences set forth in SEQ ID NOs: 221 (WFRQAPGK[EG][LR]E[FL]V[AS]), 222 (WFRQAPGK[EG][LR]EFVS), and 187 to 195. The letters in square brackets indicate the types of amino acids selectable at that site.

[0071] For example, the antigen binding protein may comprise an H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 223 (RF[AT][IV]SRDN[AS][KN]NT[LV]YLQM[NS]SL[KR][AP][DE]DT[AG][LV]YYCAA) and 196 to 203. The letters in square brackets indicate the type of amino acid selectable at that site.

[0072] For example, the antigen binding protein may comprise an H-FR4, the N-terminus of the H-FR4 being linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 224(WGQGT[LQ]VTVSS) and 204 to 206. The letters in square brackets indicate the types of amino acids selectable at that site.

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

[0074] For example, amino acid information (SEQ ID NO) of each region corresponding to the antigen-binding protein number of the present 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 application may include an antigen binding protein (e.g., a single domain antibody) having the same HCDR3 as the antigen binding protein described above (e.g., having the same HCDR1-3 as the antigen binding protein described above). 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 antigen binding protein described above.

[0080] For example, the antigen binding protein may comprise a heavy chain variable region VH, wherein 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 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 the brackets indicate the types of amino acids that can be selected at that site.

[0081] For example, the antigen-binding protein may comprise a heavy chain variable region VH, and the VH may comprise an amino acid sequence shown in any one of SEQ ID NOs: 1-80.

[0082] For example, the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3 of a heavy chain variable region VH, and the VH may comprise an amino acid sequence shown in any one of SEQ ID NOs: 1-80.

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

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

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

[0086] For example, the antibodies herein may include camelid antibodies, chimeric antibodies, humanized antibodies and / or fully human antibodies.

[0087] For example, the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-80.

[0088] The present application provides polypeptides that may comprise the antigen binding proteins of the present application.

[0089] For example, the polypeptide may further comprise a structure that improves the stability of the antigen binding protein and / or is capable of binding to an Fc receptor.

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

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

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

[0093] For example, the Fc region herein may comprise an IgG1-derived Fc and / or an IgG4-derived Fc.

[0094] For example, the Fc region herein may comprise the amino acid sequence set forth in SEQ ID NO: 227 (EPKS[ACS]DKTHTCPPCPAPE[AL][AL]GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR[DE]E[LM]TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK). The letters in brackets indicate the type of amino acid selectable at that site.

[0095] For example, the Fc region herein may comprise an amino acid sequence shown in any one of SEQ ID NOs:209-212.

[0096] The present application provides immunoconjugates which may comprise the isolated antigen-binding proteins of the present application and / or the polypeptides of the present application.

[0097] The present application provides an isolated antigen binding protein of the present application and / or a nucleic acid capable of encoding a polypeptide of the present application.

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

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

[0100] The vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used, for example, in 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 in an indirect manner, 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 known in the art and described, for example, in virology and molecular biology manuals. Conventional virus-based systems may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors for gene transfer. In some cases, genes can be introduced and integrated into the host genome using retroviral, lentiviral, and adeno-associated viral methods, resulting in long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce relatively high viral titers. Lentiviral vectors may include long terminal repeats 5'LTR and truncated 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS) and / or post-translational regulatory element (WPRE). The vectors described herein can be introduced into cells.

[0101] The present application provides host cells that may comprise an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, and / or a vector of the present application. The cells may comprise an isolated antigen binding protein, the polypeptide, the immunoconjugate, one or more nucleic acid molecules and / or one or more vectors described herein. For example, each or each type of cell may comprise one or more nucleic acid molecules or vectors described herein. For example, each or each type of cell may comprise multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described herein. For example, a vector described herein may be introduced into the host cell, such as a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / 0 cell, a HEK293T cell, a 293F cell, or a HEK293A cell, or other eukaryotic cell, such as a cell derived from a plant, a fungus, or a yeast cell. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc. For example, the cell may comprise a yeast cell. For example, the cell may comprise an E. coli cell. For example, the cell may comprise a mammalian cell. For example, the cell may comprise an immune cell.

[0102] A method of producing an isolated antigen binding protein of the present application and / or a polypeptide of the present application, said method comprising culturing a cell of the present application under conditions allowing expression of said isolated antigen binding protein and / or said polypeptide.

[0103] The composition may comprise the isolated antigen-binding protein of the present application, the polypeptide of the present application, the immunoconjugate of the present application, the nucleic acid of the present application, the vector of the present application, and / or the cell of the present application, and optionally a pharma- ceutically acceptable adjuvant. In the present application, the pharma-ceutically acceptable adjuvant may comprise a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex, and / or a non-ionic surfactant. Any conventional medium or agent is contemplated for use in the pharmaceutical composition of the present application, so long as it is not compatible with the cells described in the present application. In the present application, the pharma-ceutically acceptable excipient may comprise an additive other than the main drug in a drug formulation, also referred to as an adjuvant.

