Antibody for detecting total antibody concentration in blood and kit and application thereof

Abstract

The invention relates to an antibody for detecting total antibody concentration in blood and a kit and application thereof, the antibody provided by the invention comprises: (1) a first antibody comprising a heavy chain variable region VH1 and a light chain variable region VL1, the VH1 comprising three CDRSs in a sequence as shown in SEQ ID NO: 41; the VL1 comprises three CDRSs in a sequence as shown in SEQ ID NO: 42; and / or (2) a second antibody comprising a heavy chain variable region VH2 and a light chain variable region VL2, the VH2 comprising three CDRSs in the sequence as shown in SEQ ID NO: 43; and the VL2 comprises three CDRSs in a sequence as shown in SEQ ID NO: 44. The antibody provided by the invention has the advantages of excellent sensitivity, accuracy and stability, strong target interference resistance and the like when being used for detecting the total antibody concentration in blood.

Description

Technical Field

[0001] This invention relates to the field of antibody medical detection. Specifically, it relates to an antibody for detecting the total antibody concentration in blood, a kit for the same, and its uses. Background Technology

[0002] Tissue factor (TF), also known as prothrombin kinase, CD142, or coagulation factor 3, is a transmembrane glycoprotein composed of an extracellular domain, a transmembrane domain, and an intracellular domain. TF is an essential molecule for initiating extrinsic coagulation events and is functionally expressed on the cell surface. Under normal physiological conditions, TF is located on the adventitia of the blood vessel wall and on the fibroblasts surrounding the blood vessel, but is scarce in the medial or intima layers. Only when the integrity of the blood vessel wall is compromised is TF exposed to the circulating blood, exerting its hemostatic effect by activating the coagulation cascade.

[0003] In contrast to its limited expression in normal tissues and cells, TF has been confirmed to be overexpressed in a variety of malignant tumors, including cervical cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer, and colorectal cancer. Therefore, TF can serve as a target for antibody drugs and ADC (antibody-drug conjugate) drugs. Several ADC drugs targeting TF are currently available (e.g., the ADC drugs disclosed in WO2023 / 160651, etc.).

[0004] With the widespread clinical application of TF-ADC drugs, related monitoring during treatment is also very important. Because antibody drugs can induce the production of anti-drug antibodies (ADA), ADA can bind to therapeutic antibodies, neutralize their biological activity or accelerate their clearance, leading to a decrease in efficacy; in severe cases, it may also cause allergic reactions such as immune complex deposition, posing safety risks.

[0005] Therefore, both domestic and international drug regulatory agencies require that biologics undergo immunogenicity assessment during clinical development and post-marketing studies, specifically by detecting ADA levels in the serum of subjects. Pharmacokinetic (PK) studies are beneficial for evaluating drug safety and efficacy; however, for ADC drugs, given their high heterogeneity, multiple analytes must be evaluated to determine their pharmacokinetic (PK) characteristics. Total antibody (including intact ADC, free naked antibody (without load), and antibodies in the ADC-antidrug antibody (ADA) complex) reflects the total exposure of the ADC drug in vivo, and its concentration is commonly used to characterize the PK properties of ADC drugs. Too low a total antibody concentration may lead to insufficient efficacy, while too high a concentration may increase the risk of immunogenicity. Currently, analytical methods for biological samples include immunoassays, which often employ enzyme-linked immunosorbent assay (ELISA) or electrochemiluminescence immunoassay (ECL). However, these methods often suffer from matrix interference, insufficient sensitivity, and poor ability to capture low-affinity antibodies.

[0006] The ELISA sandwich assay involves first binding a coating antibody to an ELISA plate, then adding the target antibody which specifically binds to the coating antibody, followed by the addition of a specific secondary antibody, and finally adding an enzyme-labeled detection antibody, which then catalyzes a colorimetric reaction using substrate catalysis. This method allows the target antibody to specifically bind to the coating antibody and the secondary antibody at different binding epitopes. Furthermore, the binding of horseradish peroxidase-labeled streptavidin to the biotin-labeled secondary antibody not only provides multi-stage amplification but also offers advantages such as high specificity, high sensitivity, and stability.

[0007] As clinical work on TF-ADC drugs progresses further, there is an urgent need to accurately and reliably detect the total antibody concentration of ADC drugs in human serum and precisely capture the dynamics of drug release in order to help make effective treatment decisions and monitor treatment progress. Summary of the Invention

[0008] To meet the above requirements, this invention provides an antibody for detecting the total antibody concentration in blood, a kit containing the antibody, and its uses. As shown in the examples, the antibody of this invention possesses high specificity, high sensitivity, and high stability. Using the antibody and ELISA detection method of this invention, the total antibody concentration of TF-ADC drugs in blood can be effectively detected, thereby enabling the detection of the total antibody concentration of ADC drugs in blood after drug administration, accurately capturing drug release dynamics, and helping to monitor treatment efficacy and progress.

