Antibody with high affinity to IGF-1

Abstract

The invention provides an antibody with high affinity to IGF-1, and belongs to the technical field of antibody engineering, and the heavy chain variable region amino acid sequence of the antibody has more than 90% of identity with SEQ ID NO: 1; the amino acid sequence of the light chain variable region has more than 90% of identity with SEQ ID NO: 2, the affinity with IGF-1 is two times or more than two times that of a wild antibody, and an important raw material is provided for later preparation of targeted drugs for prolonging the life of animals such as dogs.

Description

[0001] This application is a divisional application of application number 202411132602.2 filed on August 16, 2024, entitled "An anti-IGF-1 antibody and its antigen-binding fragment". Technical Field

[0002] This invention belongs to the field of antibody engineering technology, and particularly relates to an anti-IGF-1 antibody and its antigen-binding fragment. Background Technology

[0003] IGF-1 is a polypeptide with a structure similar to insulin, a single-chain protein composed of 70 amino acids with a molecular weight of 7649 Da. IGF-1 shares 48% amino acid sequence homology with insulin and has the same disulfide bond binding site. Its structure is also similar to insulin, consisting of four domains: (NH-BCAD-COOH), B (1-29), C (30-41), A (42-62), and D (63-70). Domains A and B are structural homologs of the B and A chains of insulin, respectively. Domain C is similar to the linker peptide of proinsulin, while domain D has no corresponding counterpart in insulin. IGF-1 is a very important cell mitosis promoter in animals and humans; it is crucial for maintaining the levels of proteins related to cell differentiation, and when used in combination with some growth factors, it can promote cell differentiation and maturation.

[0004] The gene structure of IGF-1 is sequence-consistent in dogs, humans, cattle, sheep, and pigs, and it also exhibits cross-species activity. IGF-1 is expressed in a wide range of tissues and cell types and has autocrine, paracrine, and endocrine functions. After secretion, IGF-1 enters the bloodstream and immediately forms a complex with IGFBP, thereby preventing proteolysis or binding to its receptor. IGF-1 is a very important cell mitosis promoter in animals and humans; it is crucial for maintaining the levels of proteins related to cell differentiation, and when used in combination with some growth factors, it can promote cell differentiation and maturation. Currently, IGF-1 appears to have a significant impact on lifespan; altering insulin / insulin-like signaling (IIS) increases the lifespan of *C. elegans* by 100%, while altering the rapamycin target (TOR) pathway increases its lifespan by 30%, and a double mutant fusing both increases the lifespan of *Nematodea* by 5-fold.

[0005] In large breeds (Canis lupus familiaris) and giant breeds, size reproduction leads to highly elevated levels of IGF-1, a hormone that drives cell growth. High IGF-1 effectively drives these animals to grow rapidly in their youth, but high IGF-1 levels in adult dogs are thought to accelerate aging and shorten their healthy lifespan. Large breeds generally have shorter lifespans than small breeds, often by half. Giant breeds like Great Danes have an average lifespan of 7-10 years, while small breeds like Chihuahuas have an average lifespan of 14-16 years. Studies by Berryman et al. have found that large breeds secrete more IGF-1 than small breeds. IGF-1, a substance that promotes bone and muscle growth, is one reason for the rapid growth and larger size of large breeds, thus contributing to their shorter lifespan. Furthermore, IGF-1 increases the risk of certain diseases and promotes tumor development, making large breeds more susceptible to tumors and cancers than small breeds. Bertrand Jordan et al. discovered that the most significant polymorphism associated with canine body size occurs in the IGF-1 gene region. Measurements of serum IGF-1 protein concentrations in the blood of approximately 50 dogs showed a positive correlation with body weight (and the presence of the T allele). The IGF-1 locus has been shown to contribute to approximately 15% of the height difference between breeds. High IGF-1 effectively drives these animals to grow at a young age, but high IGF-1 levels in adult dogs are thought to accelerate aging and shorten their healthy lifespan. Improving the lifespan of adult dogs remains a significant challenge.

[0006] IGF-1 antibodies have been reported to inhibit the proliferation of the mouse fibroblast cell line BALB / c3T3 stimulated with 20 ng / ml human IGF-1 (Russell et al., 1984). The clinical candidate drug KM1468 is a rat monoclonal antibody that neutralizes the biological activity of human IGF-I, human IGF-II, and mouse IGF-II without neutralizing the biological activity of mouse IGF-I. KM1468 has been shown to neutralize human IGF-I and IGF-II and inhibit the growth of human prostate cancer cells in adult bone implanted in non-obese diabetic / severely combined immunodeficient mice (Goya et al., 2004). Furthermore, KM1468 has been shown to inhibit liver metastases of human colorectal cancer (Miyamoto et al., 2005). These antibodies validating IGF-1 may provide a novel approach to reducing IGF-1 levels in adult dogs. Summary of the Invention

[0007] This invention utilizes phage display technology to screen existing phage libraries for specific IGF-1 monoclonal positive strains; constructs prokaryotic expression vectors to obtain strains that specifically express IGF-1 single-domain antibodies; and prepares and purifies the IGF-1 single-domain antibodies. Furthermore, this invention also provides mutants with enhanced affinity.

