An anti-IGF-1 antibody and its antigen-binding fragment

Abstract

The present invention provides an antibody or antigen-binding fragment targeting IGF-1, belonging to the field of antibody engineering technology. The antibody or antigen-binding fragment comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; and a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. The present invention also provides antibody mutants with improved affinity, which provide important raw materials for the subsequent preparation of targeted drugs for extending the lifespan of animals such as dogs.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody engineering, and in particular relates to an anti-IGF-1 antibody and an antigen-binding fragment thereof. Background Art

[0002] IGF-1 is a polypeptide with a structure similar to insulin. It is a single-chain protein composed of 70 amino acids with a molecular weight of 7649 Da. IGF-1 shares 48% homology with insulin's amino acid sequence and possesses the same disulfide bond binding sites. Its structure is also similar to that of insulin, divided into 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 insulin B and A chains, respectively. Domain C is similar to the connecting peptide of proinsulin, while domain D has no counterpart in insulin. IGF-1 is a very important mitogen in animals and humans. It is crucial for maintaining the levels of proteins involved in cell differentiation and, when combined with certain growth factors, can promote cell differentiation and maturation.

[0003] The IGF-1 gene structure shares a consistent sequence in dogs, humans, cattle, sheep, and pigs, and exhibits cross-species activity. IGF-1 is expressed in numerous tissues and cell types and has autocrine, paracrine, and endocrine functions. Upon secretion, IGF-1 enters the bloodstream and immediately forms a complex with IGFBPs, preventing proteolysis and binding to its receptors. IGF-1 is a crucial mitogen in both animals and humans; it is crucial for maintaining levels of proteins involved in cell differentiation and, when combined with certain growth factors, can promote cell differentiation and maturation. It has been discovered that IGF-1 appears to have a significant effect on lifespan. Alterations in insulin / insulin-like signaling (IIS) increase the lifespan of Caenorhabditis elegans by 100%, while alterations in the target of rapamycin (TOR) pathway increase lifespan by 30%. Double mutants combining both extend the lifespan of nematodes by fivefold.

[0004] In large dogs (Canis lupusfamiliaris) and giant breeds, breeding for size leads to highly elevated levels of IGF-1, a hormone that drives cell growth. High IGF-1 effectively drives these animals to grow large in their youth, but elevated IGF-1 levels in adult dogs are thought to accelerate aging and shorten their healthy lifespan. Large dogs generally have shorter lifespans than small dogs, often by half. Giant breeds like the Great Dane have an average lifespan of 7-10 years, while small breeds like the Chihuahua have an average lifespan of 14-16 years. Berryman et al., in their study of dogs, found that large dogs secrete more IGF-1, a substance that promotes bone and muscle growth, than small dogs. This contributes to their rapid growth and larger size, leading to their shorter lifespans. Furthermore, IGF-1 increases the risk of certain diseases and promotes tumor development, making large dogs more susceptible to diseases such as tumors and cancer than small dogs. Bertrand Jordan et al. found that the most significant polymorphism associated with dog 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 dog weight (as well as the presence of the T allele). The IGF-1 locus has been shown to account for approximately 15% of the height variation between breeds. High IGF-1 effectively drives these animals to grow large 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 presents significant challenges.

[0005] IGF-1 antibodies have been shown to inhibit the proliferation of the mouse fibroblast cell line BALB / c3T3 stimulated with 20 ng / ml of human IGF-1 (Russell et al., 1984). The clinical candidate drug KM1468 is a rat monoclonal antibody that neutralizes the bioactivity of human IGF-I, human IGF-II, and mouse IGF-II, but not mouse IGF-I. KM1468 has been shown to neutralize both human IGF-I and IGF-I and to inhibit the growth of human prostate cancer cells implanted in adult human bone in non-obese diabetic / severe combined immunodeficient mice (Goya et al., 2004). Furthermore, KM1468 has been shown to inhibit liver metastasis of human colorectal cancer (Miyamoto et al., 2005). These IGF-1-validated antibodies may offer a novel approach for reducing IGF-1 levels in adult dogs. Summary of the Invention

[0006] The present invention utilizes phage display technology to screen existing phage libraries for specific IGF-1 monoclonal positive strains. A prokaryotic expression vector is then constructed to obtain strains that specifically express IGF-1 single-domain antibodies, which are then prepared and purified. Furthermore, the present invention provides mutants with enhanced affinity.