[0104] The present application provides reagent kits that may include the isolated antigen binding proteins of the present application, the polypeptides of the present application, the immunoconjugates of the present application, the nucleic acids of the present application, the vectors of the present application, the cells of the present application, and / or the compositions of the present application.

[0105] The present application provides a method of inhibiting the proliferation of cells overexpressing IGF-1R or a functionally active fragment thereof, which may include administering an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, a vector of the present application, a cell of the present application, a composition of the present application, and / or a reagent kit of the present application. The present application provides a method of inhibiting the MAPK pathway, which may include administering an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, a vector of the present application, a cell of the present application, a composition of the present application, and / or a reagent kit of the present application. For example, inhibiting the MAPK pathway may include inhibiting phosphorylation of a MAPK pathway protein. For example, the MAPK pathway protein may include MEK, Erk1 / 2, and Akt, 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.

[0106] The present application provides a method of inhibiting the interaction of IGF-1R or a functionally active fragment thereof with its ligand, which may include administering an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, a vector of the present application, a cell of the present application, a composition of the present application, and / or a 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 in vitro methods, ex vivo methods, non-diagnostic or non-therapeutic methods.

[0107] The present application provides methods for detecting the presence and / or content of IGF-1R or a functionally active fragment thereof, which may include administering an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, a vector of the present application, a cell of the present application, a composition of the present application, and / or a reagent kit of the present application. In the present application, the methods may include in vitro methods, ex vivo methods, non-diagnostic or non-therapeutic methods, such as determining the presence and / or expression level of IGF-1R in a sample obtained from a subject by contacting the sample with an antigen binding protein of the present application and detecting the presence and / or content of the antigen binding protein that binds to the sample.

[0108] The present application provides the use of an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, a vector of the present application, a cell of the present application, a composition of the present application, and / or a reagent kit of the present application in the manufacture of a medicament, wherein the medicament 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 expression of IGF-1R or a functionally active fragment thereof. For example, the disease or condition may include breast cancer.

[0109] The present application provides an isolated antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, a nucleic acid of the present application, a vector of the present application, a cell of the present application, a composition of the present application, and / or a reagent kit of the present application for preventing, alleviating, and / or treating 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 expression of IGF-1R or a functionally active fragment thereof. For example, the disease or condition may include breast cancer.

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

[0111] Without intending to be bound by any theory, the following examples are merely illustrative of the proteins, preparation methods and uses of the present application and are not intended to limit the scope of the present invention.

[0112] Working Example

[0113] Example 1 Immunization of Alpacas

[0114] Recombinant human IGF-1R protein (his tag) was used to immunize healthy adult alpacas. For the first immunization, 0.5 mg of recombinant human IGF-1R protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites, and for the booster immunizations, 0.25 mg of recombinant human IGF-1R protein was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple sites for a total of three boosts. Blood was collected before the first immunization and 1 week after each boost to monitor antiserum titers.

[0115] Example 2: Construction of an alpaca immune library

[0116] After immunization, peripheral blood was collected from the alpacas, lymphocytes were separated using lymphocyte separation fluid, and then TRIzol was administered. TM Total RNA was extracted using the ELISA kit, cDNA was obtained by reverse transcription using the SuperScript® III First-Strand Synthesis System for RT-PCR, and VHH genes were amplified by nested PCR. The VHH gene fragment was recovered using a gel purification kit, and then enzymatically digested with the restriction endonuclease sfiI, followed by cloning it into the phagemid vector pComb 3XSS. The constructed clone product was transformed into E. coli TG1 electroporated competent cells to construct a VHH gene library, and the plate gradient dilution method was used to obtain a library volume of 1.3 × 10 9 The total number of cfu was determined to be 100, and colony PCR results showed that the library insertion rate was 100%. After inoculation and cultivation of the gene library at logarithmic phase, M13K07 phage was used for rescue, and after rescue cultivation, the phages were collected by centrifugation and purified by PEG-NaCl to obtain a phage display library, which was used for subsequent screening.

