Pro-c3 specific antibodies and uses thereof

Abstract

The application provides a PRO-C3 specific antibody and application thereof, and belongs to the technical field of antibody engineering, and specifically, the PRO-C3 specific antibody provided by the application comprises a first antibody, a second antibody and a third antibody, the heavy chain variable region sequence of the first antibody is shown as SEQ ID No. 43, and the light chain variable region sequence of the first antibody is shown as SEQ ID No. 44; the heavy chain variable region sequence of the second antibody is shown as SEQ ID No. 45, and the light chain variable region sequence of the second antibody is shown as SEQ ID No. 46; the heavy chain variable region sequence of the third antibody is shown as SEQ ID No. 47, and the light chain variable region sequence of the third antibody is shown as SEQ ID No. 48. The PRO-C3 specific antibody provided by the application has extremely high specificity and affinity for PRO-C3, and can be used for preparation of an in-vitro detection product.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering technology, and more specifically, to PRO-C3 specific antibodies and their applications. Background Technology

[0002] The progression of liver fibrosis induced by chronic liver disease (CLD) is a key biomarker determining clinical prognosis. As the common pathological basis for cirrhosis, portal hypertension, liver failure, and hepatocellular carcinoma, the progressive accumulation of liver fibrotic scars exhibits an insidious development pattern. This slow and asymptomatic progression poses significant challenges to the development of anti-fibrotic treatment strategies: traditional assessment systems based on histological or clinical endpoints require large-scale clinical samples, long-term observation periods, and high trial costs, and the results are subject to high uncertainty; furthermore, the heterogeneous distribution of fibrosis in liver tissue easily leads to significant sampling bias in routine liver biopsies.

[0003] The current clinical "gold standard" for staging liver fibrosis still relies on liver biopsy histopathological assessment. However, this method has limitations such as the risk of bleeding due to invasive procedures and high consumption of medical resources, making it difficult to meet the needs of large-scale population screening and dynamic disease monitoring. In histological assessment systems, traditional semi-quantitative scoring systems (such as NASH-CRN, Metavir, and Ishak) are widely used, but they lack sufficient sensitivity to subtle changes in the degree of fibrosis, making it difficult to accurately capture early anti-fibrotic effects or slowly progressive lesions.

[0004] Conventional non-invasive tests (NITs), including the Fibrosis-4 (FIB-4) index and the NAFLD Fibrosis Score (NFS), are primarily used for initial screening of advanced fibrosis. Clinical studies have shown that these traditional biomarkers exhibit low diagnostic efficacy (AUROC < 0.60) in reflecting histological improvements, indicating their inability to dynamically track changes in disease activity.

[0005] Procollagen Type III N-terminal Peptide (PRO-C3), accompanying the synthesis, maturation, and deposition of type III collagen, can serve as a serological marker for liver fibrosis staging. PRO-C3 is the first independent biomarker for assessing active fibrosis and fibroblast activity, and its diagnostic, prognostic, and pharmacokinetic value has been validated in various liver disease models and treatment trials. However, current PRO-C3 detection kits are based on competitive immunoassays using single antibodies, which suffer from low specificity, limiting the accuracy and reliability of the assay.

[0006] Therefore, there is an urgent need to develop novel antibodies that bind to PRO-C3 with high specificity to improve the accuracy and reliability of detection. Summary of the Invention

[0007] The technical problem to be solved by this invention is: how to improve the specificity of the antibody for PRO-C3.

[0008] To solve the above-mentioned technical problems, the present invention provides a PRO-C3 specific antibody, wherein the PRO-C3 specific antibody is selected from any one of a first antibody, a first antibody mutant, a second antibody, a second antibody mutant, a third antibody, and a third antibody mutant, wherein...

[0009] The first antibody comprises: heavy chain CDR1 shown in SEQ ID No. 1, heavy chain CDR2 shown in SEQ ID No. 2, heavy chain CDR3 shown in SEQ ID No. 3, light chain CDR1 shown in SEQ ID No. 4, light chain CDR2 shown in SEQ ID No. 5, and light chain CDR3 shown in SEQ ID No. 6;

[0010] The first antibody mutant has at least 90% sequence identity with the first antibody and retains its specific binding ability to PRO-C3;

[0011] The second antibody comprises: heavy chain CDR1 shown in SEQ ID No. 15, heavy chain CDR2 shown in SEQ ID No. 16, heavy chain CDR3 shown in SEQ ID No. 17, light chain CDR1 shown in SEQ ID No. 18, light chain CDR2 shown in SEQ ID No. 19, and light chain CDR3 shown in SEQ ID No. 20;

[0012] The second antibody mutant has at least 90% sequence identity with the second antibody and retains its specific binding ability to PRO-C3;

[0013] Preferably, the amino acid sequence of SEQ ID No. 19 is KLS.