[0009] Solution for solving the problem: In a first aspect, the present invention provides an antibody for detecting the total antibody concentration in blood, the antibody comprising a first antibody (also referred to herein as a coating antibody) and / or a second antibody (also referred to herein as a detection antibody), wherein the first antibody comprises a heavy chain variable region VH1 and a light chain variable region VL1, wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 41; and the VL1 comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 42. The second antibody comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein VH2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 43; and VL2 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 44.

[0010] In a second aspect, the present invention provides a polynucleotide sequence encoding an antibody according to the first aspect of the present invention, a vector comprising the polynucleotide sequence, and a cell comprising the polynucleotide sequence or the vector.

[0011] In a third aspect, the present invention provides a kit comprising the antibody described in the first aspect of the present invention.

[0012] In a fourth aspect, the present invention provides the use of the antibody described in the first aspect of the present invention in the preparation of a product for detecting the total antibody concentration in blood.

[0013] In this invention, the targeted tissue factor drug conjugates include TF-ADC drugs and TF-ADC-antidrug antibodies (ADAs), such as the antiTF antibodies and camptothecin drug conjugates disclosed in WO2023 / 160651 (which is hereby incorporated herein by reference in its entirety), particularly "antibody-drug conjugate A" as defined herein.

[0014] The effects of the invention: This invention provides an antibody based on enzyme-linked immunosorbent assay (ELISA) capable of detecting the total antibody concentration in blood. It has advantages such as a wide effective quantitative range (100 ng / mL to 3200,000 ng / mL), high sensitivity, good specificity, good repeatability, strong anti-interference ability, and high stability. The antibody provided by this invention can be used to detect the total antibody content of tissue factor in blood and can be used for clinical evaluation of the pharmacokinetic characteristics of anti-human tissue factor antibodies or anti-human tissue factor antibody drug conjugates, thereby assessing their safety and efficacy. Attached Figure Description

[0015] Refer to the instruction manual. Figure 1 Reading this description will provide a better understanding of the preferred embodiments of the invention as detailed below. For illustrative purposes, the figures show presently preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the figures.

[0016] Figure 1 This is a graph of the four-parameter fitting curve for the standard curve. Detailed Implementation

[0017] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0018] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0019] As used herein, the term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value. The term is also intended to cover values ​​within the range of ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0.1% of the specified numeric value.

[0020] As used herein, the term “and / or” when used in conjunction with multiple options should be understood to mean any one of the options or any combination of two or more of the options.

[0021] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0022] As used herein, the terms “tissue factor” or “TF” are used interchangeably and, unless otherwise stated, include any variant of human tissue factor, including sequence variants, especially naturally occurring variants, allele variants, and post-translational modification variants and conformational variants, and encompassing its species homologs. Furthermore, it should be understood that the term covers TF expressed in natural or recombinant cells. An example of tissue factor is the human TF protein containing the amino acid sequence UniProtKB-P13726. In this document, unless otherwise specified, the terms “tissue factor” or “TF” refer to tissue factor derived from humans.

[0023] As used herein, the term "antibody" refers to a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. This term encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), murine antibodies, rabbit antibodies, chimeric or humanized antibodies, full-length antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0024] As used herein, the term "whole antibody" (which may be used interchangeably with "full-length antibody," "complete antibody," and "intact antibody") comprises at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated VH herein) and a heavy chain constant region. The heavy chain constant region consists of domains CH1, CH2, and CH3, and optionally CH4. Each light chain consists of a light chain variable region (abbreviated VL herein) and a light chain constant region. The light chain constant region consists of a domain CL. The variable region is a domain in the heavy or light chain of the antibody that participates in the binding of the antibody to its antigen. The constant region does not directly participate in the binding of the antibody to the antigen but exhibits various effector functions. The light chain of an antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain. Antibody heavy chains can be classified into five main types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. Several of these types can be further subdivided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different antibody types are respectively named α, δ, ε, γ, and µ.

[0025] As used herein, the term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that participates in the binding of the antibody to its antigen. The heavy chain variable region (VH) and light chain variable region (VL) consist of a hypervariable region (HVR, also known as a complementarity-determining region (CDR)) and interspersed, more conserved regions (i.e., framework regions (FR)). Specifically, each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. The terms "light chain variable region" and "VL" are used interchangeably.

[0026] As used herein, the terms "complementarity-determining region" or "CDR region" or "CDR(s)" or "hypervariant region" refer to regions within the variable domain of an antibody that are highly variable in sequence and form structurally defined loops ("hypervariant loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. CDRs of the heavy and light chains are sequentially numbered starting from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. CDRs located within the variable domain of the antibody heavy chain are also referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. Within a given amino acid sequence of a light chain variable region or heavy chain variable region, the CDR sequence can be determined using various schemes known in the art. Such schemes include, but are not limited to, the Kabat, AbM, Chothia, Contact, and IMGT definition schemes.