[0117] Example 3 Screening and Identification of Nanobodies

[0118] The nanobody phage display library constructed in Example 2 was incubated in an immunotube (Nunc-Immuno) blocked with 1% casein. TM) and incubated at room temperature upside down for 90 minutes to allow binding, then left to stand for 30 minutes to remove non-specifically binding phages. The library was then transferred to an immunotube coated with IGF-1R (hIGF-1R-his) antigen and incubated at room temperature upside down for 90 minutes to allow binding, then left to stand for 30 minutes. The immunotube was washed 10 times with PBST to remove non-binding or weakly binding phages. Specific phages were dissociated by neutralizing the immunotube with 1 mL of freshly prepared 100 mM triethylamine and 500 μL of 1 M Tris-HCL, pH 6.4, after gentle shaking, and the eluate was collected and placed on ice. The eluate was infected with logarithmic growth phase E. coli TG1, the infected E. coli TG1 was taken and gradient diluted, and LB solid medium (plate dilution method) was applied, and the plate was counted after 12-14 hours, the amount of phage elution was calculated, and monoclonals were randomly selected and amplified to obtain phages, which were identified using enzyme-linked immunosorbent assay (ELISA). The method was to coat a 96-well Greiner ELISA plate with 50 nM recombinant human IGF-1R (hIGF1R-his) protein, coat overnight at 4 °C, and then block with 1% casein. The phages awaiting identification were blocked with 0.1% casein for 1 hour, then placed in the above ELISA plate and incubated for 2 hours, washing with PBST between different blocks / incubations. After washing the plate, anti-M13-HRP (Sinobio, 11973-MM05) was added and allowed to bind at 37°C for 1 hour. After washing, TMB substrate solution (Thermo, 34029) was added to develop color. The reaction was stopped, and the OD was measured using a microplate reader. 450The results were read. Positive control wells (enriched library for each round), negative control (helper phage) and blank wells (PBS) were set up. If the OD value of the measured well was more than three times higher than that of the negative control, it was judged to be positive. Clones corresponding to the positive wells were cultured, the plasmids were extracted and sequenced, and the DNA sequences were translated into amino acid sequences and then aligned. Clones with the same CDR1, CDR2 and CDR3 sequences were considered to be the same antibody strain, while clones with different CDR sequences were considered to be different antibody strains, and a total of 43 special sequences were obtained.

[0119] TG1 with a special sequence, identified by the above ELISA binding experiment, was induced overnight with 1 μM IPTG, and the supernatant was collected by centrifugation after repeated freezing and thawing. After blocking with 0.1% BSA for 1 hour, the above supernatant sample was placed in a microplate reader coated with 5 μg / mL antigen protein and blocked with 1% casein with 1.25 μg / mL IGF1-Fc and incubated at room temperature for 1 hour. The plate was washed with PBST between different incubation procedures. 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, and the OD was measured using a microplate reader after the reaction was completed. 450 Based on the results, the blocking activity of each clone was sorted, and some sequences with the highest blocking activity were selected for subsequent purification and expression.

[0120] Example 4 Expression and purification of nanobody VHH

[0121] The positive clones with the special sequence screened in Example 3 and with high inhibitory activity were transformed into Top10F', and the expression of nanobody VHH was induced using IPTG. The cell precipitate was collected by centrifugation, and the supernatant was collected after disruption by ultrasonication, purified using TALON® Metal Affinity Resins (Takara, 635502), and the buffer was exchanged with phosphate buffer. The purified VHH had an OD 280 The antibody was quantified by measuring the amount of IgG1 and stored at 4° C. for use.

[0122] SDS-PAGE of the purified nanobody is shown in FIG.

[0123] Example 5 ELISA experiment of competitive binding of nanobody to antigen protein and its ligand

[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 with 1% casein at room temperature for 1 h. The VHH samples were 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 h. After washing the well plate three times with 0.1% PBST, horseradish peroxidase-labeled anti-human Fc tag antibody (anti-human IgG-HRP, Abcam ab6759, 1:5000, containing 1% casein) was added and incubated at room temperature for 30 min, followed by washing three times with 0.1% PBST, and color development was performed by adding TMB substrate solution (Thermo, 34028), and the OD was measured using a microplate reader. 450 The EC was calculated by fitting the curve using Graphpad Prism software. 50 The IC values ​​were calculated. The results showed that multiple nanobodies can effectively competitively bind to human IGF-1R, where the IC values ​​of two nanobodies were 50 The values ​​are shown in Table 1.

[0125] [Table 6]

[0126] Example 6 Expression and purification of nanobody-Fc fusion proteins

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

[0128] Example 7 ELISA experiment of competitive binding of nanobody-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. The plate was washed 4 times with 1×TBST and then blocked with 2% BSA at room temperature for 1.5 h. After washing the plate 4 times with 1×TBST, 50 μL of different concentrations of nanobody-Fc fusion protein (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, respectively, and incubated at 37°C for 1 h. After washing the well plate four times with 1xTBST, horseradish peroxidase-labeled streptavidin (Thermo, 21126) was added and incubated at 37°C for 1 h. After washing four times with 1xTBST, 200 μL of TMB substrate solution was added and color developed in the dark for 20 min. The reaction was stopped by adding 50 μL of 1M H2SO4, and the OD was measured using a microplate reader. 450 The IC was calculated by fitting the curves using Graphpad Prism software. 50 The values ​​were calculated. The results, as shown in Table 2, showed that IGF1R-5115 Fc could effectively competitively bind to IGF-1R, and its inhibitory activity was superior to that of the positive control antibody Teprotumumab.