[0007] Immunoglobulin or antibody

[0008] The light and heavy chains of immunoglobulins or antibodies are divided into variable (V) and constant (C) regions. The variable region of an antibody is composed of a random combination of three gene segments: V, D, and J. In humans and other mammals, the number and arrangement of these segments are extremely complex, resulting in the high variability of antibody molecules. The variable region of an antibody molecule consists of six interconnected regions: CDR1, CDR2, and CDR3, and the FR1, FR2, FR3, and FR4 between them. CDR1 and CDR2 are located at the ends of the variable region and are primarily involved in antigen recognition and binding. CDR3, located in the center of the variable region, is the primary determinant of antibody specificity. FR1, FR2, FR3, and FR4 provide support between CDR1, CDR2, and CDR3, primarily responsible for maintaining the structural stability of the variable region. Each VH and VL region consists of three CDR domains and four FR domains, arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0009] The light chains of immunoglobulins or antibodies can be classified as either kappa (κ) or lambda (λ), with each antibody typically containing two identical light chains. Based on the number of Y monomers and the type of heavy chain, mammalian antibodies can be divided into five isotypes: IgG, IgM, IgA, IgD, and IgE. These isotypes differ in their biological properties, functional regions, and ability to bind to different antigens.

[0010] Antigen-binding fragment

[0011] As used herein, "antibody" is used in the broadest sense to refer to a protein containing an antigen binding site, encompassing natural antibodies, synthetic antibodies, and modified antibodies of various structures, including but not limited to complete traditional antibodies and antigen-binding fragments.

[0012] The antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment can be but is not limited to Fab, Fab', F(ab')2, Fv, single-chain antibody (scFv), single-domain antibody (sdAb), HcAb, CDR fragment.

[0013] The "Fab" fragments described in this invention are composed of the variable regions of one light chain and one heavy chain, as well as the constant CH1 domain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. Antibody Fab fragments displayed on phage coat proteins have relatively high structural stability and can be easily converted into intact Ig antibodies, generally without compromising binding activity.

[0014] The "Fab'" mentioned in the present invention contains the variable regions of one light chain and one heavy chain, the CH1 domain, and the region between the CH1 and CH2 domains, so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0015] The "F(ab')2" described in the present invention is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0016] The "Fv" mentioned in the present invention comprises the variable regions of the heavy chain and the light chain, but lacks the constant region.

[0017] The "single-chain antibody (scFv)" described in the present invention is composed of a heavy chain variable region and a light chain variable region connected 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 multivalent.

[0018] The "sdAb" described in this invention consists of only a single antibody variable region. Because it is typically around 15 kDa and nanometer-sized, it is also known as a nanobody. Single-domain antibodies are highly stable, can bind well to antigens, and are less likely to trigger an immune response. They are also highly soluble and easy to express, meeting the various requirements of clinical trials. Their simple structure and high specificity make their modification easier, allowing them to fully leverage their advantages in clinical diagnosis and subsequent treatment of diseases.

[0019] The "HcAb" described in the present invention consists of a nanobody (VHH) and two constant domains CH2 and CH3 regions.

[0020] CDR-transplanted antibodies are chimeric antibodies that further replace the mouse framework region (FR) with a canine framework region (FR). Only three mouse CDRs are retained, and the others are canine structures.

[0021] Complementary determining region (CDR)

[0022] Herein, "complementarity determining region" or "CDR region" or "CDR" are used interchangeably, and the three CDRs of the heavy chain are referred to as CDR-H1, CDR-H2 and CDR-H3, and the three CDRs of the light chain are referred to as CDR-L1, CDR-L2 and CDR-L3.

[0023] The most commonly used coding rules for the CDRs are provided by Kabat EA et al. In addition, IMGT (Lefranc, 2003), Chothia (Al-Lazikani, 1997), etc. provide CDR coding rules, which are well known to those skilled in the art. The boundaries of the CDRs of the antibodies in this application can be determined by those skilled in the art according to any method in the art.

[0024] It should be noted that the CDR boundaries of the same antibody obtained using different definition methods may differ, that is, the CDR sequences of the same antibody variable region obtained using different definition methods may differ. Therefore, antibodies whose complementarity-determining region sequences contain the CDR sequences described in this application, but whose claimed CDR boundaries differ from the specific CDR boundaries defined in this application simply due to the use of a different CDR boundary definition method, still fall within the scope of protection of this application.

[0025] 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 region and VH region of the antibody or antigen-binding fragment are determined by the Kabat numbering rules.