[0027] Table 1 shows exemplary CDR area ranges under some different CDR definition schemes using the Kabat and Chothia numbering systems.

[0028] Table 1

[0029] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing methods or any combination thereof.

[0030] CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence. Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position according to the Kabat numbering system (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (MD), 1991, NIH Publication No. 91-3242).

[0031] As used herein, the term "sequence identity" refers to the degree of sequence similarity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. The "sequence identity percentage" can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions in both sequences containing the same amino acid residue to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence identity percentage. Optimal alignment for determining the sequence identity percentage can be performed in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region.

[0032] In this invention, the percentage of amino acid sequence identity, with respect to the antibody sequence, is determined by optimally aligning the candidate antibody sequence with the given antibody sequence, preferably according to the Kabat numbering rules. In this document, without specifying a comparison window (i.e., the target antibody region to be compared), alignment along the entire length of the given antibody sequence will be applicable. In some embodiments, sequence identity may be distributed across the entire heavy chain variable region and / or the entire light chain variable region, or the percentage of sequence identity may be limited only to the framework region, while the sequence corresponding to the CDR region remains 100% identical.

[0033] As used herein, the term "vector" refers to an artificial construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, and DNA or RNA expression vectors encapsulated in liposomes.

[0034] As used in this article, the term "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include "transformed cells," which include primary transformed cells and their progeny, regardless of passage number.

[0035] As used in this article, the term "detection antibody" refers to an antibody that has been directly labeled as detectable. If a primary antibody is directly labeled as a detectable marker, then that primary antibody is a detection antibody. If the primary antibody is indirectly labeled as a detectable marker through a secondary antibody, then that secondary antibody is a detection antibody.

[0036] Various aspects of the invention will be further described in the following subsections.

[0037] I. The antibody of the present invention After extensive screening, the inventors proposed the antibody of this invention for detecting the concentration of total antibodies (TF total antibodies) in blood, and verified its detection performance. Clinical pathological applications also demonstrate that the antibody of this invention possesses high specificity, high accuracy, and high sensitivity, meeting the desired detection / diagnostic objectives.

[0038] In a first aspect, the present invention provides an antibody for detecting the concentration of total antibodies (TF total antibodies) in blood, comprising a first antibody and / or a second antibody. In some embodiments, the first antibody comprises a heavy chain variable region VH1 and a light chain variable region VL1. In some embodiments, the second antibody comprises a heavy chain variable region VH2 and a light chain variable region VL2.

[0039] I-1 antibody CDR region In some embodiments, VH1 includes HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 41; and VL1 includes LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 42.

[0040] In some embodiments, the second antibody includes a heavy chain variable region VH2 and a light chain variable region VL2, wherein the VH2 includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 43; and the VL2 includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 44.

[0041] In some preferred embodiments, the CDR sequences of the first and / or second antibodies of the present invention are defined according to the Kabat, AbM, Chothia, Contact, or IMGT numbering systems or any combination thereof.

[0042] In some implementations, the present invention defines the CDRs according to the IMGT, Kabat, and Chothia numbering systems.

[0043] In some implementations, the complementarity-determining region of the antibody is selected from any of the following: (a) Based on the definition of the IMGT antibody encoding system, The first antibody has HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; LCDR1 as shown in SEQ ID NO:4, LCDR2 with an LTS amino acid sequence, and LCDR3 as shown in SEQ ID NO:6; and / or, The second antibody has HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11; LCDR1 as shown in SEQ ID NO:12, LCDR2 with the amino acid sequence NAK, and LCDR3 as shown in SEQ ID NO:14; (b) Based on the definition of the Chothia antibody encoding system, The first antibody has HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:18, HCDR3 as shown in SEQ ID NO:19; LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, LCDR3 as shown in SEQ ID NO:22; and / or, The second antibody has HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:24, HCDR3 as shown in SEQ ID NO:25; LCDR1 as shown in SEQ ID NO:26, LCDR2 as shown in SEQ ID NO:27, and LCDR3 as shown in SEQ ID NO:28. (c) Based on the Kabat antibody encoding system definition, The first antibody has HCDR1 as shown in SEQ ID NO:29, HCDR2 as shown in SEQ ID NO:30, HCDR3 as shown in SEQ ID NO:31; LCDR1 as shown in SEQ ID NO:32, LCDR2 as shown in SEQ ID NO:33, LCDR3 as shown in SEQ ID NO:34; and / or, The second antibody has HCDR1 as shown in SEQ ID NO:35, HCDR2 as shown in SEQ ID NO:36, HCDR3 as shown in SEQ ID NO:37; LCDR1 as shown in SEQ ID NO:38, LCDR2 as shown in SEQ ID NO:39, and LCDR3 as shown in SEQ ID NO:40.