[0130] [Table 7]

[0131] Example 8: Humanization of Nanobodies and Detection of Binding Activity of Humanized Nanobody-Fc Fusion Proteins to Antigen Proteins

[0132] The above nanobody 5115 was selected for humanization. Through database sequence analysis of the sequence, the IGHV3-23 germline gene Germline was selected as a template, and CDR grafting (retaining some sensitive site camel amino acids) was performed to obtain 5115-H0. Based on 5115-H0, further combination back mutation design was performed through experience 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, and the pcDNA3.4 plasmid was enzymatically 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, the bacterial solution was applied, and the cultured at 37°C for 16-20h. A single colony in the plate was selected and colony PCR was performed to identify positive clones by electrophoresis, a positive clone was selected and sequenced, and the correctly sequenced clone was expanded and cultured, and the plasmid was extracted. Using the ExpiCHO-S expression system, the above plasmid was expressed in ExpiCHO-S cells for 7 days as required. Finally, it was one-step purified by Protein A affinity chromatography to obtain the VHH-Fc fusion protein.

[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 for 2 h at room temperature with PBST containing 5% nonfat powder. The blocking solution was discarded, and the well plate was washed three times with PBST, after which 30 μL of different concentrations of nanoantibody-Fc fusion protein was added and incubated at room temperature for 1 h. After washing the well plate three times with PBST, 30 μL / well of anti-human Fc antibody labeled with horseradish peroxidase (abcam, ab97225) was added, incubated at room temperature for 1 h, washed three times with PBST, and 30 μL of TMB substrate solution was added to develop color in the dark for 5–30 min. After the reaction was stopped by adding 30 μL of 2 M H2SO4, the OD was measured using a microplate reader. 450The EC was calculated by fitting the curve using Graphpad Prism software. 50 The EC values ​​were calculated. The results showed that all the above Nanobody-Fc fusion proteins could effectively bind to antigen proteins, and the binding activity of some humanized Nanobodies was comparable to or better than the positive control. The EC 50 The values ​​are shown in Table 3.

[0134] [Table 8]

[0135] Example 9: Species cross-validation and selective binding validation of nanobody-Fc fusion proteins with antigenic proteins

[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 with 1% casein at room temperature for 1 h. The nanobody-Fc fusion protein was diluted to 2 μg / mL with 0.1% BSA / PBS, added to the antigen-coated 96-well plate and incubated at room temperature for 1 h. After washing the well plate three times with 0.1% PBST, horseradish peroxidase-labeled anti-human Fc tag antibody (anti-human IgG-HRP, Abcam ab6759, 1:5000, containing 1% casein) was added and incubated at room temperature for 30 min, followed by washing three times with 0.1% PBST, adding TMB substrate solution (Thermo, 34028) to develop color, and measuring OD using a microplate reader. 450 It was read as follows.

[0137] As shown in Figure 2, 5115-Fc fusion protein and humanized Nanobody-Fc fusion protein (5115-H0 Fc) had binding activity to both recombinant human and cynomolgus IGF-1R, but no binding activity to mouse IGF-1R. Furthermore, the two tested Nanobody-Fc fusion proteins had no binding activity to human insulin receptor protein, indicating good selectivity for IGF-1R antigen.

[0138] Example 10: Construction of mutants of humanized Nanobody-Fc fusion protein and detection of their binding activity to antigenic proteins

[0139] The first round of mutant construction: 5115-H4 was selected as the mother line, and its VHH gene was ligated into a dAb phage display vector to obtain a mother line plasmid. A series of primers were designed to perform single-point or double-point mutations on the mother line, respectively, to construct four affinity matured phage display libraries. Using immunotube screening (i.e. solid-phase screening), the immunotube was coated with the antigen protein recombinant human IGF-1R-His, and the affinity matured phage display library was added and incubated, washed, eluted, and the high affinity specific monoclonal antibodies were enriched through three rounds of panning. Through the above panning process and VHH ELISA detection, 15 candidate molecules were selected from them based on the affinity and sequence similarity at the VHH level, and the expression and ELISA detection of nanobody-Fc fusion proteins were performed.

[0140] The second round of mutant construction: 5115-H4-M34 and 5115-H1 were selected as the parent line, respectively, and their VHH genes were ligated into the dAb phage display vector to obtain the parent line plasmid. A series of primers were designed to perform single-point or double-point mutations on the parent line, respectively, to construct a total of eight affinity matured phage display libraries. Using immunotube screening (i.e. solid-phase screening), the immunotube was coated with the antigen protein hIGF1R-His, and the affinity matured phage display library was added and incubated, washed, eluted, and the high affinity specific monoclonal antibodies were enriched through three rounds of panning. Through the above panning process and VHH ELISA detection, 16 candidate molecules were selected from the 5115-H4-M34 parent line mutants based on the affinity and sequence similarity at the VHH level for the expression of nanobody-Fc fusion proteins, and 40 candidate molecules were selected from the 5115-H1 parent line mutants for the expression of nanobody-Fc fusion proteins and ELISA detection.