[0014] The third antibody includes: heavy chain CDR1 shown in SEQ ID No. 29, heavy chain CDR2 shown in SEQ ID No. 30, heavy chain CDR3 shown in SEQ ID No. 31, light chain CDR1 shown in SEQ ID No. 32, light chain CDR2 shown in SEQ ID No. 33, and light chain CDR3 shown in SEQ ID No. 34;

[0015] The third antibody mutant has at least 90% sequence identity with the third antibody and retains its specific binding ability to PRO-C3.

[0016] Preferably, the first antibody further includes: heavy chain FR1 shown in SEQ ID No. 7, heavy chain FR2 shown in SEQ ID No. 8, heavy chain FR3 shown in SEQ ID No. 9, heavy chain FR4 shown in SEQ ID No. 10, light chain FR1 shown in SEQ ID No. 11, light chain FR2 shown in SEQ ID No. 12, light chain FR3 shown in SEQ ID No. 13, and light chain FR4 shown in SEQ ID No. 14;

[0017] The second antibody further includes: heavy chain FR1 shown in SEQ ID No. 21, heavy chain FR2 shown in SEQ ID No. 22, heavy chain FR3 shown in SEQ ID No. 23, heavy chain FR4 shown in SEQ ID No. 24, light chain FR1 shown in SEQ ID No. 25, light chain FR2 shown in SEQ ID No. 26, light chain FR3 shown in SEQ ID No. 27, and light chain FR4 shown in SEQ ID No. 28;

[0018] The third antibody further includes: heavy chain FR1 shown in SEQ ID No. 35, heavy chain FR2 shown in SEQ ID No. 36, heavy chain FR3 shown in SEQ ID No. 37, heavy chain FR4 shown in SEQ ID No. 38, light chain FR1 shown in SEQ ID No. 39, light chain FR2 shown in SEQ ID No. 40, light chain FR3 shown in SEQ ID No. 41, and light chain FR4 shown in SEQ ID No. 42.

[0019] Preferably, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID No. 43, and the amino acid sequence of the light chain variable region of the first antibody is shown in SEQ ID No. 44.

[0020] The amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID No. 45, and the amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID No. 46.

[0021] The amino acid sequence of the heavy chain variable region of the third antibody is shown in SEQ ID No. 47, and the amino acid sequence of the light chain variable region of the third antibody is shown in SEQ ID No. 48.

[0022] Preferably, the first antibody mutant includes:

[0023] (a) A heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 43; and / or

[0024] (b) A light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 44; or

[0025] (c) The heavy chain variable region sequence shown in SEQ ID No. 43 and the light chain variable region sequence shown in SEQ ID No. 44;

[0026] The second antibody mutant includes:

[0027] (d) A heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 45; and / or

[0028] (e) A light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 46; or

[0029] (f) The heavy chain variable region sequence shown in SEQ ID No. 45 and the light chain variable region sequence shown in SEQ ID No. 46;

[0030] The third antibody mutant includes:

[0031] (g) A heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 47; and / or

[0032] (h) A light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 48; or

[0033] (i) The heavy chain variable region sequence shown in SEQ ID No. 47 and the light chain variable region sequence shown in SEQ ID No. 48.

[0034] A second aspect of the present invention provides a nucleotide molecule encoding the amino acid sequence of the PRO-C3 specific antibody described in the first aspect; or

[0035] The amino acid sequence that encodes the PRO-C3 specific antibody described in the first aspect has at least 90% sequence identity and retains antibody activity.

[0036] A third aspect of the present invention provides a carrier comprising the nucleotide molecule described in the second aspect.

