Antibody for improving extraction amount of plasma cfDNA and application thereof

Abstract

The application relates to the technical field of antibodies, and particularly provides an antibody specifically binding to double-stranded DNA and application thereof. The antibody provided by the application comprises three CDRs in a heavy chain variable region amino acid sequence shown in SEQ ID NO. 7 and three CDRs in a light chain variable region amino acid sequence shown in SEQ ID NO. 8, has high affinity to cfDNA, can effectively protect and enrich cfDNA in vitro and in vivo, and can be used for clinical detection in aspects of early screening of tumors, monitoring of micro residual lesions and dynamic evaluation of curative effects.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to an antibody that specifically binds to double-stranded DNA (dsDNA) and its application in enriching cfDNA in vivo and increasing the amount of plasma cfDNA extracted, as well as its application in in vitro liquid biopsy. Background Technology

[0002] Circulating cell-free DNA (cfDNA), as a core biomarker in the field of liquid biopsy, possesses outstanding advantages such as non-invasive sampling, convenient detection, and the ability to dynamically reflect the tumor burden status in vivo. It has broad application prospects and extremely high translational value in the field of clinical tumor diagnosis. By detecting multi-dimensional molecular signals such as gene variations, methylation epigenetic modifications, and fragment omics characteristics of peripheral blood cfDNA, abnormal molecular characteristics of early-stage tumors can be effectively identified. This overcomes the shortcomings of traditional tumor detection technologies, assisting clinicians in achieving early screening, early diagnosis, and early treatment of tumors, significantly optimizing the prognosis of cancer patients, prolonging their survival, and improving their quality of life.

[0003] In the evaluation of postoperative efficacy and long-term follow-up of tumors, minimal residual disease (MRD) detection based on cfDNA has a significant time advantage over traditional imaging detection. It can sensitively capture early signals of tumor recurrence and metastasis before visible lesion changes appear on imaging, accurately assess the clinical treatment response and long-term prognostic risk, and provide important molecular evidence for the clinical development and adjustment of individualized treatment plans.

[0004] However, in practical clinical applications, cfDNA detection technology still faces significant technical hurdles. The core limiting factor is its low abundance in peripheral blood and its susceptibility to DNase degradation and immune cell clearance, leading to insufficient extraction and limited detection sensitivity. Currently, there is a lack of tools that can safely and efficiently protect cfDNA from DNase degradation in vivo, and conventional detection techniques struggle to reliably capture low-abundance tumor-specific molecular signals, resulting in insufficient sensitivity and repeatability to meet the stringent requirements of high-precision clinical testing.

[0005] From the perspective of sample collection, while increasing the amount of clinical blood collected can slightly increase the total amount of cfDNA extracted, limitations imposed by clinical operating procedures, patient tolerance, and blood collection costs prevent a significant increase in blood volume. Therefore, this optimization method is not suitable for large-scale clinical application. Current industry technologies for improving the sensitivity of liquid biopsies are mostly focused on downstream detection and data analysis, primarily including high-depth sequencing technology, unique molecular identifier (UMI) error correction technology, and bioinformatics intelligent noise reduction algorithms. These optimization methods only correct and reduce noise in the post-detection sequencing data, representing end-stage optimization solutions and failing to fundamentally address the core issue of insufficient original abundance of peripheral blood cfDNA.

[0006] In conclusion, developing a novel in vivo enrichment technology that can effectively enhance the abundance of peripheral blood cfDNA from the source is a key technical challenge that urgently needs to be overcome in the field of liquid biopsy. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention employs screening to obtain a specific antibody capable of effectively protecting and enriching cfDNA in vivo. Therefore, the primary objective of this invention is to find an antibody capable of enriching cfDNA in vivo and increasing the amount of cfDNA extracted from plasma, thereby enabling liquid biopsy of low-abundance cfDNA.

[0008] To achieve the above objectives, the present invention specifically adopts the following technical solution: The present invention first provides an isolated anti-dsDNA antibody or its antigen-binding fragment, comprising three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO.7 and three CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO.8; Alternatively, variants with a single or multiple CDRs in the light and heavy chain CDR regions, with no more than two amino acid conserved changes in each CDR region.