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

[0142] [Table 9]

[0143] [Table 10]

[0144] Example 11. Detection of the affinity of Nanobody-Fc fusion proteins to recombinant human IGF-1R

[0145] The affinity of nanobody fusion protein to recombinant human IGF-1R was detected using BIAcore instrument. Antigen was diluted to 12.5μg / mL with NaAc buffer at pH 4.75, flow rate was set to 10μL / min, default activation chip time of EDC and NHS mixture was 420s, recombinant human IGF-1R-his was respectively coupled to 800RU level on CM5 chip using preset binding volume mode, and unbound activation groups were blocked with ethanolamine. Nanobody-Fc fusion protein diluted 2-fold in series (5 concentrations each for each fusion protein) was injected sequentially at a flow rate of 50μL / min at 25℃, with binding time of 120s and dissociation time of 600s. BIAcore T200 analysis software was used to fit the curves using 1:1 Langmuir binding model, thereby determining its kinetic constants such as binding rate constant, dissociation rate constant and binding dissociation constant.

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

[0147] [Table 11]

[0148] Example 12 ELISA experiment of competitive binding of nanobody-Fc fusion protein to antigen protein and its ligand

[0149] From the construction of the humanized and mutant libraries, nanobody-Fc fusion proteins with relatively high antigen-binding activity were selected, and their competitive binding activity to the antigen protein and its ligand 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. The plate was washed 4 times with 1×TBST and then blocked with 2% BSA at 37°C for 1.5 h. After washing the plate 4 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 different concentrations of nanobody-Fc fusion protein (starting concentration 10 μg / mL, 2-fold gradient dilution) were added to the 96-well plate, respectively, and incubated at 37°C for 1 h. After washing the well plate four times with 1xTBST, horseradish peroxidase-labeled streptavidin (Thermo, 21126) was added and incubated at 37°C for 1 h. After washing four times with 1xTBST, 200 μL of TMB substrate solution was added and color developed in the dark for 20 min. The reaction was stopped by adding 50 μL of 1M H2SO4, and the OD was measured using a microplate reader. 450 The IC was calculated by fitting the curves using Graphpad Prism software.50 The values ​​were calculated. As shown in Table 7, the above Nanobody-Fc fusion protein can effectively competitively bind to IGF-1R, and the inhibitory activity is superior to that of the positive control antibody Teprotumumab.

[0151] [Table 12]

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

[0153] The VHH gene sequence and Fc sequence of 5115-H4-M34 (IgG1 C220S L234A L235A D356E L358M or IgG1 C220A L234A L235A D356E L358M) were synthesized into a full-length sequence, and the pcDNA3.4 plasmid was enzymatically 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, the bacterial liquid was spread, and cultured at 37 ° C for 16 to 20 h. A single colony in the plate was selected and colony PCR was performed, positive clones were identified by electrophoresis, positive clones were selected and sequenced, and correctly sequenced clones were expanded and cultured, and the plasmid was extracted. HEK293 cells were transiently transfected using liposomal transfection reagent and incubated in a cell incubator at 37°C with 5% CO2 in an orbital oscillator rotating at 125 rpm. On day 5, the supernatant was collected by centrifugation, purified by Protein A affinity chromatography column, and buffer exchanged with phosphate buffer by dialysis to obtain nanobody-Fc fusion proteins 5115-H4-M34-Fc' and 5115-H4-M34-Fc", respectively.

[0154] BaF3 cells overexpressing human IGF-1R can shed the dependence of wild-type BaF3 cells on IL-3 and instead grow dependent on downstream signaling pathways induced by the binding of IGF-1 to IGF-1R. Therefore, it can be used as a cytological model to detect the blocking activity of anti-IGF-1R antibodies against the binding of IGF-1 to IGF-1R. Take a stable human IGF-1R BaF3 cell line in logarithmic growth phase, resuspend the cells using fresh RPMI1640 medium, and inoculate the cell suspension at 90 μL / well (3000 cells / well) into a 96-well cell culture plate and place it in a carbon dioxide incubator (37°C, 5% CO2). Prepare 10x nanobody-Fc fusion protein solution or control antibody solution and add it to the 96-well plate inoculated with cells at 10 μL / well, making the final concentration of protein 20 μg / mL, and dilute it 3.16 times to 9 concentrations. The final concentration of IGF-1 in the incubation system was 100 ng / mL. The cell culture plate was placed in a carbon dioxide incubator (37°C, 5% CO2) and incubated for 72 hours. The cell culture plate was then equilibrated to room temperature, and cell activity was detected using CellTiter-Glo reagent (Promega, G7573). The cell viability was calculated using the following formula: Cell viability (%) = (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%. Data were analyzed using Graphpad Prism software and fitted using nonlinear S-curve regression to obtain dose-effect curves, thereby determining the IC 50 The values ​​were calculated, and the fitting results are shown in Table 8. The results showed that the two tested Nanobody-Fc fusion proteins both had relatively strong inhibitory effects on the proliferation of BaF3 cells overexpressing human IGF-1R, and the inhibitory effects were significantly superior to those 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 nanobody-Fc fusion protein on IGF-1-induced MCF-7 cell proliferation