[0026] In one embodiment, the amino acid sequence of the heavy chain variable region of the antibody 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 positions of the CDR regions are determined according to the Kabat numbering convention, as follows:

[0027] V region CDR1 CDR2 CDR3 H 31-35 50-66 99-106 L 23-33 49-55 88-98

[0028] In one embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4. The positions of the CDR regions are determined according to the Kabat numbering convention, as follows:

[0029] V region CDR1 CDR2 CDR3 H 31-35 50-66 99-110 L 23-35 51-57 90-100

[0030] In one embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6. The positions of the CDR regions are determined according to the Kabat numbering convention, as follows:

[0031] V region CDR1 CDR2 CDR3 H 31-35 50-66 99-110 L 23-35 51-57 90-100

[0032] In one embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. The positions of the CDR regions are determined according to the Kabat numbering convention, as follows:

[0033] V region CDR1 CDR2 CDR3 H 31-35 50-66 99-103 L 23-35 51-57 90-100

[0034] In one embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10. The positions of the CDR regions are determined according to the Kabat numbering convention, as follows:

[0035] V region CDR1 CDR2 CDR3 H 31-35 50-66 99-108 L 23-35 51-57 90-100

[0036] In some embodiments, the antibodies or antigen-binding fragments of the invention comprise a heavy chain complementarity determining region (CDR) and a light chain complementarity determining region, wherein the heavy chain complementarity determining region is selected from:

[0037] 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

[0038] In some embodiments, the antibodies or antigen-binding fragments of the invention comprise a heavy chain complementarity determining region (CDR) and a light chain complementarity determining region (CDR), wherein the light chain complementarity determining region is selected from:

[0039] CDR-L1 CDR-L2 CDR-L3 1 GGDSIGSKSVQ YGTNRPA QVWDRSNKAIV 2 GGDSIGSRSVQ YGTNRPA QVWDRSNKAIV 3 GGDSIGSKDVQ YGTNRPA QVWDRSNKAIV 4 GGDSIGSKMVQ YGTNRPA QVWDRSNKAIV 5 GGDSIGSKSVQ YGTNRPA QVWDSSNKAIV 6 GGDSIGSKSVQ YGTNRPA QVWDRSNQAIV 7 TGSSSNIGRGSVA INNNRPS SSWDSSLRSAV 8 TGSSSNIGRGSVR SNSNRPS SSYDSSLRGIV 9 TGSSSNIGRGHVS ANNNRPS STWDSSLKAAV 10 TGSSPNIGRGSVA VNNNRPS SSWDTSLSDLV

[0040] In some embodiments, the heavy chain complementarity determining regions and light chain complementarity determining regions of the antibody are selected from:

[0041]

[0042]

[0043] The antibody of the present invention may be, but is not limited to, a dog-mouse chimeric antibody, a murine antibody, or a dog antibody.

[0044] In an embodiment, the monovalent Fab of the antibody of the present invention can be linked to another Fab or scFv targeting a different protein to produce a bispecific antibody. The bispecific antibody can have dual functions, such as a therapeutic function conferred by the present invention and a transport function that can bind to a receptor molecule to enhance transfer across biological barriers.

[0045] The terms "bispecific antibody", "bifunctional antibody", "bispecific antibody", "bispecific antibody" or "BsAb" used interchangeably below refer to antibodies that have two different antigen-binding sites, which can bind to two target antigens simultaneously. While exerting the targeting ability of the antibody, it also has the function of mediating another special function. The special functional effector molecules mediated can also be drugs, receptors, toxins, enzymes, cytokines, radionuclides, etc. The two arms of the bispecific antibody that bind to the antigen can be derived from Fab, Fab', Fv, scFv, dsFv, sdAb, HcAb or CDR fragments, etc.

[0046] As used herein, an "antigen-binding fragment" refers to a fragment, portion, region, or domain of an antibody that is capable of binding to an epitope. An antigen-binding fragment may contain 1, 2, 3, 4, 5, or all 6 CDR domains of such an antibody, and while capable of binding to the epitope, may exhibit different specificities, affinities, or selectivities. Preferably, an antigen-binding fragment contains all 6 CDR domains of the antibody.

[0047] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are chimeric antibodies. A chimeric antibody refers to a portion of a heavy chain and / or light chain that is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular 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, as well as fragments of such antibodies, as long as 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 CDRs) and containing one or more sequences from murine antibodies, such as FR or C region sequences.

[0048] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are murinized antibodies."Murinized antibodies"refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as canine, have been grafted onto murine framework sequences.

[0049] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are canine antibodies or whole canine antibodies.