[0008] Immunoglobulin or antibody

[0009] Immunoglobulins or antibodies have light and heavy chains divided into variable regions (V) and constant regions (C). The antibody variable region is composed of three gene segments: V, D, and J, which are randomly combined. In humans and other mammals, the number and combination of these gene segments are highly complex, resulting in extremely high variability in antibody molecules. The variable region of an antibody molecule consists of six interconnected regions: CDR1, CDR2, and CDR3, and the intervening FR1, FR2, FR3, and FR4. CDR1 and CDR2 are located at the ends of the variable region and are primarily involved in antigen recognition and binding. CDR3 is located in the center of the variable region and is the most important determinant of antibody specificity. FR1, FR2, FR3, and FR4 form the supporting structure between CDR1, CDR2, and CDR3, and are mainly responsible for maintaining the structural stability of the variable region. Each VH and VL consists of three CDR domains and four FR domains, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0010] Immunoglobulins or antibodies can be classified into kappa (κ) or lambda (λ) types, and each antibody typically contains two identical light chains. Based on the number of Y monomers and the type of heavy chain, antibodies in mammals can be classified into five isotypes: IgG, IgM, IgA, IgD, and IgE. These isotypes differ in their biological characteristics, functional regions, and ability to bind to different antigens.

[0011] Antigen-binding fragment

[0012] In this article, "antibody" is used in the broadest sense to refer to a protein that contains an antigen-binding site, encompassing natural antibodies, synthetic antibodies, and modified antibodies of various structures, including but not limited to complete conventional antibodies and antigen-binding fragments.

[0013] The antibody or its antigen-binding fragment, wherein the antigen-binding fragment may be, but is not limited to, Fab, Fab′, F(ab′)2, Fv, single-chain antibody (scFv), single-domain antibody (sdAb), HcAb, or CDR fragment.

[0014] The "Fab" described in this invention consists of a light chain, a variable region of a heavy chain, and a constant CH1 domain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. Antibody Fab fragments displayed on phage coat proteins exhibit relatively high structural stability and can be readily converted into intact Ig antibodies, generally without impairing binding activity.

[0015] The “Fab’” described in this invention contains a variable region of a light chain and a heavy chain, a CH1 domain, and a region between the CH1 and CH2 domains, so that interchain disulfide bonds can be formed between the two heavy chains of two Fab’ segments to form F(ab′)2 molecules.

[0016] The “F(ab′)2” described in this invention consists of two Fab’ segments held together by disulfide bonds between two heavy chains.

[0017] The “Fv” described in this invention includes variable regions of heavy and light chains, but lacks constant regions.

[0018] The "single-chain antibody (scFv)" described in this invention is composed of a heavy chain variable region and a light chain variable region linked by a short peptide (linker). If only a single VH and VL are used, the single-chain antibody is monovalent; if two VH and VL are used, it is bivalent; or if more than two VH and VL are used, it is polyvalent.

[0019] The "sdAb" described in this invention contains only a single antibody variable region. Because its size is typically around 15 kDa and its volume is in the nanometer range, it is also known as a nanobody. Single-domain antibodies possess strong stability, can bind well to antigens, and are not easily triggered by the body's immune response. Furthermore, they exhibit good solubility and ease of expression, meeting various requirements for clinical trials. Due to their simple structure and high specificity, single-domain antibodies are easier to modify and can fully leverage their advantages in the clinical diagnosis and subsequent treatment of diseases.

[0020] The "HcAb" described in this invention consists of a nanobody (VHH) and two constant structural domains, CH2 and CH3.

[0021] CDR transplantation antibodies are based on chimeric antibodies that further replace mouse framework regions (FRs) with canine framework regions (FRs), retaining only 3 mouse CDRs while the others are canine structures.

[0022] Complementary determining region (CDR)

[0023] In this paper, the terms "complementary determination region" or "CDR region" or "CDR" can be used interchangeably. The three CDRs of the heavy chain are called CDR-H1, CDR-H2 and CDR-H3, and the three CDRs of the light chain are called CDR-L1, CDR-L2 and CDR-L3.

[0024] The most commonly used encoding rules for the CDR are provided by Kabat EA et al. In addition, IMGT (Lefranc, 2003), Chothia (Al-Lazikani, 1997), and others have also provided CDR encoding rules, which are well known to those skilled in the art. The CDR of the antibody in this application can be determined by those skilled in the art according to any scheme in the art.

[0025] It should be noted that the boundaries of the CDRs of the same antibody obtained based on different definition methods may differ, that is, the CDR sequences of the variable region of the same antibody obtained under different definition methods are different. Therefore, when the complementarity determination region sequence of the latter antibody contains the sequence of the CDR described in this application, but the antibody's claimed CDR boundary is different from the specific CDR boundary defined in this application only because a different CDR boundary definition method was used, the antibody still falls within the protection scope of this application.

[0026] In a specific embodiment of the present invention, the CDR is defined according to the Kabat numbering system, and the number and position of the CDR amino acid residues in the VL and VH regions of the antibody or antigen binding fragment are determined by the Kabat numbering rules.

[0027] The amino acid sequence of the heavy chain variable region of the wild-type antibody of this invention is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2. The location of its CDR region is determined by the Kabat numbering rules, as follows:

[0028] V region CDR1 CDR2 CDR3 H 31-35 50-66 99-106 L 23-33 49-55 88-98

[0029] The heavy chain complementarity-determining region (CDR-H) and light chain complementarity-determining region (CDR-H) of the wild-type antibody are as follows:

[0030] The CDR-H1 amino acid sequence is NYGMS.