[0009] Furthermore, when antibody CDRs are encoded according to the Kabat encoding rules, the antibody or antigen-binding fragment contains the following CDR sequence: i. The amino acid sequences of the complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NO. 1, 2, and 3, respectively; ii. The amino acid sequences of the complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region are shown in SEQ ID NO. 4, 5, and 6, respectively; Alternatively, variants with one or more CDRs having no more than two amino acid conservation changes per CDR region, similar to the six CDR regions of i-ii above.

[0010] Furthermore, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the sequences of which are selected from the following: a) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO.7; b) The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO.8.

[0011] In some embodiments, the sequence of the antibody or antigen-binding fragment is selected from the full antibody sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10.

[0012] Furthermore, the antibody or antigen-binding fragment is also linked to a coupling portion, which is selected from one or more of, for example, enzymes, luciferins, carrier proteins, and biotin. In some respects, the antibody or antigen-binding fragment may be selectively linked to the coupling portion via a linker; in other respects, the linker is a peptide or polypeptide.

[0013] Furthermore, the antibody or antigen-binding fragment is selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies.

[0014] The present invention also provides an isolated polynucleotide encoding any of the aforementioned antibody or antigen-binding fragments.

[0015] The present invention also provides a recombinant vector comprising the aforementioned polynucleotide; In some respects, the recombinant vector is a cloning vector or an expression vector.

[0016] The present invention also provides a product comprising any of the aforementioned antibody or antigen-binding fragments; preferably, contained in a suitable container.

[0017] The present invention also provides a pharmaceutical composition comprising any of the antibody or antigen-binding fragments described above; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0018] This invention also provides various applications of any of the aforementioned antibody or antigen-binding fragments: 1) Application in enriching cfDNA in vivo and increasing the amount of plasma cfDNA extracted; 2) Application in the preparation of reagents for enriching cfDNA in vivo and increasing the extraction yield of plasma cfDNA; 3) Application in in vitro liquid biopsy; 4) Application in the preparation of reagents for in vitro liquid biopsy.

[0019] The present invention also provides a method for enriching cfDNA in vivo, comprising the step of administering any of the aforementioned antibodies to a test individual.

[0020] The present invention also provides a method for in vitro liquid biopsy, including the aforementioned method steps for enriching cfDNA in vivo, and further including the steps of extracting cfDNA from the subject individual and performing in vitro detection on the cfDNA.

[0021] Beneficial technical effects of the present invention: The antibody of this invention has advantages such as high specificity, high affinity, high protection, and low immune response. It can specifically bind to and protect cfDNA in the blood circulation, prolong its half-life, increase the amount of extractable cfDNA nucleic acid in plasma, and enhance the tumor signal detection capability from the source. It solves the technical problem of the difficulty in stable detection of low-abundance tumor mutations in liquid biopsy. Specifically, in various aspects: 1) The antibody of this invention can protect and enrich cfDNA in vivo, thereby increasing the abundance of the detectable substance from the in vivo source. The antibody of this invention can specifically bind to double-stranded DNA in circulating blood, block nuclease degradation and immune clearance pathways, effectively prolong the in vivo half-life of cfDNA, and increase the extractable amount of plasma cfDNA by 3 to 4 times within 2 hours after administration, breaking through the bottleneck of low abundance cfDNA detection.

[0022] 2) The antibody detection platform of this invention is highly versatile and widely applicable. The antibody-mediated enrichment strategy enhances cfDNA abundance at the sample level and is compatible with mainstream nucleic acid molecular detection platforms such as qPCR, ddPCR, and NGS. It significantly improves the analytical sensitivity (LoD), specificity, and experimental repeatability of the detection system without requiring large-scale modifications to existing detection procedures.

[0023] 3) This invention addresses a core clinical pain point and has clear translational value. This invention fundamentally solves the key technical problem of insufficient detection sensitivity due to low cfDNA abundance in liquid biopsy scenarios such as early tumor screening, minimal residual disease (MRD) monitoring, and efficacy evaluation, possessing significant clinical application value and industrialization prospects.