[0050] Sequence identity

[0051] As used herein, "identity" refers to the relationship between the sequences of two or more proteins or polypeptide molecules, as determined by aligning and comparing the sequences to determine the percentage of identical residues between amino acids, and can be calculated based on the size of the smallest molecule to be compared. Optimal alignment was performed using the default gap weights provided by the program.

[0052] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophilicity). Generally, conservative amino acid substitutions will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. Therefore, sequences resulting in a sequence identity greater than 90% are considered to still fall within the scope of the present invention. Specifically, the latter sequence has a sequence identity of 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) with the sequences listed in the application, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

[0053] Recombination and expression

[0054] The present invention provides an isolated nucleic acid comprising a polynucleotide sequence encoding the antibody.

[0055] The present invention provides a recombinant expression vector comprising the above nucleic acid molecule.

[0056] When using prokaryotic cells as hosts, recombinant expression vectors generally contain a strong promoter that enables transcription (for example, 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), a ribosome binding site for initiating translation, and a transcription / translation termination sequence.

[0057] When eukaryotic cells are used as hosts, recombinant expression vectors can utilize promoters derived from the genome of mammalian cells (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).

[0058] In addition, the recombinant expression vectors of the present invention also include plasmids (for example, 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 (for example, λgt4.λB, λ-Charon, λΔz1 and M13, etc.) or viruses (for example, SV40, etc.).

[0059] The present invention provides a host cell, which comprises the above nucleic acid molecule or the above recombinant expression vector.

[0060] 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.

[0061] In one embodiment of the present invention, the host cell can be a fungus, such as Aspergillus sp., a yeast cell such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, a lower eukaryotic cell and a higher eukaryotic cell such as an insect cell.

[0062] In one embodiment of the present invention, the host cell can be from a plant and / or a mammal. 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, baby 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 the antibody protein according to the present invention.

[0063] In the present invention, the method of transforming into a host cell includes any method for introducing a nucleic acid into an organism, cell, tissue or organ, and can be performed using standard techniques selected according to the type of host cell as known in the art. The method includes but is not limited to electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, and transformation mediated by PEG, dextran sulfate, lipofectamine or desiccation / inhibition.

[0064] Application and mode of action

[0065] Use of the antibody or antigen-binding fragment of the present invention in any of the following:

[0066] 1) preparing a drug for treating or preventing IGF-1-mediated diseases in dogs;

[0067] 2) preparing a drug for extending the lifespan of dogs;

[0068] 3) Prepare a kit for identifying IGF-1.

[0069] In one embodiment, antibody conjugates, multispecific antibodies (preferably bispecific antibodies), fusion proteins or pharmaceutical compositions can be used to prepare drugs for prolonging the lifespan of animals, especially dogs, and the main component comprises the antibody or antigen-binding fragment thereof described in the present invention.

[0070] Preferably, the antibody conjugate further comprises a coupling portion coupled to the antibody or antigen-binding fragment thereof, wherein the coupling portion is selected from a purification tag, a cytotoxic agent, a detectable label, a radioactive isotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol, etc.

[0071] Preferably, the multispecific antibody further comprises antibodies or antigen-binding fragments targeting other antigens and / or other antigenic epitopes.

[0072] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0073] 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, target of rapamycin (TOR) pathway inhibitors, rapamycin, acarbose, and aspirin.

[0074] A diagnostic kit comprising a reagent for detecting the presence and / or level of IGF-1 in a sample from a subject, wherein the reagent comprises an antibody or antigen-binding fragment that specifically binds to IGF-1. Preferably, the antibody is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof.

[0075] Furthermore, the kit further comprises a detection agent selected from the group consisting of a chemiluminescent label, an electrochemiluminescent label, a chromophore, a fluorescent label, a fluorescein-type label, an umbelliferone, a lissamine, a cyanine, a Texas Red, a paramagnetic label, a radioactive label, biotin, streptavidin / biotin, avidin / biotin, a hapten, digoxigenin, a metal complex, a metal, an enzyme, colloidal gold, or a combination thereof.

[0076] Furthermore, the detection method or principle is selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single molecule immunoassay technology (Simoa), flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot, protein chip, positron emission tomography and / or single photon emission computed tomography. Preferably, the kit includes reagents, materials, containers and / or devices required for the detection selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single molecule immunoassay technology, flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot and / or protein chip. Preferably, the enzyme-linked immunosorbent assay is selected from direct enzyme-linked immunosorbent assay, indirect enzyme-linked immunosorbent assay, direct sandwich enzyme-linked immunosorbent assay and indirect sandwich enzyme-linked immunosorbent assay.