[0031] The amino acid sequence of CDR-H2 is GITSTGGTTYYADAVKG.

[0032] The amino acid sequence of CDR-H3 is GWFSSFDY;

[0033] The CDR-L1 amino acid sequence is GGDSIGSKSVQ.

[0034] The CDR-L2 amino acid sequence is YGTNRPA.

[0035] The amino acid sequence of CDR-L3 is QVWDRSNKAIV;

[0036] The CDR is defined according to the Kabat numbering system.

[0037] The antibody with high affinity for IGF-1 provided by the present invention has an amino acid mutation at one or more positions relative to the wild-type antibody in its CDR, and its affinity for IGF-1 is 2 times or more than that of the wild-type antibody.

[0038] Preferably, the antibody CDR has one, two, three, four, five, six, seven, or eight amino acid mutations relative to the wild-type antibody.

[0039] Preferably, the CDR-H1 amino acid sequence of the mutant is selected from NYGMS, DYGMS, EYGMS, and NYAMS.

[0040] Preferably, the CDR-H2 amino acid sequence of the mutant is selected from GITSTGGTTYYADAVKG, AITSTGGTTYYADAVKG, GITSNGGTTYYADAVKG, GITSTGTTTYYADAVKG, GITATGGTTYYADAVKG, AITATGTTTYYADAVKG, and GILSTGGTTYYADAVKG.

[0041] Preferably, the CDR-H3 amino acid sequence of the mutant is selected from GWFSSFDY, AWFSSFDY, GWFTSFDY, and AWFTSFDY.

[0042] Preferably, the CDR-L1 amino acid sequence of the mutant is selected from GGDSIGSKSVQ, GGDSIGSRSVQ, GGDSIGSKDVQ, and GGDSIGSKMVQ.

[0043] Preferably, the CDR-L3 amino acid sequence of the mutant is selected from QVWDRSNKAIV, QVWDRSNQAIV, and QVWDSSNKAIV.

[0044] Preferably, the antibody heavy chain complementarity-determining region of the present invention is selected from:

[0045] CDR-H1 CDR-H2 CDR-H3 1 NYGMS GITSTGGTTYYADAVKG GWFSSFDY 2 DYGMS GITSTGGTTYYADAVKG GWFSSFDY 3 EYGMS GITSTGGTTYYADAVKG GWFSSFDY 4 NYAMS GITSTGGTTYYADAVKG GWFSSFDY 5 NYGMS AITSTGGTTYYADAVKG GWFSSFDY 6 NYGMS GITSNGGTTYYADAVKG GWFSSFDY 7 NYGMS GITSTGTTTYYADAVKG GWFSSFDY 8 EYGMS GITSTGTTTYYADAVKG GWFTSFDY 9 NYGMS GITATGGTTYYADAVKG GWFSSFDY 10 NYGMS GITSTGGTTYYADAVKG AWFSSFDY 11 NYGMS GITSTGGTTYYADAVKG GWFTSFDY 12 EYAMS AITATGTTTYYADAVKG AWFTSFDY 13 NYGMS GILSTGGTTYYADAVKG GWFSSFDY 14 DYYMY RINIDGTTTWYSNAVKG EVFRGGARSPED 15 TYSMS GISNGGSVTYYTDAVKG GQYGSTWYGGDY 16 SYAMN WIRSDGRRTYYADAVKG GAKDY 17 SYAMS GINSGGSSTSYADAVKG RPVGTGNFEY

[0046] In some embodiments, the antibody light chain complementarity-determining region of the present invention is selected from:

[0047]

[0048]

[0049] In some embodiments, the heavy chain complementarity-determining region and the light chain complementarity-determining region of the antibody are selected from:

[0050] CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 1 NYAMS GITSTGGTTYYADAVKG GWFSSFDY GGDSIGSRSVQ YGTNRPA QVWDRSNKAIV 2 NYAMS GITSTGGTTYYADAVKG GWFSSFDY GGDSIGSKSVQ YGTNRPA QVWDRSNQAIV 3 NYGMS AITSTGGTTYYADAVKG GWFSSFDY GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV 4 NYGMS AITSTGGTTYYADAVKG GWFSSFDY GGDSIGSKMVQ YGTNRPA QVWDRSNKAIV 5 NYGMS AITSTGGTTYYADAVKG GWFSSFDY GGDSIGSKSVQ YGTNRPA QVWDSSNKAIV 6 NYGMS AITSTGGTTYYADAVKG GWFSSFDY GGDSIGSKSVQ YGTNRPA QVWDRSNKAIV 7 NYGMS GITSNGGTTYYADAVKG GWFSSFDY GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV 8 NYGMS GITSNGGTTYYADAVKG GWFSSFDY GGDSIGSKMVQ YGTNRPA QVWDRSNKAIV 9 NYGMS GITSTGTTTYYADAVKG GWFSSFDY GGDSIGSRSVQ YGTNRPA QVWDRSNKAIV 10 NYGMS GITSTGTTTYYADAVKG GWFSSFDY GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV 11 EYGMS GITSTGTTTYYADAVKG GWFTSFDY GGDSIGSRSVQ YGTNRPA QVWDRSNKAIV 12 EYGMS GITSTGTTTYYADAVKG GWFTSFDY GGDSIGSKSVQ YGTNRPA QVWDSSNKAIV 13 NYGMS GITSTGGTTYYADAVKG GWFSSFDY GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV 14 NYGMS GITSTGGTTYYADAVKG GWFSSFDY GGDSIGSKSVQ YGTNRPA QVWDSSNKAIV 15 NYGMS GITATGGTTYYADAVKG GWFSSFDY GGDSIGSKMVQ YGTNRPA QVWDRSNKAIV 16 NYGMS GITATGGTTYYADAVKG GWFSSFDY GGDSIGSKSVQ YGTNRPA QVWDSSNKAIV 17 NYGMS GITSTGGTTYYADAVKG AWFSSFDY GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV 18 NYGMS GITSTGGTTYYADAVKG AWFSSFDY GGDSIGSKMVQ YGTNRPA QVWDRSNKAIV 19 NYGMS GITSTGGTTYYADAVKG AWFSSFDY GGDSIGSKSVQ YGTNRPA QVWDSSNKAIV 20 NYGMS GITSTGGTTYYADAVKG GWFTSFDY GGDSIGSRSVQ YGTNRPA QVWDRSNKAIV 21 NYGMS GITSTGGTTYYADAVKG GWFTSFDY GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV 22 NYGMS GITSTGGTTYYADAVKG GWFTSFDY GGDSIGSKMVQ YGTNRPA QVWDRSNKAIV 23 NYGMS GITSTGGTTYYADAVKG GWFTSFDY GGDSIGSKSVQ YGTNRPA QVWDSSNKAIV 24 EYGMS GITSTGTTTYYADAVKG GWFTSFDY GGDSIGSKSVQ YGTNRPA QVWDRSNKAIV 25 EYAMS AITATGTTTYYADAVKG AWFTSFDY GGDSIGSRSVQ YGTNRPA QVWDRSNKAIV 26 EYAMS AITATGTTTYYADAVKG AWFTSFDY GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV 27 EYAMS AITATGTTTYYADAVKG AWFTSFDY GGDSIGSKMVQ YGTNRPA QVWDRSNKAIV 28 EYAMS AITATGTTTYYADAVKG AWFTSFDY GGDSIGSKSVQ YGTNRPA QVWDSSNKAIV 29 EYAMS AITATGTTTYYADAVKG AWFTSFDY GGDSIGSKSVQ YGTNRPA QVWDRSNQAIV 30 EYAMS AITATGTTTYYADAVKG AWFTSFDY GGDSIGSKSVQ YGTNRPA QVWDRSNKAIV 31 NYGMS GILSTGGTTYYADAVKG GWFSSFDY GGDSIGSKMVQ YGTNRPA QVWDRSNKAIV 32 EYGMS GITSTGTTTYYADAVKG GWFTSFDY GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV.

[0051] The antibodies described in this invention may be, but are not limited to, canine-mouse chimeric antibodies, murine antibodies, or canine antibodies.

[0052] In embodiments, the monovalent Fab of the antibody of the present invention can be linked to another Fab or scfv targeting different proteins to generate a bispecific antibody. The bispecific antibody can have dual functions, such as the therapeutic function imparted by the present invention and the transport function that can bind to receptor molecules to enhance transboundary transport across biological barriers.

[0053] The following interchangeable terms, "bispecific antibody," "bifunctional antibody," "bispecific antibody," or "BsAb," refer to antibodies that have two different antigen-binding sites, allowing them to bind to two target antigens simultaneously. In addition to their antibody targeting function, they also mediate another specific function. The mediated specific effector molecule can be a drug, receptor, toxin, enzyme, cytokine, radionuclide, etc. The two arms of a bispecific antibody that bind to the antigen can be derived from Fab, Fab′, Fv, scFv, dsFv, sdAb, HcAb, or CDR fragments, etc.

[0054] The term "antigen-binding fragment" as used herein refers to a fragment, portion, region, or domain of an antibody capable of binding to an epitope. An antigen-binding fragment may contain one, two, three, four, five, or all six CDR domains of the antibody, and may exhibit varying specificities, affinities, or selectivity despite its ability to bind to the epitope. Preferably, the antigen-binding fragment contains all six CDR domains of the antibody.

[0055] In some embodiments, the antibodies and their antigen-binding fragments of the present invention are chimeric antibodies. A chimeric antibody is defined as one in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity. The present invention provides variable region antigen-binding sequences from canine antibodies. Therefore, the chimeric antibodies mentioned in the present invention include antibodies having one or more canine antigen-binding sequences (such as CDR) and containing one or more sequences from mouse antibodies, such as FR or C region sequences.

[0056] In some embodiments, the antibodies and antigen-binding fragments of the present invention are murine antibodies. "Murine antibody" refers to an antibody in which a CDR sequence derived from another mammalian species, such as a dog, has been transplanted onto a mouse frame sequence.

[0057] In some embodiments, the antibody and its antigen-binding fragment of the present invention are canine antibodies or whole-canine antibodies.

[0058] In some embodiments of the present invention, the canine anti-heavy chain amino acid sequence is shown in SEQ ID NO:3.