[0024] 4) The antibody of this invention is also engineered with Fc, which improves the protective effect and enhances its safety in vivo. This invention involves site-directed mutation of the antibody's Fc segment, reducing the clearance of the antibody-cfDNA complex by immune cells and significantly eliminating antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and Fc receptor-mediated effector function. This reduces the risk of non-specific immune activation in vivo and improves the antibody's protective effect against cfDNA and its safety for clinical use. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1. SPR sensing curves of the interaction between gradient concentrations of 42.G7 antibody and dsDNA molecules.

[0027] Figure 2. FRET fluorescence method for detecting the DNA enzymatic protection ability of each group of antibodies.

[0028] Figure 3. Statistical analysis of the fold increase in cfDNA extraction in each group after drug administration to tumor-bearing mice. Detailed Implementation

[0029] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The following terms or definitions are provided merely to aid in understanding the invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0031] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0032] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0033] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0034] The terms "approximately" and "generally" in this invention refer to a range of accuracy that, as would be understood by those skilled in the art, still guarantees the technical effects of the discussed features. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0035] Terms such as "or more," "at least," and "more than," for example, "at least one," should be understood to include, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more of the stated values. This also includes any larger numbers or fractions in between.

[0036] Conversely, the term "not exceeding" includes every value less than the stated value. For example, "not exceeding 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 5 4, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also includes any smaller numbers or fractions in between.

[0037] The terms "multiple," "at least two," "two or more," and "at least the second" should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70. 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also includes any larger numbers or fractions in between.

[0038] Furthermore, the terms first, second, third, (a), (b), (c), and similar terms used in the specification and claims are for distinguishing similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described in this invention can be implemented in a different order than that described or illustrated in this invention.

[0039] The isolated anti-dsDNA antibody or antigen-binding fragment of the present invention is a specific antibody with high affinity for double-stranded DNA; the sequence contains three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO.7 and three CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO.8.

[0040] In this invention, the term "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody, any antigen-binding fragment thereof, or a single chain thereof. Therefore, the term "antibody" includes proteins or peptides containing at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. This embodiment, including but not limited to, includes the complementarity-determining region (CDR), variable region, constant region, frame (FR) region, or any portion thereof of the heavy or light chain or its ligand-binding moiety, or at least a portion of the binding protein.

[0041] In this invention, the term "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, the fragment binds to the same antigen recognized by the intact antibody.

[0042] In this invention, the term "isolated," such as "isolated antibody or its binding fragment," refers to molecules isolated from other antibodies or fragments thereof present in natural sources; it can also refer to nucleic acids or peptides that are substantially free of cellular material, viral material, or cell culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated" is intended to include those that do not exist in their natural state and will not exist in their natural state. The term "isolated" is also used in this invention to refer to cells, polypeptides, or nucleic acids isolated from other cellular proteins or tissues. Isolated antibodies are intended to include purified and recombinant polypeptides.

[0043] The term "variable" indicates that certain portions of the variable region in an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their respective antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of both the natural heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly bound together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity.

[0044] The terms "complementarity-determining domain" or "complementarity-determining region" ("CDR") are interchangeable terms for the hypervariable regions of the VL and VH. A CDR is the target protein binding site of an antibody chain carrying the specific antibody for that target protein. Each VL or VH contains three CDRs (CDR1-3, numbered sequentially from the N-terminus), totaling approximately 15-20% of the variable domain. CDRs can be designated by their region and sequence. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. The CDR is structurally complementary to the target protein's epitope and is therefore directly responsible for binding specificity. The remaining extensions of the VL or VH (the so-called framework regions) exhibit less variation in their amino acid sequence.

[0045] In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR ring, Kabat based on antibody sequence variability, the international ImMunoGeneTics database (IMGT), and the NorthCDR definition based on nearest-neighbor propagation clustering utilizing a large number of crystal structures, etc.

[0046] However, it should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different. For example, the residue ranges of the CDR regions using Kabat, AbM, Chothia, IMGT, and Contact numbering under different assignment systems are shown in Table 1 below.