[0037] A fourth aspect of the present invention provides a host cell, the host cell comprising the nucleotide molecule described in the second aspect or the carrier described in the third aspect, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

[0038] A fifth aspect of the present invention provides a PRO-C3 detection kit, the PRO-C3 detection kit comprising the PRO-C3 specific antibody described in the first aspect; and / or

[0039] The nucleotide molecules described in the second aspect; and / or

[0040] The carrier described in the third aspect; and / or

[0041] The host cell described in the fourth aspect.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The PRO-C3 specific antibody provided by this invention has extremely high affinity and selectivity for PRO-C3. It can specifically bind to the PRO-C3 standard peptide and has a very low cross-reactivity rate with the extended and truncated PRO-C3 peptides, which can effectively avoid cross-reactivity. During detection, it can effectively avoid interference from incompletely cleaved or over-cleaved fragments in clinical samples. Attached Figure Description

[0044] Figure 1 The results of mouse serum titer detection in Example 1 of this invention;

[0045] Figure 2 This refers to the specific detection results of the cell supernatants of the three cell lines in Example 1 of the present invention;

[0046] Figure 3 The results of SDS-PAGE electrophoresis of antibodies produced by the three cell lines in Example 1 of this invention;

[0047] Figure 4 The results of SEC-HPLC detection of antibodies produced by the three cell lines in Example 1 of this invention;

[0048] Figure 5 This refers to the specificity detection results of the antibody in Example 2 of the present invention;

[0049] Figure 6 The isoelectric point measurement results are shown in Example 5 of this invention.

[0050] Figure 7 Typical calibration curves were established for each pairing in Example 7;

[0051] Figure 8 The results are from the serum sample test in Example 7. Detailed Implementation

[0052] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0053] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0054] Terminology Explanation:

[0055] Antibodies are immunoglobulin molecules composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Based on the differences between the light and heavy chains, antibodies can be defined as five classes: IgM, IgD, IgG, IgA, and IgE. The antibody heavy chain consists of a variable region (VH) and a constant region (CH). The constant region consists of three domains (CH1, CH2, and CH3). The antibody light chain consists of a variable region (VL) and a constant region (CL), with the constant region consisting of one domain (CL). The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions called complementation-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of seven parts arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" is not limited to any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0056] Kd value: also known as dissociation rate constant, reflects the affinity of an antibody for its substrate. The smaller the Kd value, the stronger the affinity. Generally, the Kd value can be used to describe the affinity and specificity of an antibody for its substrate.

[0057] Ka value: the binding rate constant, which reflects the affinity of the antibody for the substrate. It is the opposite of Kd value. The larger the Ka value, the stronger the affinity. Usually, Ka = 1 / Kd.

[0058] Vector: As used in this article, a vector refers to a nucleotide delivery vehicle that can insert nucleotides. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors include, but are not limited to: plasmids, phage particles, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to: retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0059] Host cells: The host cells referred to in this article are cells that can be used to introduce the vector, including prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells, animal cells such as CHO cells, and human cells such as HEK293 cells.

[0060] As described in the background section, existing PRO-C3 antibodies have the drawback of low specificity, which fails to meet the requirements of clinical testing. In view of this, specific embodiments of the present invention provide a highly specific PRO-C3 antibody.

[0061] The PRO-C3 specific antibody is selected from any one of the following: a first antibody, a first antibody mutant, a second antibody, a second antibody mutant, a third antibody, and a third antibody mutant. The heavy chain variable region sequence of the first antibody is shown in SEQ ID No. 43, and the light chain variable region sequence of the first antibody is shown in SEQ ID No. 44; the heavy chain variable region sequence of the second antibody is shown in SEQ ID No. 45, and the light chain variable region sequence of the second antibody is shown in SEQ ID No. 46; the heavy chain variable region sequence of the third antibody is shown in SEQ ID No. 47, and the light chain variable region sequence of the third antibody is shown in SEQ ID No. 48.

[0062] More specifically, the heavy chain FR1 of the first antibody is shown in SEQ ID No. 7, the heavy chain CDR1 is shown in SEQ ID No. 1, the heavy chain FR2 is shown in SEQ ID No. 8, the heavy chain CDR2 is shown in SEQ ID No. 2, the heavy chain FR3 is shown in SEQ ID No. 9, the heavy chain CDR3 is shown in SEQ ID No. 3, and the heavy chain FR4 is shown in SEQ ID No. 10; the light chain FR1 of the first antibody is shown in SEQ ID No. 11, the light chain CDR1 is shown in SEQ ID No. 4, the light chain FR2 is shown in SEQ ID No. 12, the light chain CDR2 is shown in SEQ ID No. 5, the light chain FR3 is shown in SEQ ID No. 13, the light chain CDR3 is shown in SEQ ID No. 6, and the light chain FR4 is shown in SEQ ID No. 14.