[0059] In some embodiments of the present invention, the canine anti-light chain amino acid sequence is shown in SEQ ID NO:4.

[0060] In some embodiments of the present invention, the amino acid sequence of the canine-mouse chimeric antibody heavy chain is shown in SEQ ID NO:5.

[0061] In some embodiments of the present invention, the light chain amino acid sequence of the canine-mouse chimeric antibody is shown in SEQ ID NO:6.

[0062] Sequence identity

[0063] The term "identity" as used in this article refers to the relationship between the sequences of two or more protein or polypeptide molecules, such as determining the percentage of identical residues between amino acids by aligning and comparing sequences, and can be calculated based on the size of the smallest molecule to be compared. Optimal alignment is performed using the default gap weights provided by the program.

[0064] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with another amino acid residue having a side chain R group having similar chemical properties (e.g., charge or aqueousity). Generally, conservative amino acid substitutions will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other due to a conservative substitution, the percentage sequence identity can be increased to correct for the conservatism of the substitution. Methods for making this adjustment are well known to those skilled in the art. Therefore, sequences resulting from such changes with a sequence identity greater than 90% are considered to still fall within the scope of protection of this invention. Specifically, the latter sequence has 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology with the sequences SEQ ID NO:1 and SEQ ID NO:2 listed in the application.

[0065] Reorganization and Expression

[0066] The present invention provides an isolated nucleic acid comprising a polynucleotide sequence encoding the antibody described herein.

[0067] The present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0068] When prokaryotic cells are used as the host, recombinant expression vectors typically contain strong promoters that enable transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), ribosome-binding sites for initiating translation, and transcription / translation termination sequences.

[0069] When eukaryotic cells are used as the host, recombinant expression vectors can utilize promoters derived from mammalian cell genomes (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter).

[0070] In addition, the recombinant expression vectors of the present invention also include plasmids (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pcDNA3.4, pET series and pUC19, etc.), bacteriophages (e.g., λgt4, λB, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., SV40, etc.).

[0071] The present invention provides a host cell comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector.

[0072] In one embodiment of the present invention, the host cell may be a prokaryotic cell, such as E. coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp.

[0073] In one embodiment of the invention, the host cell can be a fungus, such as Aspergillus sp., yeast cells such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurospora crassa, lower eukaryotic cells, and higher eukaryotic cells such as insect cells.

[0074] In one embodiment of the invention, the host cell may be derived from plants and / or mammals. Preferred examples of host cells include, but are not limited to, PER.C6 cells, monkey kidney cells 7 (COS7, particularly simian COS cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, young hamster kidney (BHK) cells, Madin-Darby canine kidney (MDCK) cells, myeloma cell lines, HuT78 cells, 293T cells, 293F cells, and other mammalian host cells that produce antibody proteins according to the invention.

[0075] In this invention, the methods for conversion into host cells include any method for introducing nucleic acids into an organism, cell, tissue, or organ, which can be performed using standard techniques selected according to the type of host cell, as is known in the art. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated conversion, and conversion mediated by PEG, dextran sulfate, lipofectamine, or drying / inhibition.

[0076] Applications and modes of operation

[0077] The use of the antibody or antigen-binding fragment of the present invention in any of the following:

[0078] 1) To prepare drugs for the treatment or prevention of IGF-1-mediated diseases in dogs;

[0079] 2) To prepare drugs that prolong the lifespan of dogs;

[0080] 3) Prepare a kit for identifying IGF-1.

[0081] In one embodiment, antibody conjugates, multispecific antibodies (preferably bispecific antibodies), fusion proteins, or pharmaceutical compositions can be used to prepare drugs that prolong the lifespan of animals, especially dogs, with the main components comprising the antibodies or antigen-binding fragments described in this invention.

[0082] Preferably, the antibody-drug conjugate further comprises a conjugation portion conjugated to the antibody or its antigen-binding fragment, the conjugation portion being selected from purified tags, cytotoxic agents, detectable markers, radioactive isotopes, luminescent substances, colored substances, enzymes, or polyethylene glycol, etc.

[0083] Preferably, the multispecific antibody further comprises an antibody or antigen-binding fragment targeting other antigens and / or other antigenic epitopes.

[0084] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0085] Preferably, the pharmaceutical composition is used alone or in combination with one or more drugs. More preferably, it further comprises an additional therapeutic agent selected from one or more of the following: IGF-1 pathway inhibitors, IGF-1 inhibitors, IGF-1R inhibitors, rapamycin target (TOR) pathway inhibitors, rapamycin, acarbose, and aspirin.

[0086] A diagnostic kit comprising a reagent for detecting the presence and / or level of IGF-1 in a subject sample, the reagent comprising an antibody or antigen-binding fragment that specifically binds to IGF-1, preferably said antibody being a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

[0087] Furthermore, the kit also includes a detection reagent selected from chemiluminescent labels, electrochemiluminescent labels, chromophores, fluorescent labels, fluorescein-type labels, umbelliferone, lissamine, cyanin, Texas red, paramagnetic labels, radioactive labels, biotin, streptavidin / biotin, avidin / biotin, hapten, digoxigenin, metal complexes, metals, enzymes, colloidal gold, or combinations thereof.