[0044] In some implementations, based on the IMGT, Kabat, and Chothia numbering system, the complementarity-determining regions of the first antibody (coating antibody) are shown in Table 2, and the complementarity-determining regions of the second antibody (detection antibody) are shown in Table 3.

[0045] Table 2

[0046] Table 3

[0047] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing methods or any combination thereof.

[0048] I-2 antibody variable region In some embodiments, the present invention also provides an antibody comprising a heavy chain variable region and a light chain variable region for detecting total antibody concentration in blood, wherein the first antibody comprises a heavy chain variable region VH1 and a light chain variable region VL1. In some embodiments, the second antibody comprises a heavy chain variable region VH2 and a light chain variable region VL2.

[0049] In some embodiments, the VH1 comprises an amino acid sequence that has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99%) identity with the amino acid sequence shown in SEQ ID NO: 41.

[0050] In some embodiments, the VH1 comprises the amino acid sequence shown in SEQ ID NO: 41. In some specific embodiments, the amino acid sequence of the VH1 is as shown in SEQ ID NO: 41.

[0051] SEQ ID NO: 41 (First / coated antibody heavy chain variable region): DVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWHWIRQFPGTKLEWMGYISYSDSTAYNPSLKNRISITHDTSKNQFFLKLNSVTTEDTATYYCVGRQRGLDYALDYWGQGTSVTVSS In some embodiments, the VL1 comprises an amino acid sequence that has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99%) identity with the amino acid sequence shown in SEQ ID NO: 42.

[0052] In some embodiments, the VL1 comprises the amino acid sequence shown in SEQ ID NO: 42, and in some specific embodiments, the amino acid sequence of the VL1 is as shown in SEQ ID NO: 42.

[0053] SEQ ID NO: 42 (First / Coated antibody light chain variable region): QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAADAATYYCQQWSSNPPTFGAGTKLELK In some embodiments, the first antibody includes a heavy chain variable region VH1 and a light chain variable region VL1, wherein the amino acid sequence of VH1 is shown in SEQ ID NO: 41 and the amino acid sequence of VL1 is shown in SEQ ID NO: 42.

[0054] In some embodiments, the VH2 comprises an amino acid sequence that has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99%) identity with the amino acid sequence shown in SEQ ID NO: 43.

[0055] In some embodiments, the VH2 comprises the amino acid sequence shown in SEQ ID NO: 43, and in some specific embodiments, the amino acid sequence of the VH2 is as shown in SEQ ID NO: 43.

[0056] SEQ ID NO: 43 (Second / Detection antibody heavy chain variable region): QVQLQQSGAELMKPGASVKLSCKATGYIITGYWIEWVKQRPGHGLEWIGEILPGSGSIKYNEKFKGKATFIVDTSSNTAYMQLSSLTTDDSAIYYCARGYGNYFPYWGQGTLVTVSA In some embodiments, the VL2 comprises an amino acid sequence that has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99%) identity with the amino acid sequence shown in SEQ ID NO: 44.

[0057] In some embodiments, the VL2 comprises the amino acid sequence shown in SEQ ID NO: 44, and in some specific embodiments, the VL2 amino acid sequence is as shown in SEQ ID NO: 44.

[0058] SEQ ID NO: 44 (Second / Detection antibody light chain variable region): DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLQVYNAKTLAGGVPSRFSGGGSGTQFSLEINSLQPEDFGTYYCQHHYGTPYTFGSGTKLEIK In some embodiments, the second antibody includes a heavy chain variable region VH2 and a light chain variable region VL2, the amino acid sequence of which is shown in SEQ ID NO: 43 and the amino acid sequence of which is shown in SEQ ID NO: 44.

[0059] In some embodiments, the antibody includes a first antibody and a second antibody, wherein the amino acid sequence of VH1 of the first antibody is shown in SEQ ID NO: 41, the amino acid sequence of VL1 is shown in SEQ ID NO: 42, the amino acid sequence of VH2 of the second antibody is shown in SEQ ID NO: 43, and the amino acid sequence of VL2 is shown in SEQ ID NO: 44.

[0060] I-3 antibody heavy and light chains In some embodiments, the antibodies of the present invention may comprise heavy chain constant regions and / or light chain constant regions. The heavy chain constant regions contained in the antibodies of the present invention can be any isotype or subtype, such as the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4 isotypes. The light chain constant regions contained in the antibodies of the present invention can be κ light chain constant regions or λ light chain constant regions.

[0061] In some embodiments, the antibody of the present invention is a full-length antibody composed of two heavy chains and two light chains.

[0062] In some embodiments, the first antibody comprises a heavy chain H1 and a light chain L1. In some embodiments, H1 comprises an amino acid sequence having at least 80% (e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99%) identity with the amino acid sequence shown in SEQ ID NO:7.

[0063] In some embodiments, H1 comprises the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the amino acid sequence of H1 is as shown in SEQ ID NO:7.