[0063] More specifically, the heavy chain FR1 of the second antibody is shown in SEQ ID No. 21, the heavy chain CDR1 is shown in SEQ ID No. 15, the heavy chain FR2 is shown in SEQ ID No. 22, the heavy chain CDR2 is shown in SEQ ID No. 16, the heavy chain FR3 is shown in SEQ ID No. 23, the heavy chain CDR3 is shown in SEQ ID No. 17, and the heavy chain FR4 is shown in SEQ ID No. 24; the light chain FR1 of the second antibody is shown in SEQ ID No. 25, the light chain CDR1 is shown in SEQ ID No. 18, the light chain FR2 is shown in SEQ ID No. 26, the light chain CDR2 is shown in SEQ ID No. 19, the light chain FR3 is shown in SEQ ID No. 27, the light chain CDR3 is shown in SEQ ID No. 20, and the light chain FR4 is shown in SEQ ID No. 28.

[0064] More specifically, the amino acid sequence of SEQ ID No. 19 is KLS.

[0065] More specifically, the heavy chain FR1 of the third antibody is shown in SEQ ID No. 35, the heavy chain CDR1 is shown in SEQ ID No. 29, the heavy chain FR2 is shown in SEQ ID No. 36, the heavy chain CDR2 is shown in SEQ ID No. 30, the heavy chain FR3 is shown in SEQ ID No. 37, the heavy chain CDR3 is shown in SEQ ID No. 31, and the heavy chain FR4 is shown in SEQ ID No. 38; the light chain FR1 of the third antibody is shown in SEQ ID No. 39, the light chain CDR1 is shown in SEQ ID No. 32, the light chain FR2 is shown in SEQ ID No. 40, the light chain CDR2 is shown in SEQ ID No. 33, the light chain FR3 is shown in SEQ ID No. 41, the light chain CDR3 is shown in SEQ ID No. 34, and the light chain FR4 is shown in SEQ ID No. 42.

[0066] More specifically, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID No. 43, and the amino acid sequence of the light chain variable region of the first antibody is shown in SEQ ID No. 44;

[0067] The amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID No. 45, and the amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID No. 46.

[0068] The amino acid sequence of the heavy chain variable region of the third antibody is shown in SEQ ID No. 47, and the amino acid sequence of the light chain variable region of the third antibody is shown in SEQ ID No. 48.

[0069] More specifically, the first antibody mutant comprises: (a) a heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 43; and / or (b) a light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 44; or (c) the heavy chain variable region sequence as in (a) and the light chain variable region sequence as in (b). The second antibody mutant comprises: (d) a heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 45; and / or (e) a light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 46; or (f) the heavy chain variable region sequence as in (d) and the light chain variable region sequence as in (e). The third antibody mutant includes (g) a heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 47; and / or (h) a light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 48; or (i) the heavy chain variable region sequence as in (g) and the light chain variable region sequence as in (h).

[0070] The present invention also provides a nucleotide molecule that encodes the amino acid sequences shown in SEQ ID No. 43 to SEQ ID No. 48 or encodes an amino acid sequence that has at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 43 to SEQ ID No. 48.

[0071] A specific embodiment of the present invention also provides a carrier, the carrier comprising the aforementioned nucleotide molecules.

[0072] A specific embodiment of the present invention also provides a host cell, wherein the host cell comprises the aforementioned nucleotide molecules or carriers.

[0073] A specific embodiment of the present invention also provides a PRO-C3 detection kit, wherein the PRO-C3 detection kit includes the aforementioned PRO-C3 specific antibody and / or nucleotide molecules and / or vector and / or host cells.

[0074] More specifically, the test samples for the aforementioned PRO-C3 test kit can be selected from blood, serum, plasma, cerebrospinal fluid, semen, saliva, or urine. The test can be performed by an immunoassay method, including immunoblotting, colloidal gold-based, luminescent, fluorescent, chemiluminescent, or electrochemiluminescent detection methods, preferably heterogeneous sandwich immunoassay.