[0088] Further, the detection method or principle is selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single-molecule immunoassay (SimoA), flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blotting, protein chip, positron emission tomography (PET), and / or single-photon emission computed tomography (SPECT). Preferably, the kit includes reagents, materials, containers, and / or devices required to perform the detection selected from chemiluminescence assay, electrochemiluminescence assay, ELISA, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single-molecule immunoassay, flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blotting, and / or protein chip. Preferably, the ELISA assay is selected from direct ELISA, indirect ELISA, direct sandwich ELISA, and indirect sandwich ELISA.

[0089] Specifically, the antibody or its antigen-binding fragment binds to IGF-1, preferably canine IGF-1 (accession number: A0A8I3NBV2). This invention constructs a recombinant canine IGF-1 protein with the following specific amino acid sequence:

[0090] MGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSALE (SEQ ID NO: 7).

[0091] Compared with the prior art, the present invention has the following beneficial effects:

[0092] This invention provides multiple antibodies targeting IGF-1 and their antigen-binding fragments; and optimizes the variable region CDR on the preferred sequence, so that the affinity with IGF-1 is increased by at least 2 times compared with wild-type antibodies, providing important raw materials for the subsequent preparation of targeted drugs and qualitative / quantitative detection kits. Attached Figure Description

[0093] Figure 1 The results of SDS-PAGE for the expression and purification of recombinant canine-mouse chimeric antibody (wild type).

[0094] Figure 2 Western Blot results for expression purification (wild type) of recombinant canine antibody.

[0095] Figure 3 SDS-PAGE results for prokaryotic expression purification of antigen IGF-1 protein.

[0096] Figure 4 To detect the EC50 of recombinant canine-mouse chimeric antibody and IGF-1 antigen using an indirect ELISA method 50 Value result.

[0097] Figure 5 To detect ECGs of recombinant canine anti-IGF-1 antigen using an indirect ELISA method 50 Value result.

[0098] Figure 6 The results of Western Blot analysis for high-throughput expression of antibody mutants. Detailed Implementation

[0099] This invention does not impose any particular limitation on the preparation method of the recombinant vector; any conventional recombinant vector preparation method in the art can be used. In this invention, the gene can be synthesized by a biotechnology company. This invention does not impose any particular limitation on the separation and purification method; any conventional protein separation and purification method in the art can be used; preferred technical solutions are described in the embodiments.

[0100] The binding kinetic parameters include the dissociation equilibrium kinetic parameter KD. KD is the equilibrium dissociation constant between the antibody and its antigen, i.e., the ratio of koff / kon. KD is inversely proportional to affinity. The KD value is related to the antibody concentration (the amount of antibody required for a specific experiment), therefore, the lower the KD value (the lower the concentration), the higher the antibody affinity.

[0101] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0102] Example 1: Expression and purification of anti-IGF-1 wild-type antibody

[0103] Gene fragments of the heavy and light chain variable regions (SEQ ID NO:1 and SEQ ID NO:2) of the wild-type antibody B8 against IGF-1 were synthesized by Genscript Biotech Co., Ltd. and constructed into the pUC57 vector. These fragments were then constructed into the mouse and canine constant regions, respectively. Specific primers were designed using SnapGene software, and the primer sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. The antibody variable region gene fragments were amplified using conventional PCR. The PCR reaction system (25 μL) consisted of: 25 μL of 2×PCR enzyme mixture, 2 μL of upstream primer, 2 μL of downstream primer, 1 μL of template, and 20 μL of ddH2O. The PCR reaction program was: 98℃ pre-denaturation for 3 min, (98℃ denaturation for 15 s, 63℃ annealing for 15 s, 72℃ extension for 15 s) for 30 cycles, and a final extension at 72℃ for 3 min. The vector containing the mouse / dog constant region was linearized using specific primers. The PCR product was recovered by gel electrophoresis. The antibody gene fragment was homologously recombinated with the linearized expression vector. After incubation at 37°C for 30 min, the mixture was immediately placed on ice. The recombinant product was transformed into competent *E. coli* DH5α strain using a heat shock method. The transformed product was plated on LB agar plates containing 100 μg / ml ampicillin (Amp). Positive clones were picked and identified by colony PCR. The plasmid was extracted and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The correctly sequenced strain was cultured and plasmid was extracted using the Tiangen endotoxin-free plasmid large-scale extraction kit (DP117-TA). For detailed operating procedures, please refer to the corresponding instruction manual.

[0104] One day before transfection, 293F cells were passaged at a density of 1–1.5 × 10⁻⁶. 6 Cells / ml, cell viability greater than 98%. Add 1 mg of recombinant plasmid to 10 ml of Opti-MEM medium, then add 2 ml of PEI transfection reagent, mix well and let stand for 15 min. Add the transfection mixture to 1 L of 293F cells. Monitor cell count and viability daily. When cell viability drops below 60% on days 5-7 of cell expression, collect and purify the sample. Centrifuge at 8000 rpm for 20 min, collect the supernatant, and filter. Pack Protein A packing material into a chromatography column, pass through 10 column volumes of ultrapure water to wash away residual ethanol. Equilibrate the column with 10 column volumes of PBS. Add the collected supernatant to the chromatography column, load 3-5 times, wash with PBS, and elute with glycine solution (pH 3.0, 0.1 mol / L). Neutralize the eluent with an appropriate volume of Tris-HCl (pH 8.0, 1 mol / L) until the solution is neutral. The purified sample was subjected to SDS-PAGE electrophoresis to verify the molecular weight and purity of the protein. The protein sample was mixed with protein loading buffer (-DTT, +DTT), placed in a metal bath, boiled at 95°C for 10 min, and then loaded onto the gel. The protein concentration was determined by BCA method to estimate the yield of the target protein obtained after affinity purification.