[0077] In particular, the antibody or antigen-binding fragment thereof binds to IGF-1, preferably canine IGF-1 (Accession No.: A0A8I3NBV2). The present invention constructs a canine IGF-1 recombinant protein, the specific amino acid sequence of which is:

[0078] MGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSALE (SEQ ID NO: 11).

[0079] In some embodiments of the present invention, the canine antibody heavy chain amino acid sequence is shown in SEQ ID NO:12.

[0080] In some embodiments of the present invention, the canine anti-light chain amino acid sequence is shown in SEQ ID NO:13.

[0081] In some embodiments of the present invention, the amino acid sequence of the heavy chain of the dog-murine chimeric antibody is shown in SEQ ID NO:14.

[0082] In some embodiments of the present invention, the amino acid sequence of the light chain of the dog-murine chimeric antibody is shown in SEQ ID NO:15.

[0083] Compared with the prior art, the present invention has the following beneficial effects:

[0084] The present invention provides multiple antibodies and antigen-binding fragments thereof targeting IGF-1; and optimizes the variable region CDR based on the preferred sequence, so that the affinity with IGF-1 is increased by at least 2 times, providing important raw materials for the subsequent preparation of targeted drugs and qualitative / quantitative detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 This is the SDS-PAGE result of prokaryotic expression and purification of antigen IGF-1 protein.

[0086] Figure 2 The SDS-PAGE results of the expression and purification of the recombinant dog-mouse chimeric antibody.

[0087] Figure 3 This is the Western Blot result of the expression and purification of recombinant canine antibody.

[0088] Figure 4 Indirect ELISA was used to detect the EC of recombinant canine-mouse chimeric antibody and IGF-1 antigen. 50 Value result.

[0089] Figure 5 Indirect ELISA was used to detect EC of recombinant canine anti-IGF-1 antigen 50 Value result.

[0090] Figure 6 Western Blot results of high-throughput expression of B8 recombinant chimeric antibody. DETAILED DESCRIPTION

[0091] The present invention does not specifically limit the preparation method of the recombinant vector; conventional methods for preparing recombinant vectors in the art can be employed. In the present invention, the gene can be synthesized by a biotechnology company. The present invention does not specifically limit the separation and purification method; conventional protein separation and purification methods in the art can be employed. Preferred technical solutions are described in the Examples.

[0092] The binding kinetic parameters include the dissociation equilibrium kinetic parameter, KD. KD is the equilibrium dissociation constant between an antibody and its antigen, i.e., the ratio of koff / kon. KD is inversely proportional to affinity. The KD value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), so the lower the KD value (the lower the concentration), the higher the affinity of the antibody.

[0093] The technical solutions provided by the present invention are 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.

[0094] Example 1 Screening of natural canine phage antibody library

[0095] In the existing natural canine phage antibody library (capacity 1.06×10 10 , Pujian Bio) for panning:

[0096] (1) Phage display antibody library screening

[0097] Immunotube solid-phase panning is performed. The positively screened target protein is coated on the surface of an immunotube. The library phage undergoes negative protein screening to remove nonspecifically bound phage. Phages after negative screening undergo panning, including blocking, phage-antigen reaction, elution, and titer determination. Four rounds of panning are repeated, and polyclonal phage ELISA validation is performed to determine specific enrichment.

[0098] (2) Monoclonal screening of products after phage display panning

[0099] The best product after one or several rounds of panning was selected for screening of no less than 288 monoclonal clones. Antigen protein 1 was used as the test group, and its tag protein 2 was used as the control group. Finally, clones with a test group > 3 times the control group were selected as positive clones. Sequencing analysis was performed on the obtained positive clones to remove erroneous sequences, double-peak sequences, and repeated sequences. Positive clones with different sequences were verified after two rounds of amplification. The clone sequences that were still positive after the second verification were finally determined to be positive clones. The results of the secondary ELISA test are shown in Table 1, and the microplate reader was used to read (450nm-620nm), where IGF1-HIS was expressed as Ag, NC-His was expressed as NC1, and PBS was expressed as NC2. The results show that the obtained positive clones were coated with protein for ELISA detection.

[0100] Table 1

[0101]

[0102]

[0103] After secondary verification, 21 positive sequences targeting IGF1-HIS were finally obtained; 5 antibodies that were more specifically able to bind to IGF-1 were further screened out, and their amino acid sequences are shown in Table 2.