[0075] The technical solutions of the present invention are further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. In some cases, terms with a conventional understanding are limited herein for clarification or ease of reference, and such limitations should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0076] Example 1

[0077] Antibody generation and purification

[0078] The PRO-C3 immunogenic peptide was synthesized in vitro. Its amino acid sequence is shown in SEQ ID No. 49. Amino acids 1-3 form the linker sequence, and amino acids 4-16 are derived from the carboxyl-terminal fragment of the N-terminal propeptide of type III collagen after ADAMTS-2 cleavage. 5 mg of the PRO-C3 immunogenic peptide was dissolved in 1250 μL of coupling buffer (10 mM PBS, containing 0.2 M EDTA·2Na, pH=7.4). The carrier protein keyhole hemocyanin (KLH) was dissolved in the same coupling buffer to prepare a solution with a final concentration of 10 mg / mL. 500 μL of the KLH solution was added to 83 μL of freshly prepared water-soluble SMCC-sulfo solution (4.8 mg / mL), and the mixture was gently inverted and incubated at 37°C for 30 minutes. After the reaction, unreacted SMCC-sulfo was removed using a desalting column. The activated KLH solution was then mixed with the completely dissolved peptide solution and reacted at room temperature for 2 hours. The conjugated product was dialyzed in 10 mM PBS (pH=7.4) buffer, and the immunogenicity concentration was determined using the BCA method. After aliquoting, the product was stored at -20°C.

[0079] Female Balb / c mice aged 6-8 weeks were selected, labeled, and immunized. For the primary immunization, Freund's complete adjuvant emulsified immunogen was administered at a dose of 60 μg per mouse via multiple subcutaneous injections. Booster immunizations were administered using Freund's incomplete adjuvant emulsified immunogen at a dose of 30 μg per mouse, also via multiple subcutaneous injections. The interval between the primary and first booster immunizations was 2 weeks, and subsequent booster immunizations were spaced 2-3 weeks apart. Starting with the third immunization, ocular blood was collected one week after each immunization, and antiserum titers were determined using an indirect ELISA method. The mouse serum titer results are shown below. Figure 1As shown. Continuous immunization was performed until the titer stabilized. #B2 mice were given a shock immunization (50 μg immunogen / mouse) 3 days before cell fusion.

[0080] Mice used for fusion and feeder layer preparation were euthanized by cervical dislocation, and their bodies were disinfected with 75% ethanol. The thymus of the feeder mice was removed in a laminar flow hood, and feeder cells were prepared by gently grinding with a sterile ground glass slide. The resulting cells were then suspended in pre-warmed culture medium for later use. The spleens of immunized mice were harvested using the same method, and spleen cells were obtained by grinding. After counting, the cells were mixed with SP2 / 0 cells cultured to the logarithmic growth phase at a 3:1 ratio, centrifuged at 250×g for 5 minutes at room temperature, and the supernatant was discarded. 1 mL of PEG solution was slowly added to the cell pellet, and the mixture was gently mixed for 3 minutes. Immediately afterwards, 40 mL of fresh culture medium was added to terminate the fusion, and the cells were centrifuged again and the supernatant was discarded. The fused cells were resuspended in feeder cell suspension containing HAT selective medium, and seeded at 200 μL per well in 96-well plates and cultured in a cell culture incubator.

[0081] After 4 days of culture, the medium was completely replaced, and the cells were cultured for another 3 days. The antibody titer in the cell supernatant was detected using an indirect ELISA method. The specific steps were as follows: 100 μL of biotin-labeled PRO-C3 screening peptide biotin-CPTGPQNYSP (SEQ ID No. 50) at a concentration of 10 ng / mL was coated onto a 96-well plate pre-coated with streptavidin. After incubation at room temperature for 30 minutes, the supernatant was discarded, and the cells were washed 5 times. 100 μL of cell culture supernatant was added to each well, vortexed, and incubated at 37°C for 1 hour. The supernatant was discarded, and the cells were washed 5 times. 100 μL of HRP-labeled goat anti-mouse IgG antibody diluted 1:10000 was added, and the cells were incubated at 37°C for 1 hour. The supernatant was then discarded, and the cells were washed. 100 μL of TMB chromogenic solution was added to each well, and the reaction was carried out at room temperature in the dark for 4 minutes. 50 μL of 0.2 M dilute sulfuric acid was then added to terminate the reaction, and the absorbance at 450 nm was measured using a microplate reader. Hybridoma cells with high absorbance were selected for subcloning. The above detection steps were repeated after each subcloning, and monoclonal cell clusters with high absorbance were selected for further culture. After 3-4 rounds of subcloning, a stable monoclonal hybridoma cell line secreting the target antibody was obtained.