[0105] The results are as follows Figure 1 , 2 As shown, recombinant canine-mouse chimeric IGF-1 antibody B8 and canine anti-B8 were successfully expressed and purified.

[0106] Example 2: Expression, purification, and ELISA detection of the EC50 of recombinant antibody and antigen 50

[0107] 3.1 Expression and purification of IGF-1 antigen

[0108] GenScript Biotechnology Co., Ltd. was commissioned to synthesize the canine IGF-1 gene fragment and construct it into the pET32a E. coli expression vector. Based on bioinformatics analysis including signal peptide, transmembrane, and glycosylation, spatial structure prediction, and referencing homologous protein information, the Gly49-Ala118 segment of the IGF-1 protein was extracted and recombinantly expressed using the E. coli expression system. The plasmid was transformed into BL21(DE3) cells, and plates were incubated upside down at 37°C for 12-16 hours. One plump colony was picked and added to 5 ml of Kan-resistant (50 μg / mL) LB medium, and incubated overnight at 37°C and 220 rpm. The next day, 5 ml of the bacterial culture was inoculated into 500 ml of Kan-resistant (50 μg / mL) TB medium and incubated at 37°C and 220 rpm. When the OD600 of the bacterial culture reached 1.2, IPTG at a final concentration of 0.5 mM was added to induce E. coli expression. Expression was induced for 5 h at 37℃ and 220 rpm in a shaker. The bacteria were harvested using a high-speed centrifuge at 13000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells were resuspended in PBS and pipetted until no granular or lumpy cells remained. The bacterial cells were sonicated and centrifuged at 13000 rpm for 15 min at 4℃ to collect inclusion bodies. The inclusion body precipitate was washed with buffer A (50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 2 M Urea) by sonication. The inclusion bodies were dissolved in buffer B (50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 10 mM DTT, 2 mM sodium deoxycholate, 8 M Urea) overnight at 4℃. Centrifuge at 13000 rpm for 15 min, filter the supernatant, remove the Ni-NTA gravity column (1 ml Ni-NTA), wash with 5 column volumes of filtered pure water, and rinse with 3 column volumes of Lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM imidazole, pH 7.4). Load the sample using a peristaltic pump, first thoroughly rinsing the peristaltic pump tubing with pure water, then rinsing with Lysis buffer. Then, use the peristaltic pump to load the filtered supernatant into the Ni-NTA gravity column and collect the flow-through. Wash the Ni-NTA with 100 ml of Washbuffer (50 mM Tris-HCl, 100 mM NaCl, 20 mM imidazole, pH 7.4) and collect the wash buffer. The target protein was eluted with elution buffer (50mM Tris-HCl, 100mM NaCl, 300mM imidazole, pH 7.4), 1 ml was added each time, and the mixture was incubated for 5 min. The eluent was collected. The molecular weight and purity of the protein were verified by SDS-PAGE electrophoresis. The protein sample was mixed with protein loading buffer (DTT), placed in a metal bath, boiled at 95°C for 10 min, and then loaded onto the gel.Protein concentration was determined using the BCA method to estimate the yield of the target protein after affinity purification. The supernatant was diluted to 0.1-1.0 mg / ml, placed in a dialysis bag, and incubated in a gradient refolding buffer. Dialysis was performed slowly at 4°C for 24-36 h, followed by dialysis into PBS. Superdex 200 was used to further improve the purity of the target protein. The purified protein was then digested with TEV to obtain IGF-1 protein without the fusion tag.

[0109] The results are as follows Figure 3 As shown, IGF-1 protein was successfully expressed and purified.

[0110] 3.2 Indirect ELISA method for detecting ECGs of recombinant antibody and IGF-1 antigen 50 value

[0111] One day in advance, 2 μg / ml IGF-1 protein was coated onto 96-well ELISA plates at 4°C. The next day, the coating solution was discarded, and 300 μL / well of 2% BSA solution was added for blocking. The plates were blocked at 37°C for 60 min, and washed four times with PBST. The purified recombinant antibody was serially diluted with PBS to corresponding concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml, and added to the blocked ELISA plates at 100 μL / well. The plates were incubated at 37°C for 60 min. The enzyme-labeled secondary antibody was diluted 1:20000 and added to the ELISA plates. The plates were incubated at 37°C for 60 min, followed by TMB color development for 20 min. The color development was then stopped, and the absorbance at 450 and 650 nm was measured. Sample dilution buffer was added as a negative control.

[0112] The results are as follows Figure 4-5 As shown, the EC of recombinant chimeric antibody B8 50 The value was 0.558, indicating the EC50 value of recombinant canine anti-B8. 50 The value is 2.513.