[0064] SEQ ID NO:7 (First / Coated Antibody Heavy Chain Full-Length Sequence): DVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWHWIRQFPGTKLEWMGYISYSDSTAYNPSLKNRISITHDTSKNQFFLKLNSVTTEDTATYYCVGRQRGLDYALDYWGQGT SVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINP CPPKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIE RTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK In some embodiments, L1 comprises an amino acid sequence that has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more) identity with the amino acid sequence shown in SEQ ID NO:8.

[0065] In some embodiments, L1 comprises the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the amino acid sequence of L1 is as shown in SEQ ID NO:8.

[0066] SEQ ID NO:8 (First / Coated antibody light chain full-length sequence): QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAADAATYYCQQWSSNPPTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC In some embodiments, the second antibody comprises a heavy chain H2 and a light chain L2. In some embodiments, H2 comprises an amino acid sequence having at least 80% (e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99%) identity with the amino acid sequence shown in SEQ ID NO:15.

[0067] In some embodiments, H2 comprises the amino acid sequence shown in SEQ ID NO:15. In some embodiments, the amino acid sequence of H2 is shown in SEQ ID NO:15.

[0068] SEQ ID NO:15 (Second / Detection antibody heavy chain full-length sequence): QVQLQQSGAELMKPGASVKLSCKATGYIITGYWIEWVKQRPGHGLEWIGEILPGSGSIKYNEKFKGKATFIVDTSSNTAYMQLSSLTTDDSAIYYCARGYGNYFPYWGQGTLV TVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPC PPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIE RTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK In some embodiments, L2 comprises an amino acid sequence that has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more) identity with the amino acid sequence shown in SEQ ID NO:16.

[0069] In some embodiments, L2 comprises the amino acid sequence shown in SEQ ID NO:16.

[0070] SEQ ID NO:16 (Full-length light chain sequence of the second / detection antibody): DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLQVYNAKTLAGGVPSRFSGGGSGTQFSLEINSLQPEDFGTYYCQHHYGTPYTFGSGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC II. Preparation of Antibodies The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0071] III. Polynucleotides, vectors, and host cells The present invention provides a polynucleotide sequence encoding the antibody described above, a vector containing the polynucleotide sequence, a cell containing the polynucleotide sequence or the vector, and an antibody conjugate containing the antibody described above.

[0072] In some embodiments, the present invention provides a nucleic acid molecule comprising a polynucleotide encoding at least one CDR region and typically all three CDR regions of a heavy chain VH or light chain VL sequence of the first or second antibody described above. In some further embodiments, the present invention provides a nucleic acid molecule comprising a polynucleotide encoding a complete or substantially complete variable region sequence of the heavy chain and / or light chain of the first or second antibody described above. As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0073] In some embodiments, the present invention provides one or more vectors comprising the nucleic acids of the present invention, including cloning vectors and expression vectors. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors that can be used in the present invention include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC).

[0074] In some embodiments, the present invention provides a host cell comprising the vector of the present invention. Suitable host cells for cloning or expressing the vector encoding an antibody include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression in bacteria such as *Escherichia coli*, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified. In yet another embodiment, the host cell is a eukaryotic cell. In yet another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments. Examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (293HEK or 293 cells); Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0.

[0075] In some embodiments, the present invention provides antibody conjugates comprising the antibodies described above, the antibody conjugates further comprising a coupling portion coupled to the antibody of the present invention, the coupling portion comprising a detectable marker or enzyme. The detectable markers described in the present invention can be any substance detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g.,...). 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers (e.g., colloidal gold or colored glass or plastics (e.g., polystyrene, polypropylene, latex, etc.)), and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioisotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In a preferred embodiment, the coupling portion is selected from fluorescent or luminescent markers, enzymes capable of producing detectable products, gold nanoparticles / nanorads, etc. In other embodiments, the coupling portion is selected from biotin. In some implementations, the antibody conjugate is a biotin-labeled secondary antibody.

[0076] IV. Reagent kits, detection methods and applications IV-1 Reagent Kit In some embodiments, the present invention provides a kit for detecting the total antibody concentration in blood, the kit comprising the antibody or antibody-drug conjugate described in any of the preceding embodiments.

[0077] In some preferred embodiments, the kit is an ELISA kit.

[0078] In some embodiments, the first antibody is a coated antibody, and the second antibody is a detection antibody conjugated to a detectable marker. In a specific embodiment, the second antibody is conjugated to biotin (Bio).

[0079] In some embodiments, the kit may also contain one or more other reagents, including but not limited to HRP-labeled streptavidin, coating buffer, washing buffer, analysis buffer, blocking buffer, chromogenic buffer, stop solution, reconstitution solution of antibody-drug conjugate, or standards.