[0104] Table 2

[0105]

[0106]

[0107] Example 2 Expression and purification of anti-IGF-1 antibodies

[0108] GenScript Biotech Co., Ltd. was commissioned to synthesize the heavy and light chain variable region gene fragments of the anti-IGF-1 antibodies B8, C5, E1, E4, and F8, respectively, and construct them into the pUC57 vector. The five antibodies were constructed with mouse and canine constant regions, respectively. Specific primers were designed using SnapGene software and synthesized by Shanghai Sangon Biotechnology Co., Ltd. Conventional PCR was used to amplify the variable region gene fragments. The PCR reaction system (25 μL) consisted of 25 μL of 2× PCR enzyme mix, 2 μL of upstream primer, 2 μL of downstream primer, 1 μL of template, and 20 μL of ddH2O. The PCR reaction program was as follows: 98°C initial denaturation for 3 min, 30 cycles of (98°C denaturation for 15 s, 63°C annealing for 15 s, and 72°C extension for 15 s), and a complete extension at 72°C for 3 min. The vector containing the mouse / canine constant region was linearized using specific primers. The PCR product was recovered by gel extraction, and the antibody gene fragment was homologously recombined with the linearized expression vector. The reaction was carried out at 37°C for 30 minutes and immediately placed on ice. The recombinant product was transformed into competent E. coli DH5α strain using the heat shock method and plated on LB plates containing 100 μg / ml ampicillin (Amp). Positive clones were selected and identified by colony PCR. The plasmids were then extracted and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The strains that were sequenced correctly were expanded and plasmids were extracted using the Tiangen Endotoxin-Free Plasmid Extraction Kit (DP117-TA). For specific steps, please refer to the corresponding instructions.

[0109] One day before transfection, culture 293F cells at a density of 1-1.5×10 6Cells / ml, with a cell viability greater than 98%. Add 1 mg of recombinant plasmid to 10 ml of Opti-MEM medium, followed by 2 ml of PEI transfection reagent, mix well, and let stand for 15 minutes. Add the transfection mixture to 1 L of 293F cells. Count and monitor the number of viable cells and cell viability daily. Collect and purify samples when the cell viability drops below 60% on day 5-7 of cell expression. Centrifuge at 8000 rpm for 20 minutes, collect the supernatant, and filter. Load the Protein A filler into a chromatography column and pass it through 10 column volumes of ultrapure water to remove any residual ethanol. After washing, equilibrate the column with 10 column volumes of PBS. Add the collected supernatant to the column and apply the sample 3-5 times. Wash with PBS and elute with glycine solution (pH 3.0, 0.1 mol / L). Neutralize the eluate with an appropriate volume of Tris-HCl (pH 8.0, 1 mol / L) until the solution is neutral. Purified samples were subjected to SDS-PAGE electrophoresis to verify protein molecular weight and purity. Protein samples were mixed with protein loading buffer (-DTT, +DTT), placed in a metal bath, and boiled at 95°C for 10 minutes before running the sample on the gel. Protein concentration was determined by the BCA assay to estimate the yield of the target protein obtained after affinity purification.

[0110] The results are as follows Figure 2 、 3 As shown, IGF-1 recombinant dog-mouse chimeric antibody and canine anti-B8, C5, E1, E4, and F8 were successfully expressed and purified.

[0111] Example 3 Expression and purification of antigen and ELISA detection of EC between recombinant antibody and antigen 50