[0082] 10 ng / mL of biotinylated PRO-C3 screening peptide biotin-CPTGPQNYSP (SEQ ID No. 50) was coated onto a pre-coated streptavidin 96-well plate and incubated at room temperature for 30 minutes, followed by washing. Serially diluted PRO-C3 standard peptide PTGPQNYSP (SEQ ID No. 51), PRO-C3 extended peptide PTGPQNYSPQ (SEQ ID No. 52), and PRO-C3 truncated peptide PTGPQNYS (SEQ ID No. 53) were added to the wells: row A started with a peptide concentration of 1000 ng / mL, and was serially diluted 1:3 to row G; row H only contained an equal volume of dilution as a control. 10-fold diluted hybridoma cell culture supernatant was added to each well, and after vortexing and mixing, the plate was incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-mouse IgG antibody diluted 1:10000 was added, followed by incubation and washing again. TMB chromogenic buffer was added and reacted for 4 minutes. The reaction was terminated with 0.2M dilute sulfuric acid, and the absorbance was read at 450 nm. Hybridoma cell lines with high binding activity to the target peptide and low cross-reactivity with extended and truncated peptides were screened. Finally, 11 hybridoma cell lines capable of stably secreting highly specific PRO-C3 monoclonal antibodies were obtained. The specificity detection results of the cell supernatant of three of these cell lines are as follows: Figure 2 As shown in the diagram. The antibody produced by cell line #13 is the primary antibody, the antibody produced by cell line #22 is the secondary antibody, and the antibody produced by cell line #25 is the tertiary antibody. The heavy chain variable region sequence of the primary antibody is shown in SEQ ID No. 43, and the light chain variable region sequence of the primary antibody is shown in SEQ ID No. 44; the heavy chain variable region sequence of the secondary antibody is shown in SEQ ID No. 45, and the light chain variable region sequence of the secondary antibody is shown in SEQ ID No. 46; the heavy chain variable region sequence of the tertiary antibody is shown in SEQ ID No. 47, and the light chain variable region sequence of the tertiary antibody is shown in SEQ ID No. 48.

[0083] After culturing and sequencing the three cell lines, the light and heavy chain variable regions of the resulting antibodies were codon-optimized for the CHO-S cell expression system. These sequences were then synthesized and cloned into the pcDNA3.4 expression vector, with insertion sites at the XbaI and EcoRV restriction sites, respectively. The synthesized products were transformed into *E. coli* DH5α competent cells, positive clones were screened, and plasmids were extracted. Subsequently, the light and heavy chain plasmids were transfected into CHO-S cells for recombinant expression via transient transfection. The purified antibodies were obtained by purification using Protein G affinity chromatography, dialysis, and a DEAE anion exchange column. SDS-PAGE electrophoresis combined with Coomassie brilliant blue staining analysis showed that under reducing conditions (R), all three monoclonal antibodies exhibited a heavy chain band of approximately 50 kDa and a light chain band of 25 kDa; under non-reducing conditions (NR), the complete molecular weight of the antibodies was approximately 150 kDa. Further analysis using SEC-HPLC revealed that the monomer content of the purified antibodies was higher than 99% in all three cell lines. The SDS-PAGE electrophoresis results of the antibodies produced by the three cell lines are shown below. Figure 3 As shown, Figure 4 Figure A shows the SEC-HPLC detection results of the purified primary antibody, with a relative peak area of ​​99.62%. Figure 4 Figure B shows the SEC-HPLC detection results of the purified secondary antibody, with a relative peak area of ​​100%. Figure 4 C in the figure shows the SEC-HPLC detection results of the purified third antibody, with a relative peak area of ​​99.72%.

[0084] Example 2

[0085] Specific detection

[0086] The specificity of the three monoclonal antibodies (first antibody, second antibody and third antibody) prepared in this invention was evaluated by competitive chemiluminescent immunoassay and compared with the reference antibody (obtained by recombinant expression based on the NB61N-62 antibody sequence in WO2020 / 245404A1).

[0087] Take approximately 20 μg of peptide and mix it with 1 mg of streptavidin-labeled magnetic beads, and incubate at room temperature for 1 hour. After the reaction, wash three times with PBS buffer, and finally resuspend the magnetic beads in PBS buffer containing BSA to a concentration of 0.2 mg / mL for later use.