[0080] In some embodiments, the kit further includes an instruction manual specifying that the kit is used to detect the presence of total tissue factor antibodies in a sample (qualitative detection) and / or to determine the concentration of total tissue factor antibodies in a sample (quantitative detection).

[0081] IV-2 detection method In some embodiments, the present invention provides a method for detecting the total antibody concentration in blood, the method being an ELISA method, the method comprising: (1) Coating and incubation: Add the coating working solution containing the coating antibody to the analytical plate, seal the plate, and incubate. (2) Wash the plate, take out the analytical plate, shake the plate dry, wash the plate with washing solution, and pat it dry on absorbent paper; (3) Blocking incubation: Block the analytical plate with blocking solution, seal the plate, and incubate at room temperature; (4) Wash the plate, take out the analytical plate, shake the plate dry, wash the plate with washing solution, and pat it dry on absorbent paper; (5) Sample incubation: Dilute the standard curve sample, quality control sample and test sample with analytical buffer, add them to the analytical plate, seal the plate and incubate; (6) Wash the plate, take out the analytical plate, shake the plate dry, wash the plate with washing solution, and pat dry on absorbent paper; (7) Detection and incubation: Add detection antibody solution, seal plate, and incubate; (8) Wash the plate, remove the analytical plate, shake the plate dry, wash the plate with washing solution, and pat dry on absorbent paper; (9) Add SA-HRP working solution, seal the plate, and incubate; (10) Wash the plate, remove the analytical plate, shake the plate dry, wash the plate with washing solution, and pat dry on absorbent paper; (11) Develop color: Add TMB colorimetric solution to the analytical plate, incubate, and terminate the pre-reading; (12) Terminate by adding the ELISA stop solution to the analysis plate; (13) Reading the board; (14) Data saving and printing.

[0082] In some implementations, the detection antibody is a bio-detection antibody.

[0083] In some preferred embodiments, the working concentration of the first antibody is 1~5µg / mL (e.g., 1µg / mL, 2µg / mL, 3µg / mL, 4µg / mL, 5µg / mL, etc.); in a preferred embodiment, the working concentration of the first antibody is 1~3µg / mL; in a preferred embodiment, the working concentration of the first antibody is 2µg / mL.

[0084] In some preferred embodiments, the incubation in step (1) is carried out at 0~10°C for 16~90 hours; in a preferred embodiment, it is carried out at 2~8°C for 16~18 hours.

[0085] In some preferred embodiments, the incubation time in step (3) is 100-150 min; in a preferred embodiment, the incubation time is 110-130 min.

[0086] In some preferred embodiments, the incubation time in step (5) is 100-150 min; in a preferred embodiment, the incubation time is 110-130 min.

[0087] In some preferred embodiments, the incubation time in step (7) is 40 to 100 minutes; in a preferred embodiment, the incubation time is 50 to 70 minutes.

[0088] In some preferred embodiments, the incubation time in step (9) is 20-60 min; in a preferred embodiment, the incubation time is 30-40 min.

[0089] In some preferred embodiments, the working concentration of the second antibody conjugated with the detectable marker is 0.1~3µg / mL (e.g., 0.1µg / mL, 0.5µg / mL, 1µg / mL, 1.5µg / mL, 2µg / mL, 2.5µg / mL, 3µg / mL, etc.); in some preferred embodiments, the working concentration of the second antibody conjugated with the detectable marker is 0.5~2µg / mL; in some preferred embodiments, the working concentration of the second antibody conjugated with the detectable marker is 1µg / mL.

[0090] In some implementations, the number of plate washing cycles in the detection method of the present invention is 3 to 5.

[0091] This invention provides the use of the antibodies or antibody conjugates described in any of the preceding claims in the preparation of products for detecting total antibody concentrations in blood. In some embodiments, the product is a detection reagent, detection plate, or kit. In some embodiments, the total antibody is TF total antibody. In this invention, TF total antibody includes antibodies in intact TF-ADC, free naked antibody (without load), and TF-ADC-antidrug antibody (ADA) complexes.

[0092] In this invention, TF-ADC drug, namely antibody-drug conjugate A, refers to an ADC having the structure of formula (I), for details please refer to WO2023 / 160651, which is hereby incorporated herein by reference in its entirety.

[0093] TF-ADC drugs have the following structure (I): Formula (I), Ab represents anti-TF antibody. Where q is the antibody-drug ratio (i.e., the ratio of the drug portion coupled to the Ab portion to the Ab portion). In some implementations, q is an integer from 1 to 8, more preferably, q is 2, 3, 4, 5, 6, 7 or 8, and in some preferred implementations, the ADC has an average DAR value of approximately 8; In some embodiments, the Ab comprises three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:45 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO:46; or more preferably comprises the heavy chain variable region of SEQ ID NO:45 and the light chain variable region of SEQ ID NO:46, and even more preferably comprises the heavy chain of SEQ ID NO:5 and the light chain of SEQ ID NO:13.