[0158] Logarithmic growth phase human breast cancer MCF-7 cells were digested with trypsin, resuspended in RPMI1640 medium containing 10% FBS, seeded into 96-well cell culture plates at a density of 4000 cells / well, and cultured overnight in a carbon dioxide incubator (37°C, 5% CO2) with the walls attached, after which the culture medium was replaced with 50μL of serum-free RPMI1640 medium and continued to be cultured in a carbon dioxide incubator for 5 hours. 2x nanobody-Fc fusion protein solution or control antibody solution was prepared using RPMI1640 medium containing 2% FBS and 20ng / mL IGF-1, with the highest concentration of 20μg / mL, diluted 5-fold, totaling 9 concentrations, and 50μL was added to the corresponding wells of the 96-well plate seeded with the above cells. The cell culture plate was placed in a carbon dioxide incubator (37°C, 5% CO2) and incubated for 6 days, after which the cell viability was detected using a 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 the IC 50 The values ​​were calculated, and the fitting results are shown in Table 9. The results show that the two tested Nanobody-Fc fusion proteins both have relatively strong inhibitory effects on IGF-1-induced MCF-7 cell proliferation, and the inhibitory effects are significantly superior to those 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 nanobody-Fc fusion protein on IGF-1-induced MCF-7 cell signaling pathway

[0162] MCF-7 cells in logarithmic growth phase were taken and inoculated into a 6-well plate (approximately 4.5*10e5 cells) and cultured overnight at 37°C in a 5% CO2 cell incubator. The culture medium was discarded, the cells were washed with 2mL of DPBS, and fresh cell culture medium without FBS was added and cultured overnight at 37°C in a 5% CO2 cell incubator. Nanobody-Fc fusion protein, positive control or negative control antibody (final concentration 2μg / mL) was added to each well, gently mixed evenly, and then incubated at 37°C for 10 minutes, IGF-1 (final concentration 50ng / mL) was added, and incubated at 37°C for 20min. The culture medium was discarded, the cells were washed with 2mL of DPBS, and the washing solution was discarded, and 150μL of cell lysis solution containing protease inhibitors was added to each well. The cells were lysed on ice for 30 min, collected and transferred to a 1.5 mL centrifuge tube, and centrifuged at 4°C, 12,000 rpm for 20 min. The protein concentration of the supernatant was measured using a BCA reagent kit, and then 4x injection buffer containing DTT was added after adjusting the protein concentration. The mixture was mixed uniformly and heated at 100°C for 5 min. Western Blot method was used to detect Erk1 / 2 phosphorylation levels and total Erk1 / 2 protein levels in each sample. The primary antibodies were Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (D13.14.4E) XP® 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 with the blank cell control, IGF-1 stimulation effectively improved the phosphorylation level of Erk1 / 2, and the nanobody-Fc fusion proteins 5115-Fc and 5115-H0 pretreatment could both effectively inhibit IGF-1-induced Erk1 / 2 phosphorylation, and the inhibitory effect was superior to that of the positive control Teprotumumab.

[0163] Furthermore, MCF-7 cells in the logarithmic growth phase were taken and inoculated into a 6-well plate (about 4.5*10e5 cells) and cultured overnight at 37°C in a 5% CO2 cell incubator. The culture medium was discarded, the cells were washed with 2mL of DPBS, and fresh cell culture medium without FBS was added and cultured overnight at 37°C in a 5% CO2 cell incubator. Nanobody-Fc fusion protein, positive control or negative control antibody was added to each well, with a final concentration of 5μg / mL or 0.5μg / mL, and after gentle homogeneous mixing, incubated at 37°C for 30 minutes, IGF-1 (final concentration 300ng / mL or 30ng / mL) was added and incubated at 37°C for 20min. The culture medium was discarded, the cells were washed with 2mL of DPBS, and the washing liquid was discarded, and then 150μL of cell lysis solution containing protease inhibitors was added to each well. The cells were lysed on ice for 30 min, collected and transferred to a 1.5 mL centrifuge tube, and centrifuged at 4°C, 12,000 rpm for 20 min. The protein concentration of the supernatant was measured using a BCA reagent kit, and then 4x injection buffer containing DTT was added after adjusting the protein concentration. The mixture was mixed uniformly and heated at 100°C for 5 min.The phosphorylated and total MEK protein levels, phosphorylated and total Erk1 / 2 protein levels, and phosphorylated and total Akt protein levels of each sample were detected by Western Blot method. The primary antibodies were Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (D13.14.4E) XP® Rabbit mAb (CST, #8544), p44 / 42 MAPK (Erk1 / 2) (137F5) Rabbit mAb (CST, #4695), Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (CST, #4060), and Akt (pan) (40D4) Mouse. The primary antibodies were anti-rabbit IgG-HRP second antibody (CST, #7074) or anti-mouse IgG-HRP (CST, #7076). The detection results are shown in Figure 4. Compared with the blank cell control, IGF-1 stimulation effectively improved the phosphorylation levels of MEK, Erk1 / 2 and Akt in MCF-7 cells, and the phosphorylation levels of the proteins increased with increasing IGF-1 concentration. There was no significant difference in the total protein levels of MEK, Erk1 / 2 and Akt in each treatment group. When pretreated with the nanobody-Fc fusion protein 5115-H4-M34 Fc' at 5 μg / mL and 0.5 μg / mL, both could effectively inhibit IGF-1-induced MEK, Erk1 / 2 and Akt phosphorylation, and the inhibitory effect on protein phosphorylation under stimulation with 30 ng / mL IGF-1 was equivalent to that of the positive control, and the inhibitory effect on protein phosphorylation under stimulation with 300 ng / mL IGF-1 was superior to that of the positive control Teprotumumab.