[0112] 3.1 Expression and purification of IGF-1 antigen

[0113] GenScript Biotech Co., Ltd. was commissioned to synthesize a gene fragment for canine IGF-1 and construct it into the pET32a E. coli expression vector. Combining bioinformatics analysis of signal peptides, transmembrane domains, and glycosylation, along with spatial structure prediction and reference to homologous protein information, the Gly49-Ala118 region of the IGF-1 protein was truncated and recombinantly expressed in an E. coli expression system. The plasmid was transformed into BL21(DE3) cells, and the plates were incubated upside down in a 37°C incubator for 12-16 hours. One plump colony was then transferred to 5ml of LB medium containing Kan-resistant (50μg / mL) and shaken overnight at 37°C at 220 rpm. The next day, 5ml of the bacterial solution was inoculated into 500ml of TB medium containing Kan-resistant (50μg / mL) and shaken at 37°C at 220 rpm. When the OD600 of the bacterial solution reached 1.2, 0.5mM IPTG was added to induce expression in the E. coli. Induce expression for 5 hours at 37°C and 220 rpm in a shaker. Use a high-speed centrifuge at 13,000 rpm for 15 minutes to harvest the bacteria, discard the supernatant medium, and collect the cells. Resuspend the cells in PBS and pipette until there are no granules or clumps of cells. Ultrasonicate the cells and centrifuge at 13,000 rpm for 15 minutes at 4°C to collect the inclusion bodies. Ultrasonicate and clean the inclusion body precipitate with buffer A (50mM Tris-HCl, 1mM EDTA, 100mM NaCl, 1% Triton X-100, 2M Urea). Solubilize the inclusion bodies with buffer B (50mM Tris-HCl, 1mM EDTA, 100mM NaCl, 10mM DTT, 2mM sodium deoxycholate, 8M Urea) and dissolve overnight at 4°C. Centrifuge at 13,000 rpm for 15 minutes, filter the supernatant, remove the Ni-NTA gravity column (1 ml Ni-NTA), rinse 5 column volumes with filtered pure water, rinse 3 column volumes with Lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM imidazole, pH 7.4), and load the sample using a peristaltic pump. The peristaltic pump tubing is first rinsed extensively with pure water and then rinsed with Lysis buffer. Then, use a peristaltic pump to load the filtered supernatant onto the Ni-NTA gravity column and collect the flow-through. Wash the Ni-NTA with 100 ml of Wash buffer (50 mM Tris-HCl, 100 mM NaCl, 20 mM imidazole, pH 7.4) and collect the wash solution. Elute the target protein with elution buffer (50mM Tris-HCl, 100mM NaCl, 300mM imidazole, pH 7.4) by adding 1ml at a time, incubate for 5 minutes, and collect the eluate. Verify the protein's molecular weight and purity by SDS-PAGE electrophoresis. Mix the protein sample with protein loading buffer (DTT), place it in a metal bath, and boil it at 95°C for 10 minutes before running the sample on the gel.Protein concentration was determined by BCA assay 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 placed in a gradient renaturation buffer. Slowly dialyzed at 4°C for 24-36 hours, finally dialyzed into PBS. Superdex200 was used to further improve the purity of the target protein. The purified protein was then digested with TEV enzyme to obtain the IGF-1 protein without the fusion tag.

[0114] The results are as follows Figure 1 As shown, IGF-1 protein was successfully expressed and purified.

[0115] 3.2 Indirect ELISA for EC detection of recombinant antibodies and IGF-1 antigen 50 value

[0116] One day in advance, coat 96-well microtiter plates with 2 μg / ml IGF-1 protein at 4°C. The next day, discard the coating solution and block with 300 μL / well of 2% BSA at 37°C for 60 min. Wash the plates four times with PBST. Then, serially dilute the purified recombinant antibody with PBS to the corresponding concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml and add 100 μL / well to the blocked microtiter plates. Incubate at 37°C for 60 min. Add enzyme-labeled secondary antibody at a 1:20,000 dilution to the plates and incubate at 37°C for 60 min. Develop with TMB for 20 min, stop color development, and measure absorbance at 450 and 650 nm. Add sample dilution as a negative control.

[0117] The results are as follows Figure 4 As shown, the EC of the recombinant chimeric antibody B8 50 The EC value of the recombinant chimeric antibody C5 was 0.558. 50 The EC value of the recombinant chimeric antibody E1 was 8.109. 50 The EC value of the recombinant chimeric antibody F8 was 38.31. 50 The value was 53.65, while the recombinant chimeric antibody E4 showed no significant binding activity to the antigen IGF-1.

[0118] The results are as follows Figure 5 As shown, the EC of recombinant canine anti-B8 50 The EC value of recombinant canine anti-C5 was 2.513. 50 The EC value of recombinant canine anti-E4 was 4.395. 50 The EC value of recombinant canine anti-F8 was 47.39. 50 The value was 2.773, while the recombinant canine anti-E1 showed no significant binding activity to the antigen IGF-1.

[0119] Different constant regions affect antibody structure and stability. In terms of production yield, canine-mouse chimeric antibodies can reach 60 mg / L, while canine antibodies can only reach 10 mg / L. These constant region variations also affect antigen binding activity. E1, when constructed onto a mouse constant region vector, showed binding activity to the antigen IGF-1, but when constructed onto a canine constant region vector, it showed no binding activity. E4, when constructed onto a mouse constant region vector, showed no binding activity, but when constructed onto a canine constant region vector, it did. Similarly, variations in F8 revealed a nearly 20-fold difference in EC50 values ​​when constructed onto mouse and canine constant region vectors, respectively. The difference between B8 and C5 was within 5-fold.