[0094] SEQ ID NO:45 (Antibody-Drug Conjugate A Heavy Chain Variable Region): QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGLEWMGMIDPSDSYTSYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCTRGSGPGLFAYWGQGTLVTVSS SEQ ID NO:46 (Variable region of the light chain of Antibody-Drug Conjugate A): DIQETQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSVSGTDFTLTISSLQPEDFATYFCQQGNTLPYTFGQGTKVEIK SEQ ID NO:5 (Heavy chain of Antibody-Drug Conjugate A): QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGLEWMGMIDPSDSYTSYAQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCTRGSGPGLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:13 (Light chain of Antibody-Drug Conjugate A): DIQETQSPSSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSVSGTDFTLTISSLQPEDFATYFCQQGNTLPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In this invention, antibody-drug conjugate A refers to an ADC having the structure of the above formula (I), wherein the Ab is an anti-TF antibody composed of two heavy chains of SEQ ID NO:5 and two light chains of SEQ ID NO:13, and the ADC has an average DAR value of approximately 8.

[0095] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Experiments in the embodiments or test examples that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material / commodity manufacturer; reagents not specifying a specific source are commercially available conventional reagents.

[0096] Example 1: Preparation of Coated Antibody and Detection Antibody A coating antibody (first antibody) and a detection antibody (second antibody) for detecting total antibody concentration in blood were obtained by immunizing mice and screening hybridomas. Sequencing confirmed that the heavy chain sequence of the first antibody is shown in SEQ ID NO:7, and the light chain sequence is shown in SEQ ID NO:8. The heavy chain sequence of the second antibody is shown in SEQ ID NO:15, and the light chain sequence is shown in SEQ ID NO:16.

[0097] The amino acid sequence was converted into a DNA sequence using eukaryotic degenerate codons. The light and heavy chain DNA were then integrated via molecular cloning to construct an antibody expression plasmid. This plasmid was transiently transfected into 293E suspension cells or Chinese hamster ovary cells (CHO cell line) using liposomes. After transfection, the 293E cells were amplified in shake flasks and cultured in fed-batch culture. The antibody was then obtained from the fermentation broth supernatant. The antibody was purified using protein A affinity chromatography, anion exchange chromatography, and cation exchange chromatography to obtain a first antibody (coating antibody) and a second antibody (detection antibody) for the following experiments.

[0098] Example 2: Method for quantitative detection of total antibody concentration of antibody-drug conjugate A in human serum based on ELISA platform The reagents and materials used are as follows: Coating buffer: BupH™ Carbonate-Bicarbonate Buffer Pack is prepared by mixing one packet of buffer salt with pure water in a specific ratio, with the ratio being 500 mL of pure water per packet. Washing solution: Contains 0.05% (v / v) Tween 20 and 1×PBS; Analysis buffer: containing 1% (w / v) BSA, 0.05% (v / v) Tween 20, and 1×PBS; Blocking solution: 3% (w / v) BSA, 1×PBS; Antibody working solution: Prepare a 2µg / mL primary antibody solution using coating buffer; Antibody solution for detection: Prepare a 1 µg / mL second antibody solution using analytical buffer, wherein the second antibody is conjugated with biotin (Bio-second antibody solution). SA-HRP working solution: sourced from R&D Company, catalog number DY998, diluted 1:200 with analytical buffer; or diluted 1:1600 with analytical buffer using SA-HRP working solution.

[0099] TMB colorimetric solution: prepared by mixing 6 mL of KPL TMB peroxidase substrate and 6 mL of KPL peroxidase substrate solution B.

[0100] Reconstitution of Antibody-Drug Conjugate A for Injection: At room temperature, using a sterile syringe, slowly inject 6 mL of sterile water into each vial of Antibody-Drug Conjugate A, minimizing foam formation. Gently shake the vial until completely dissolved. Do not shake vigorously until the foam subsides. The concentration of Antibody-Drug Conjugate A after reconstitution is 20 mg / mL. (It is recommended to aliquot the reconstituted solution and store it at -20°C, avoiding repeated freeze-thaw cycles (no more than 5 times recommended). Reconstitution and aliquoting are recommended to be performed under Class A conditions (clean bench or biosafety cabinet).) Matrix: A mixture of human serum prepared from at least 10 untreated individuals from different batches is used as the matrix.

[0101] Preparation of standard curve, blank control and quality control samples: Standard curves and quality control samples were prepared using antibody-drug conjugate A (antibody-drug conjugate A for injection, 20 mg / mL). The standard curve included seven concentration levels, with one anchor point.

[0102] The quality control samples are prepared as follows (Table 4), wherein the diluent is a mixture of human serum. Those skilled in the art can increase, decrease or adjust the volume proportionally as needed.