[0047] Table 1. CDR residue ranges defined by different assignment systems

[0048] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0049] The boundaries of the CDRs of the antibodies of the present invention can be determined artificially according to any scheme or combination thereof in the art. Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the above-described ways. That is, the CDR sequence in the present invention includes any three CDRs based on Table 1 from the amino acid sequence of the heavy chain variable region shown in SEQ ID NO.7 and any three CDRs based on Table 1 from the amino acid sequence of the light chain variable region shown in SEQ ID NO.8. For example, in some specific embodiments of the present invention, when the antibody CDRs are encoded according to the Kabat encoding rules, the CDR sequences are: i. the amino acid sequences of the complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are as shown in SEQ ID NO.1, 2, and 3, respectively; ii. the amino acid sequences of the complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region are as shown in SEQ ID NO.4, 5, and 6, respectively. Therefore, according to one embodiment of the present invention, the heavy chain and light chain variable domains of the antibody of the present invention have the following as shown in Table 2: Table 2. CDR sequence of antibody 42.G7 heavy chain

[0050] As shown in one particular embodiment, the antibody having the above-described CDR comprises the VH and VL sequences listed in Table 3 below.

[0051] Table 3. Light and heavy chain sequences of the 42.G7 antibody.

[0052] To eliminate or reduce the immune effector function of antibodies (such as ADCC, ADCP, and CDC), this invention mutates the Fc sequence of the antibody, for example, by introducing the L234A / L235A / P329G mutation (LALA-PG) into the Fc region of the heavy chain. Therefore, a more specific modified antibody full sequence disclosed in this invention is shown in Table 4 below.

[0053] Table 4. Full sequence of the modified 42.G7 antibody It is understood that the antibodies of the present invention may also contain one or more groups that are generally unrelated to antibodies. For example, antibodies may contain a resilient linker sequence, or may be modified to add a coupling moiety. In some embodiments, the coupling moiety may be selected from enzymes, luciferins, carrier proteins, and biotin, for example, for ease of detection or in vivo delivery, etc., and those skilled in the art can make appropriate selections based on the actual application scenario of the antibody.

[0054] Furthermore, the method for preparing the antibody of the present invention is well known in the art. Given that the antibody sequence of the present invention is known, different methods within the art can be selected to prepare the corresponding antibody, such as through recombinant expression, and the specific preparation method is not limited.

[0055] The present invention will be explained below through specific embodiments.

[0056] Example 1: Screening, identification, and modification of anti-dsDNA monoclonal antibodies This invention involves isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood of patients with high titers of IgG anti-dsDNA antibodies in systemic lupus erythematosus (SLE), then using the human-mouse hybrid myeloma cell line CB-F7 as the fusion parent, performing cell fusion with PEG1500, and using HAT selective medium for hybridoma screening and culture.

[0057] Initial screening was performed using dsDNA-ELISA. Positive clones were further verified by Crithidia luciliae immunofluorescence assay and Farr radioimmunoassay to confirm high affinity binding to dsDNA, and to confirm that the antibody specifically binds to dsDNA only. Simultaneously, histone ELISA confirmed that the antibody did not bind non-specifically to histones. After three rounds of subcloning using the limiting dilution method, the positive hybridoma cells were finally obtained as a monoclonal hybridoma cell line 42.G7, which stably secretes human anti-dsDNA monoclonal antibody.

[0058] Total RNA was extracted from 42.G7 hybridoma cells and reverse transcribed into cDNA using Oligo(dT) primers. Primers were designed based on the conserved region sequences of human immunoglobulins, and the heavy chain variable region (VH) and light chain variable region (VL) genes were amplified by PCR. The PCR products were cloned into a sequencing vector, and the consistent sequences were obtained by Sanger sequencing, and the corresponding amino acid sequences were deduced.

[0059] Sequence identification revealed that the 42.G7 antibody of this invention is a human IgG1 κ type antibody, and its light / heavy chain amino acid sequences are as follows: Heavy chain sequence (SEQ ID NO.7): QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYRFYWVRQPPGKGLEWIGIYPGSGRTYYARKFVKGRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARRRGYHWYVPGDSFDYWGQGTLVTVSS.

[0060] Light chain sequence (SEQ ID NO.8): EIVMTQSPDSLAVSLGERATINCRTSQGISDYLNWWYQQKPGQPPNLLIYGVSYQNTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHWYRSLTFFGQGTKVEIK.

[0061] According to the Kabat coding rules, the CDR sequence of the antibody is as follows: HCDR1 (SEQ ID NO.1): GYRFY; HCDR2 (SEQ ID NO. 2): IYPGSGRTYYARKFVKG; HCDR3 (SEQ ID NO.3): RRGYHWYVPGDSFDY; LCDR1(SEQ ID NO.4):RTSQGISDYLNW; LCDR2 (SEQ ID NO.5): GVSYQNT; LCDR3 (SEQ ID NO. 6): QHWYRSLTF.

[0062] Furthermore, to eliminate the binding of antibody Fc to the Fcγ receptor and complement C1q of immune cells, this invention further modified the antibody sequence by Fc mutation based on the measured VH and VL amino acid sequences, constructing a recombinant antibody expression vector containing the LALA-PG mutation. Specifically, the L234A / L235A / P329G mutation was introduced into the heavy chain Fc region. The expression vector was then transferred into host cells for recombinant expression, yielding a 42.G7 recombinant anti-dsDNA monoclonal antibody. The supernatant of the recombinant antibody expression was purified by Protein A affinity chromatography to obtain a 42.G7 antibody with a purity >95%. This modification effectively prevents the antibody-DNA complex from being cleared by the body's immune system.

[0063] Modified heavy chain sequence (SEQ ID NO.9): QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYRFYWVRQPPGKGLEWIGIYPGSGRTYYARKFVKGRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARRRGYHWYVPGDSFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0064] Modified light chain sequence (SEQ ID NO.10): EIVMTQSPDSLAVSLGERATINCRTSQGISDYLNWWYQQKPGQPPNLLIYGVSYQNTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHWYRSLTFFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0065] Example 2: Evaluation of SPR affinity between antibody and dsDNA molecules In this embodiment, surface plasmon resonance (SPR) technology was used to quantitatively detect the binding kinetic parameters and equilibrium affinity between the modified 42.G7 antibody and double-stranded DNA (dsDNA), verifying the antibody's ability to bind to dsDNA.

[0066] 1. Equipment and reagents 1) Detection instrument: Biacore 8K molecular interaction analyzer (Cytiva); 2) Sensor Chip: Series S Sensor Chip SA; 3) Immobilization ligand: double-stranded dsDNA, solvent: ultrapure water, working concentration: 0.5 μg / mL; 4) Running buffer: 20 mM Tris-HCl, 100 mM NaCl, 0.005% Tween 20, pH 7.5.

[0067] 2. Operating Procedures 1) Ligand immobilization: After chip pretreatment, dsDNA was introduced into channel 2 for coupling and immobilization, and the final immobilization response value was 126.1 RU; channel 1 was used as a blank reference channel.

[0068] 2) Gradient sample detection: The 42.G7 antibody was diluted to five concentration gradients: 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.63 nM. The samples were injected sequentially and allowed to bind for 120 s, followed by dissociation by passing through running buffer for 180 s. 3) Data processing: Background signal was subtracted using a blank flow channel + blank buffer as dual references. Biacore 8KEvaluation Software 5.0 was used to fit the sensing curve using a 1:1 combined model and calculate the kinetic and affinity parameters.

[0069] 3. Experimental Results The SPR sensing curve results are as follows: Figure 1 As shown in the figure, the five curves, from high to low, correspond to five 42.G7 antibody concentration gradients of 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.63 nM, respectively. The molecular interaction kinetics fitting parameters between the antibody and double-stranded DNA are shown in Table 5, with the chi-square value (Chi) representing the fitted parameters. 2 =0.171RU 2 The model fits the data excellently. Kinetic parameters: binding rate constant ka = 2.18 × 10⁻⁶. 6 M⁻¹・s⁻¹, dissociation rate constant kd = 7.13 × 10⁻ 4 s⁻¹, equilibrium dissociation constant KD = 3.28 × 10⁻ 10 M.

[0070] Table 5. Molecular interaction kinetic fitting parameters between G7 antibody and double-stranded DNA.

[0071] These results demonstrate that the 42.G7 antibody achieves sub-nanomolar ultra-high affinity; its high ka value and extremely low kd value enable it to rapidly capture dsDNA and form a stable complex that is not easily dissociated in the circulating environment. Furthermore, its steric hindrance prevents DNase from degrading cfDNA. Combined with the LALA-PG mutation in the Fc region to eliminate ADCC / CDC effects, it also reduces immune clearance and synergistically enhances the in vivo cfDNA enrichment effect.