[0088] Take approximately 0.2 mg of the antibody to be labeled, add the labeling agent at a molar ratio of antibody to acrid ester of 1:10, and react at room temperature in the dark for 2 hours. After the reaction is complete, desalt the labeled product using a desalting column equilibrated with PB buffer, determine the protein concentration, and then aliquot for use.

[0089] PRO-C3 standard peptide, PRO-C3 extended peptide, and PRO-C3 truncated peptide were serially diluted with working solution, and competitive chemiluminescence detection was performed using the reagents prepared above. Results are as follows: Figure 5 As shown, the reference antibody NB61N-62 and the extended peptide of PRO-C3 have a cross-reactivity rate of about 8%; while the three monoclonal antibodies (first antibody, second antibody and third antibody) obtained in this invention did not show obvious cross-reactivity with the extended peptide and the truncated peptide, indicating that they have high specificity and can effectively avoid interference from incompletely enzymatically digested precursor fragments in clinical samples.

[0090] Example 3

[0091] Affinity Measurement

[0092] Biolayer interferometry (BII) was used to analyze antibody affinity using the ForteBio Octet K2 system. Biotinylated PRO-C3 screening peptide Biotin-CPTGPQNYSP (SEQ ID No. 50) was coated onto the surface of a streptavidin biosensor at a concentration of 50 ng / mL, with a curing time of 0.4 seconds. The three selected antibodies (primary, secondary, and tertiary antibodies) were prepared into six concentration gradients (2 nM, 6 nM, 18 nM, 54 nM, 162 nM, and 486 nM) for assay. All samples were loaded and analyzed sequentially from low to high concentration. By real-time monitoring of the binding and dissociation processes, the binding rate constant (Ka) and dissociation rate constant (Kd) of each antibody were calculated, and the affinity constant (Kd) was further fitted to obtain the affinity constant (K). D The results are shown in Table 1.

[0093] Table 1

[0094]

[0095] As shown in Table 1, the PRO-C3 monoclonal antibodies obtained by screening in this invention all have a high affinity, with the second antibody having a 27% higher affinity than the reference antibody.

[0096] Example 4

[0097] Antibody subtype assay

[0098] Antibody subtypes were identified using enzyme-linked immunosorbent assay (ELISA) and the Mouse Monoclonal Antibody Isotyping kit. The identification results are shown in Table 2.

[0099] Table 2

[0100]

[0101] As shown in Table 2, all three PRO-C3 antibodies obtained by screening in this invention are IgG1 subtypes.

[0102] Example 5

[0103] Isoelectric point determination

[0104] Antibody isoelectric point analysis was performed using Novex™ isoelectric point focusing preforms (pH range 3–10) and a matching buffer system.

[0105] The results are as follows Figure 6 As shown, by Figure 6 It can be seen that the isoelectric points of the first antibody, the second antibody, and the third antibody are 7.4~7.5, 6.7~7.0, and 7.2~7.4, respectively.

[0106] Example 6

[0107] Thermal stability analysis

[0108] The melting temperature (Tm) of the antibody was determined using the Protein Thermal Shift™ dye assay kit (catalog number: 4461146, ThermoFisher) in conjunction with the Roche LightCycler 480 system. The program settings are as follows:

[0109] Step 1: Hold at 37℃ for 2 minutes; Step 2: Hold at 95℃ for 2 minutes. Heating mode: continuous; Heating rate: 1℃ / second. The results showed that the Tm values ​​of the first antibody, second antibody, and third antibody were 73.22℃, 70.99℃, and 73.23℃, respectively, all higher than 70℃, demonstrating good thermal stability. Among them, the thermal stability of the first and third antibodies was slightly better than that of the reference antibody NB61N-62 (Tm = 71.53℃), while that of the second antibody was comparable.

[0110] Example 7

[0111] Application of PRO-C3 specific antibodies

[0112] Based on the principle of magnetic microparticle chemiluminescent immunoassay using a double-antibody sandwich method, the three PRO-C3 monoclonal antibodies (first antibody, second antibody, and third antibody) provided in this invention can all be used to prepare a chemiluminescent immunoassay kit for detecting PRO-C3 content in human serum, suitable for the clinical auxiliary diagnosis of liver fibrosis. Specifically, the kit includes the following steps:

[0113] Take approximately 0.2 mg of antibody and add biotinylation reagent at a molar ratio of antibody to biotin of 1:(5-15). Incubate at room temperature for 2 hours. After the reaction, dialyze to remove salt using phosphate buffer. The dialysate volume should be at least 20 times that of the labeled reaction system. Change the dialysate every 4 hours, for a total of at least 3 changes. Measure the protein concentration and set aside. Take an appropriate amount of streptavidin-modified magnetic microspheres, wash three times with PB buffer, resuspend to 1-10 mg / mL, mix with an equal volume of biotin-labeled antibody solution, and incubate at room temperature for 2 hours. Finally, dilute the microspheres to 0.2 mg / mL using PBS buffer containing BSA and set aside.