Claims

1. 1. An antigen-binding protein comprising: IC of about 1.0 μg / mL or lower in competitive ELISA detection ５０ can inhibit competitive binding of IGF-1R to its ligand at a value Antigen-binding proteins.

2. The IGF-1R comprises an IGF-1R derived from a primate.

2. The antigen-binding protein of claim 1.

3. The IGF-1R ligands include IGF-1 and / or IGF-2, and functionally active fragments thereof.

3. An antigen-binding protein according to claim 1 or 2.

4. 1. An antigen-binding protein comprising: the antigen binding protein is capable of competitively binding to IGF-1R with a reference antibody, the reference antibody comprising HCDR1-3, the HCDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 85, 100 and 131, respectively; Antigen-binding proteins.

5. The reference antibody comprises a heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO: 1; 5. The antigen-binding protein of claim 4.

6. 1. An antigen-binding protein comprising: the antigen binding protein comprises a HCDR3, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 217 or 218; Antigen-binding proteins.

7. The antigen binding protein comprises an HCDR3, the HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 130-153.

7. An antigen-binding protein according to any one of claims 1 to 6.

8. the antigen binding protein comprises a HCDR2, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 215 or 216; 8. An antigen-binding protein according to any one of claims 1 to 7.

9. The antigen binding protein comprises an HCDR2, the HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 96-129.

9. An antigen-binding protein according to any one of claims 1 to 8.

10. The antigen binding protein comprises an HCDR1, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 213 or 214.

10. An antigen-binding protein according to any one of claims 1 to 9.

11. The antigen binding protein comprises an HCDR1, the HCDR1 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 81-95.

11. An antigen-binding protein according to any one of claims 1 to 10.

12. a heavy chain variable region VH, the VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 217 or 218, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 215 or 216, and the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 213 or 214; 12. An antigen-binding protein according to any one of claims 1 to 11.

13. The antibody comprises a heavy chain variable region VH, the VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising an amino acid sequence shown in any one of SEQ ID NOs: 130 to 153, the HCDR2 comprising an amino acid sequence shown in any one of SEQ ID NOs: 96 to 129, and the HCDR1 comprising an amino acid sequence shown in any one of SEQ ID NOs: 81 to 95; 13. An antigen-binding protein according to any one of claims 1 to 12.

14. The antigen binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are selected from the group consisting of the amino acid sequences: 【Table 1】 【Table 2】 14. An antigen-binding protein according to any one of claims 1 to 13.

15. The present invention relates to a method for producing a HCDR1 comprising the steps of: (a) administering a HCDR1 to a patient having an HCDR1 comprising administering a HCDR1 to a patient having an HCDR1; and (b) administering a patient having an HCDR1 comprising administering a HCDR1 to a patient having an HCDR1; and (c) administering a patient having an HCDR1 comprising administering a HCDR1 to a patient having an HCDR1. The present invention relates to a method for producing a HCDR1 comprising administering a HCDR1 to a patient having an HCDR1.

15. An antigen-binding protein according to any one of claims 1 to 14.

16. H-FR2, wherein the H-FR2 is located between HCDR1 and HCDR2, and the H-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 221, 222, and 187 to 195; 16. An antigen-binding protein according to any one of claims 1 to 15.

17. and H-FR3, the H-FR3 being located between HCDR2 and HCDR3, and the H-FR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 223 and 196 to 203; 17. An antigen-binding protein according to any one of claims 1 to 16.