[0072] Example 3: In vitro protection of cfDNA by antibodies 1. Detection of the protective effect of antibody against cfDNA using qPCR method To verify the protective ability of the 42.G7 antibody against cfDNA in vitro, four parallel systems were set up in this embodiment: blank control group, enzyme digestion control group, 11.D5 antibody protection group (another dsDNA-specific antibody in the initial screening) and 42.G7 antibody protection group. The composition of each system is shown in Table 6.

[0073] Table 6. Grouping of in vitro protective experiments against cfDNA by antibodies

[0074] 10 ng of peripheral blood cfDNA from healthy individuals was used as the substrate. According to the grouping, the corresponding buffer and antibody were added first, and the mixture was incubated at 37°C for 10 min. Then, DNase I was added to the enzyme digestion control, control antibody group, and antibody protection group samples, and incubated at 37°C for 60 min. After the enzyme digestion reaction, the cfDNA from each group was recovered and purified using the QIAamp Circulating Nucleic Acid Kit, and eluted with 30 μL of AVE buffer. The recovered products were quantitatively detected using TaqMan qPCR. The primer and probe sequences used are shown in Table 7.

[0075] Table 7. qPCR primer and probe sequences

[0076] The qPCR reaction was performed according to the standard procedure: pre-denaturation at 95℃ for 30 seconds; followed by 40 cycles of 95℃ for 10 seconds and 60℃ for 30 seconds. Each sample was tested in triplicate. The results are shown in Table 8, and the mean Ct value was used for result interpretation.

[0077] Table 8. Ct values ​​of samples detected by qPCR

[0078] The comparative results showed that the mean Ct value of the blank control group was approximately 30.34, indicating that the cfDNA remained intact; the Ct value of the enzyme digestion control group was >40, suggesting that the cfDNA had been completely degraded by DNase I; the mean Ct value of the 42.G7 antibody protection group was 32.55, indicating that the cfDNA was significantly preserved; the mean Ct value of the 11.D5 control antibody group was 34.67, showing that its protection against DNase I digestion was weaker than that of the 42.G7 antibody. These results indicate that the 42.G7 antibody can specifically bind to dsDNA and form a spatial protection, effectively blocking the enzymatic degradation of cfDNA by DNase I, and possesses excellent in vitro DNA protection capabilities.

[0079] 2. Verification of DNA protection ability using FRET fluorescence method This section uses FRET-labeled DNA substrates to further verify the protective effect of the 42.G7 antibody on DNA. The DNA substrate is labeled with a fluorophore and a quencher at both ends, respectively; in the intact state, the fluorescence is quenched. When the DNA is cleaved by DNase I, the fluorophore and quencher separate, releasing a detectable fluorescent signal. The antibody binds to DNA, inhibiting the enzymatic cleavage process, and the intensity of the fluorescent signal is negatively correlated with the protective effect. The experimental groups and reaction systems are shown in Table 9.

[0080] Table 9. FRET experimental reaction system

[0081] After thorough mixing of each system, it was immediately placed in a fluorescence microplate reader for detection. The detection conditions were: constant temperature of 37℃, excitation wavelength of 535 nm, emission wavelength of 556 nm, continuous monitoring for 60 minutes, and fluorescence readings every 5 minutes.

[0082] The results are as follows Figure 2 As shown, the negative control group (NC) only showed background fluorescence signal; the positive enzyme digestion control group (PC) fluorescence signal increased rapidly over time, reaching a peak of 5500-6000 at 30 minutes; the fluorescence signal of the 42.G7 antibody group was significantly inhibited, with a fluorescence intensity peak of 2600-2800 at 60 minutes, which was only about 50% of that of the positive control group; while the control 11.D5 antibody group had a fluorescence intensity peak of 3400-3500 at 60 minutes, and its protective effect was significantly weaker than that of the 42.G7 antibody.

[0083] The above results indicate that the 42.G7 antibody of the present invention can stably bind to DNA and form a steric hindrance effect, significantly inhibiting DNase I cleavage of DNA.