[0114] Take approximately 0.2 mg of antibody and add it to acridine ester solution at a molar ratio of antibody to acridine ester of 1:(5~15). Incubate at room temperature in the dark for 2 hours. After the reaction is complete, desalt the labeled product using a desalting column equilibrated with PB buffer, determine the protein concentration, and then aliquot for use.

[0115] In the dual-antibody sandwich detection system of the present invention, when the capturing antibody is any antibody capable of binding to the amino-terminal propeptide of type III collagen, the detection antibody may be a monoclonal antibody of the first antibody, the second antibody, or the third antibody of the present invention; when the capturing antibody is the first antibody, the second antibody, or the third antibody of the present invention, the detection antibody may be any antibody capable of binding to the amino-terminal propeptide of type III collagen.

[0116] In this embodiment, commercially available type III collagen N-terminal propeptide antibodies A and B are used as capture antibodies, and are paired with the first, second, and third monoclonal antibodies of this invention as detection antibodies, forming a total of four pairing combinations. Typical calibration curves established for each pairing are shown below. Figure 7 As shown.

[0117] Further analysis was conducted using the four paired combinations described above on serum samples from 19 healthy individuals and 19 patients with liver fibrosis. The results are shown in Table 3. Figure 8 As shown, compared with the PIIINP chemiluminescence kit and CHI3L1 chemiluminescence kit produced by Meikang Biotechnology Co., Ltd., the detection system constructed by the four pairs of the present invention has higher sensitivity and larger area under the curve (AUC), indicating that the kit based on the first antibody, the second antibody or the third antibody performs better than the conventional PIIINP and CHI3L1 detection kits in the diagnosis of liver fibrosis.

[0118] Table 3

[0119]

[0120] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A PRO-C3 specific antibody, characterized in that, The PRO-C3 specific antibody is a second antibody, which includes: heavy chain CDR1 shown in SEQ ID No. 15, heavy chain CDR2 shown in SEQ ID No. 16, heavy chain CDR3 shown in SEQ ID No. 17, light chain CDR1 shown in SEQ ID No. 18, light chain CDR2 shown in SEQ ID No. 19, and light chain CDR3 shown in SEQ ID No.

20.

2. The PRO-C3 specific antibody as described in claim 1, characterized in that, The second antibody further includes: heavy chain FR1 shown in SEQ ID No. 21, heavy chain FR2 shown in SEQ ID No. 22, heavy chain FR3 shown in SEQ ID No. 23, heavy chain FR4 shown in SEQ ID No. 24, light chain FR1 shown in SEQ ID No. 25, light chain FR2 shown in SEQ ID No. 26, light chain FR3 shown in SEQ ID No. 27, and light chain FR4 shown in SEQ ID No.

28.

3. The PRO-C3 specific antibody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID No. 45, and the amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID No.

46.

4. The PRO-C3 specific antibody as described in claim 1, characterized in that, The second antibody includes: (a) A heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 45; and (b) A light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 46; or (c) The heavy chain variable region sequence shown in SEQ ID No. 45 and the light chain variable region sequence shown in SEQ ID No.

46.

5. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the amino acid sequence of any of the PRO-C3 specific antibodies according to claims 1 to 4; or The amino acid sequence that has at least 90% sequence identity with the PRO-C3 specific antibody of any one of claims 1 to 4 and retains antibody activity.

6. A carrier, characterized in that, The carrier comprises the nucleotide molecule as described in claim 5.

7. A host cell, characterized in that, The host cell comprises the nucleotide molecule of claim 5 or the vector of claim 6, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

8. A PRO-C3 detection kit, characterized in that, The PRO-C3 detection kit includes: The PRO-C3 specific antibody according to any one of claims 1 to 4; and / or The nucleotide molecule of claim 5; and / or The carrier as described in claim 6; and / or The host cell as described in claim 7.

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