[0120] Example 4 Antibody Affinity Modification and Screening

[0121] High-throughput expression and screening of mutants of anti-IGF-1 antibodies. According to the ELISA results of Example 3, the recombinant chimeric antibody B8 was selected as the template for affinity modification. The process of selecting the mutation sites of the heavy and light chains was as follows: HBnet and FastaDesign were used to calculate the mutant structure and energy of the heavy and light chains, respectively, and then AlphaFold3 was used to predict the structure of the mutant complex, and Rosetta and MD were used to calculate the binding energy, respectively. The mutation sites were selected for free combination screening. Mutation primers were designed, expression vectors were constructed, and the correct plasmids were verified by sequencing for transfection. 293F cells were plated in 96-well deep-well plates 24 hours in advance at a cell density of 1.5×10 6 Cells / ml and cell viability greater than 98%, 0.5 ml per well, placed in a high-speed shaking shaker at 37 ° C, 5% CO2, and 1000 rpm for overnight culture. The next day, transfection was performed by preparing a new 96-well plate. 50 μL of Opti-Mem, 2 ul of PEI, and 500 ng of antibody plasmid were added to each well. After gentle vortexing and standing at room temperature for 15 minutes, the Opti-Mem, PEI, and plasmid mixture was added to the 96-well plate containing 293F cells. After mixing, the plate was returned to a high-speed shaking shaker at 37 ° C, 5% CO2, and 1000 rpm. 24 hours after transfection, 0.5 ml of cell culture medium was added to each well of the 96-well deep-well plate (the culture medium must be preheated at 37 ° C in advance). On the 5th to 6th day after transfection, several wells were randomly selected to measure the number of viable cells and cell viability. If the cell viability is less than 50%, the supernatant can be centrifuged and collected. SDS-PAGE electrophoresis and Western Blot were then performed to verify the expression of the antibody and SPR affinity test. Figure 6 As shown, all high-throughput screened antibody mutants were expressed normally.

[0122] In this example, mutations were performed on the antibody heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2. The wild type (SEQ ID NO: 1) and mutants (SEQ ID NO: 2-SEQ ID NO: 33) are shown in Table 3.

[0123] Table 3

[0124]

[0125]

[0126] Example 5 Determination of Kinetic Constants of Antibody Affinity

[0127] The kinetic constants for the interaction between the antibodies of the present invention and IgF-1 were determined using Biacore 8K (GE Healthcare). Approximately 20-100 response units (RU) of anti-IGF-1 antibody were captured on a Series S Sensor Chip Protein A chip using a multi-cycle capture method at 25°C. IGF-1 protein (0, 100 nM) was passed through the chip at a flow rate of 5 μL / min, with binding for 100 seconds and dissociation for 120 seconds. Kinetic parameters were calculated using a 1:1 binding model fitted in Biacore Evaluation Software (GE Healthcare). Experiments were repeated at least twice.

[0128] In this example, the KD values ​​of the corresponding antibodies were measured according to the antibody numbers shown in Table 3. The results are shown in Table 4.

[0129] Table 4

[0130]

[0131]

[0132] The antibody mutants obtained by screening of the present invention have significantly improved affinity for IGF-1 compared with the wild type, preferably at least 2 times higher.

Claims

1. An anti-IGF-1 antibody, characterized in that Comprising a heavy chain complementarity determining region (CDR-H) and a light chain complementarity determining region (CDR-L), wherein: The amino acid sequence of CDR-H1 is NYGMS, the amino acid sequence of CDR-H2 is GITSTGGTTYYADAVKG, and the amino acid sequence of CDR-H3 is GWFSSFDY; The amino acid sequence of CDR-L1 is GGDSIGSKSVQ, the amino acid sequence of CDR-L2 is YGTNRPA, and the amino acid sequence of CDR-L3 is QVWDRSNKAIV; The CDRs are defined according to the Kabat numbering system.

2. The antibody according to claim 1, characterized in that The antibody comprises a heavy chain variable region (VH), and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

1.

3. The antibody according to claim 1, characterized in that The antibody comprises a light chain variable region (VL), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

4. The antibody according to any one of claims 1 to 3, wherein The antibody is a murinized antibody, a dog-mouse chimeric antibody or a full canine antibody.

5. An isolated nucleic acid comprising a polynucleotide sequence encoding the antibody of any one of claims 1-4. A recombinant expression vector comprising the nucleic acid according to claim 5 . A host cell comprising the nucleic acid of claim 5 or the vector of claim 6.

8. A pharmaceutical composition, wherein The pharmaceutical composition comprises: the antibody according to any one of claims 1 to 4, or the antibody produced by the host cell according to claim 7, and a veterinary acceptable carrier.

9. Use of the antibody according to any one of claims 1 to 4 in preparing a kit for identifying IGF-1.

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