[0084] Example 4: Pharmacodynamic study of antibody enrichment of cfDNA in mice 1. cfDNA enrichment experiment in healthy mice To verify the protective and enrichment effect of the 42.G7 antibody on cfDNA in vivo, SPF-grade BALB / c mice were randomly divided into three groups of three mice each. The grouping and administration regimens are shown in Table 10.

[0085] Table 10. Experimental grouping and administration regimens of healthy mice

[0086] Blood was collected from the orbital venous plexus of mice before and 2 hours after drug administration, and whole blood was collected using EDTA-K2 anticoagulant tubes. All blood samples were processed within 1 hour: first, the samples were centrifuged at 2000×g, 4℃ for 20 minutes, and the supernatant plasma was collected; then, the samples were centrifuged at 12000×g, 4℃ for 10 minutes to completely remove cell debris, and the plasma was retained.

[0087] 50 μL of plasma was collected, and cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit. The DNA was then quantified by qPCR. The results are shown in Table 11.

[0088] Table 11. Results of cfDNA extraction from healthy mice

[0089] The results showed that there was no significant difference in the baseline plasma cfDNA levels between the two groups of mice before administration; 2 hours after administration, the cfDNA content in the PBS control group fluctuated only slightly, with changes of less than 1.5 times; the 11.D5 antibody increased by 1.48 to 2.75 times; while the plasma cfDNA content in the 42.G7 antibody group increased significantly, with increases of 3.19 to 5.89 times.

[0090] These results indicate that the 42.G7 antibody can bind to cfDNA more effectively in mice, thereby delaying its degradation and clearance in vivo, prolonging its half-life, and significantly increasing the extractable amount of cfDNA in plasma.

[0091] 2. cfDNA enrichment experiment in tumor-bearing mice To further verify the enrichment effect of the 42.G7 antibody on cfDNA in a mouse tumor model, this example also used CT26 mouse colon cancer cells to construct a BALB / c subcutaneous tumor-bearing mouse model. When the tumor volume grew to approximately 300-500 mm³, the mice were randomly divided into five groups of six mice each. The grouping and drug administration regimens are shown in Table 12.

[0092] Table 12. Experimental grouping and administration regimens of tumor-bearing mice

[0093] Blood was collected from the orbital venous plexus of mice before and 2 hours after drug administration, using EDTA-K2 anticoagulant tubes. All blood samples were processed within 1 hour: first, the samples were centrifuged at 2000×g, 4℃ for 20 minutes, and the supernatant plasma was collected; then, they were centrifuged at 12000×g, 4℃ for 10 minutes to thoroughly remove cell debris, retaining the plasma. 50 μL of plasma was used to extract cfDNA using the QIAamp Circulating Nucleic Acid Kit, and quantified by qPCR.

[0094] Quantitative results show ( Figure 3 In the PBS control group, cfDNA showed only physiological fluctuations and no significant enrichment effect. Two hours after administration of the 42.G7 antibody at a dose of 4 mpk, the average extraction amount of plasma cfDNA in tumor-bearing mice increased by 4.1 times; two hours after administration of the 8 mpk dose, the average extraction amount of plasma cfDNA in tumor-bearing mice increased by 5.2 times, both of which were significantly different from the PBS group (P < 0.001). In contrast, the control 11.D5 antibody at doses of 4 mpk and 8 mpk increased the average extraction amount of plasma cfDNA in tumor-bearing mice by 1.5 times and 2.2 times, respectively, with no significant difference from the PBS group (P > 0.05).

[0095] The above results confirm that the 42.G7 antibody can efficiently enrich cfDNA in vivo, and the enrichment effect increases with increasing dosage.

[0096] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An isolated anti-dsDNA antibody, characterized in that, The heavy chain and light chain sequences of the antibody are selected from SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. An isolated polynucleotide, characterized in that, The polynucleotide encodes the antibody of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is a cloning vector or an expression vector.

5. A product characterized in that, It contains the antibody as described in claim 1.

6. Any of the following applications of the antibody according to claim 1: 1) Application in the preparation of reagents for enriching cfDNA in vivo and increasing the extraction yield of plasma cfDNA; 2) Application in the preparation of reagents for in vitro liquid biopsy.

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