[0113] Example 3 Antibody Affinity Modification and Screening

[0114] High-throughput expression and screening of mutants of the anti-IGF-1 antibody. Based on the ELISA results of Example 3, recombinant chimeric antibody B8 was selected as the template for affinity modification. The selection process for mutation sites in the heavy and light chains was as follows: HBnet and FastaDesign were used to calculate the mutant structures and energies of the heavy and light chains, respectively, to predict the structure of the mutant complex. Rosetta and MD were used to calculate the binding energy, respectively. Mutation sites were selected for free assortment screening. Mutation primers were designed, expression vectors were constructed, and plasmids verified by sequencing were transfected. 293F cells were seeded in 96-well deep-well plates 24 hours in advance, with a cell density of 1.5 × 10⁶ cells / well. 6Cells with a cell count / ml and viability greater than 98% were cultured overnight at 37°C, 5% CO2, and 1000 rpm using 0.5 ml of culture medium per well. The next day, transfection was performed. A new 96-well plate was prepared, with 50 μL of Opti-Mem, 2 μL of PEI, and 500 ng of antibody plasmid added to each well. After gentle shaking and incubation at room temperature for 15 min, the Opti-Mem, PEI, and plasmid mixture was added to the 96-well plate containing 293F cells. After mixing, the plate was returned to the 37°C, 5% CO2, and 1000 rpm high-speed shaking incubator. 24 h post-transfection, 0.5 ml of cell culture medium (preheated to 37°C) was added to each well of the 96-well plate. On days 5-6 post-transfection, several wells were randomly selected to measure cell viability and viability. Cells with viability below 50% were centrifuged and the supernatant collected. Subsequent SDS-PAGE electrophoresis and Western blotting were performed to verify antibody expression and SPR affinity assays. Results are as follows: Figure 6 As shown, all antibody mutants screened by high-throughput screening were expressed normally.

[0115] In this embodiment, mutations were performed on antibodies in the heavy chain variable region as shown in SEQ ID NO:1 and the light chain variable region as shown in SEQ ID NO:2. Wild type (serial number 1) and mutants (serial numbers 2-33) are shown in Table 1.

[0116] Table 1

[0117]

[0118]

[0119] Example 4 Determination of the kinetic constant of antibody affinity

[0120] The kinetic constants of the interaction between the antibody of this invention and IgF-1 were determined using a Biacore 8K (GE Healthcare) instrument. At 25°C, a multi-cycle capture method was used to capture approximately 20–100 response values ​​(RU) of anti-IGF-1 antibody on a Series S Sensor Chip ProteinA chip. IGF-1 protein (0, 100 nM) was flowed through the chip at a flow rate of 5 μL / min for 100 seconds of binding and 120 seconds of dissociation. The experimental results were used to calculate the kinetic parameters using a 1:1 binding model fitted in Biacore Evaluation Software (GE Healthcare). The experiment was repeated at least twice.

[0121] In this embodiment, the KD values ​​of wild-type and mutant antibodies were determined according to the antibody serial numbers shown in Table 3, and the results are shown in Table 2.

[0122] Table 2

[0123]

[0124]

[0125] The antibody mutants obtained by screening in this invention have significantly improved affinity for IGF-1 compared with wild type, preferably at least 2 times higher.

Claims

1. An antibody with high affinity for IGF-1, characterized in that, The complementarity-determining region (CDR) of the antibody has one or more amino acid mutations relative to the wild-type antibody, and its affinity for IGF-1 is 2 times or more that of the wild-type antibody. The heavy chain complementarity-determining region (CDR-H) and light chain complementarity-determining region (CDR-H) of the wild-type antibody are as follows: The CDR-H1 amino acid sequence is NYGMS. The amino acid sequence of CDR-H2 is GITSTGGTTYYADAVKG. The amino acid sequence of CDR-H3 is GWFSSFDY; The CDR-L1 amino acid sequence is GGDSIGSKSVQ. The CDR-L2 amino acid sequence is YGTNRPA. The amino acid sequence of CDR-L3 is QVWDRSNKAIV; The CDR is defined according to the Kabat numbering system.

2. The antibody according to claim 1, characterized in that, The heavy chain complementarity-determining region (CDR-H) and light chain complementarity-determining region (CDR-H) of the antibody are selected from:

3. The antibody according to claim 1, characterized in that, The antibody contains a heavy chain variable region (VH), the amino acid sequence of which has more than 90% but less than 100% identity with the amino acid sequence shown in SEQ ID NO:

1.

4. The antibody according to claim 1, characterized in that, The antibody comprises a light chain variable region (VL), the amino acid sequence of which has more than 90% but less than 100% identity with the amino acid sequence shown in SEQ ID NO:

2.

5. The antibody according to any one of claims 1-4, wherein, The antibody is a murine antibody, a canine-mouse chimeric antibody, or a whole-canine antibody.

6. An isolated nucleic acid comprising a polynucleotide sequence encoding an antibody according to any one of claims 1-5.

7. A recombinant expression vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the vector of claim 7.

9. A pharmaceutical composition, wherein, The pharmaceutical composition comprises: the antibody of any one of claims 1-5, or the antibody produced by the host cell of claim 8, and a veterinary-acceptable carrier.

10. The use of the antibody according to any one of claims 1-5 in any of the following: 1) To prepare drugs for the treatment or prevention of IGF-1-mediated diseases in dogs; 2) To prepare drugs that prolong the lifespan of dogs; 3) Prepare a kit for identifying IGF-1.

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