18. The present invention relates to a method for the preparation of a HCDR3 comprising administering to a patient a HCDR3 comprising administering to said patient a HCDR3 comprising administering to said patient a HCDR4 comprising administering to said patient a HCDR3 comprising administering to said patient a HCDR4 comprising administering to said patient a HCDR3 comprising administering to said patient a HCDR4 comprising administering to said patient a HCDR3 18. An antigen-binding protein according to any one of claims 1 to 17.

19. The present invention 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; 19. An antigen-binding protein according to any one of claims 1 to 18.

20. A heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 225 or 226; 20. An antigen-binding protein according to any one of claims 1 to 19.

21. The heavy chain variable region VH comprises an amino acid sequence represented by any one of SEQ ID NOs: 1 to 80; 21. An antigen-binding protein according to any one of claims 1 to 20.

22. comprising an antibody or an antigen-binding fragment thereof, 22. An antigen-binding protein according to any one of claims 1 to 21.

23. comprising a single domain antibody or an antigen-binding fragment thereof, 23. An antigen-binding protein according to any one of claims 1 to 22.

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; 23. The antigen-binding protein of claim 22.

25. The antibody comprises a camelid antibody, a chimeric antibody, a humanized antibody and / or a fully human antibody.

23. The antigen-binding protein of claim 22.

26. Contains an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 80; 26. An antigen-binding protein according to any one of claims 1 to 25.

27. A polypeptide comprising: Comprising an antigen-binding protein according to any one of claims 1 to 26. Polypeptides.

28. The polypeptide further comprises a structure that enhances the stability of the antigen binding protein and / or is capable of binding to an Fc receptor.

28. The polypeptide of claim 27.

29. The polypeptide further comprises an immunoglobulin Fc region. A polypeptide according to any one of claims 27 to 28.

30. The antigen binding protein is linked directly or indirectly to the Fc region.

30. The polypeptide of claim 29.

31. the C-terminus of the VH of the antigen binding protein is linked directly or indirectly to the N-terminus of the Fc region; A polypeptide according to any one of claims 29 to 30.

32. The Fc region comprises an IgG1-derived Fc and / or an IgG4-derived Fc. A polypeptide according to any one of claims 29 to 31.

33. The Fc region comprises the amino acid sequence set forth in SEQ ID NO: 227; A polypeptide according to any one of claims 29 to 32.

34. The Fc region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 209 to 212; A polypeptide according to any one of claims 29 to 33.

35. 1. An immune complex comprising: Comprising an isolated antigen binding protein according to any one of claims 1 to 26 and / or a polypeptide according to any one of claims 27 to 34. immune complex.

36. A nucleic acid comprising: Encoding the isolated antigen binding protein of any one of claims 1 to 26 and / or the polypeptide of any one of claims 27 to 34. Nucleic acid.

37. A vector comprising:

37. The nucleic acid of claim 36, vector.

38. A cell, The invention comprises 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 immunoconjugate according to claim 35, a nucleic acid according to claim 36 and / or a vector according to claim 37. cell.

39. A method for producing an isolated antigen binding protein according to any one of claims 1 to 26 and / or a polypeptide according to any one of claims 27 to 34, comprising the steps of:

40. The method of claim 38, further comprising culturing the cell of claim 38 under conditions that allow expression of the isolated antigen binding protein and / or the polypeptide. method.

40. 1. A composition comprising:

35. A method for the treatment of a disease comprising administering to a patient an antigen-binding protein comprising administering to said patient an antigen-binding protein comprising said antigen-binding protein, ... composition.

41. A reagent kit comprising: The invention comprises 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 immunoconjugate according to claim 35, a nucleic acid according to claim 36, a vector according to claim 37, a cell according to claim 38 and / or a composition according to claim 40. Reagent kit.

42. 1. A method of inhibiting the proliferation of a cell overexpressing IGF-1R or a functionally active fragment thereof, inhibiting the MAPK pathway and / or inhibiting the interaction of IGF-1R or a functionally active fragment thereof with its ligand, comprising: Administering 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. method.

43. Inhibiting the MAPK pathway includes inhibiting phosphorylation of MAPK pathway proteins.

43. The method of claim 42.

44. The MAPK pathway proteins include MEK, Erk1 / 2 and Akt, or functionally active fragments thereof; 44. The method according to any one of claims 42 to 43.

45. The IGF-1R ligands include IGF-1 and / or IGF-2, and functionally active fragments thereof.

45. The method according to any one of claims 42 to 44.

46. 1. A method for detecting the presence and / or content of IGF-1R or a functionally active fragment thereof, comprising: Administering 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. method.

47. 20. 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 immunoconjugate 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 medicament, comprising The drug is used to prevent, alleviate and / or treat a disease or condition, use.

48. The disease or condition includes a tumor.

48. The use according to claim 47.

49. The disease or condition includes a tumor associated with expression of IGF-1R or a functionally active fragment thereof. Use according to any one of claims 47 to 48.

50. The disease or condition includes breast cancer.

50. The use according to any one of claims 47 to 49.

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