[0103] Table 4

[0104] Sample preparation: On the day of the experiment, the samples to be tested should be thawed and mixed at room temperature. All samples need to be diluted 5-fold (MRD) with analytical buffer before analysis. For samples requiring additional dilution, dilute with mixed human serum first, then dilute 5-fold (MRD) with analytical buffer.

[0105] Termination solution: Source: Solarbio Life Sciences, catalog number C1058; Antibody-drug conjugate A: 120 mg / vial, supplied by Dongyao Pharmaceutical. In this invention, it is prepared into a 20 mg / mL solution using sterile distilled water.

[0106] In the method of the invention, a first antibody is used as a coating protein to bind to a 96-well analytical plate to capture the total antibody-drug conjugate A in the sample. Then, a second antibody is added to detect the total antibody-drug conjugate A bound to the plate. Finally, SA-HRP is added to bind to the first antibody, and TMB solution is added for color development. The absorbance measured after color development is directly proportional to the amount of total antibody-drug conjugate A in the sample.

[0107] The steps for detecting the total antibody concentration in a sample are as follows: 1. Coating incubation: Add coating working solution to a 96-well analytical plate, 100 µL / well, seal the plate, and incubate at 2~8℃ for 16~18 hours.

[0108] 2. Wash the plate; remove the 96-well analytical plate, shake the plate dry, wash the plate 3 times with 300 µL / well washing solution, and pat dry on absorbent paper.

[0109] 3. Blocking and incubation: Block the 96-well analytical plate with 300 µL / well blocking solution, seal the plate, and incubate at room temperature and 500 rpm for 110~130 min.

[0110] 4. Wash the plate; take out the 96-well analytical plate, shake the plate dry, wash the plate 3 times with 300 µL / well washing solution, and pat dry on absorbent paper.

[0111] 5. Sample incubation: Dilute the standard curve sample, quality control sample, and test sample 5 times with analytical buffer, add 100 µL / well to a 96-well analytical plate, seal the plate, and incubate at room temperature and 500 rpm for 110~130 min.

[0112] 6. Wash the plate; remove the 96-well analytical plate, shake the plate dry, wash the plate 3 times with 300µL / well washing solution, and pat dry on absorbent paper.

[0113] 7. Detection and incubation: Add detection antibody solution, 100µL / well, seal the plate, and incubate at room temperature and 500 rpm for 50~70 min.

[0114] 8. Wash the plate; remove the 96-well analytical plate, shake the plate dry, wash the plate 3 times with 300µL / well washing solution, and pat dry on absorbent paper.

[0115] 9. SA-HRP incubation: Add SA-HRP working solution, 100 µL / well, seal the plate, and incubate at room temperature and 500 rpm for 30~40 min.

[0116] 10. Wash the plate; remove the 96-well analytical plate, shake the plate dry, wash the plate 3 times with 300µL / well washing solution, and pat dry on absorbent paper.

[0117] 11. Color development: Add 100 µL / well TMB color development solution to the 96-well analytical plate and incubate at room temperature in the dark for about 3 minutes. It is recommended to pre-read at 370 nm wavelength until the OD value of STD01 is greater than 1.5 and then terminate the process.

[0118] 12. Termination: Add 100 µL / well of ELISA stop solution to the 96-well analysis plate.

[0119] 13. Reading the board: Use Softmax Pro GxP7.0.3 to read the board at a wavelength of 450 nm (correction wavelength is 630 nm).

[0120] 14. Data Processing: SoftMax Pro GxP v7.0.3 was used for data processing. A standard curve was established by using 4-parameter logical fitting (4-PL, Marquardt) and weighting 1 / Y². The sample concentration was calculated using Watson LIMS 7.5 SP1.

[0121] The standard curve needs to meet the following criteria: 1) The density of the duplicate wells %CV ≤ 20.0% (the acceptance criterion for the standard curve points corresponding to ULOQ and LLOQ is ≤ 25.0%). 2) The bias% of the average concentration of the duplicate wells is within ±20.0% (the acceptance standard for the corresponding standard points of ULOQ and LLOQ is within ±25.0%). 3) At least 75% (≤6) of the non-zero, non-anchor point standard curve points must meet the above criteria to produce an acceptable standard curve. If fewer than 75% (<6) of the standard curve points meet the acceptance criteria, the analysis batch fails.

[0122] 4) Standard curve points (including anchor points without abnormalities) should not be removed simply so that the quality control samples can meet their acceptance criteria.

[0123] 5) Anchor points should not be arbitrarily removed unless doing so would help curve fitting and allow the standard points to pass the acceptance criteria.

[0124] Quality control samples: For analytical batches other than accuracy and precision, the analytical batch shall include two sets of quality control samples for each of the three concentration levels (HQC, MQC and LQC) of the standard preparation. The acceptance criteria for quality control samples are: %CV ≤ 20.0%; %Bias within ±20.0%.

[0125] At least two-thirds of the total number of quality control samples, and at least half of the concentration level of each quality control sample, must meet the above acceptance criteria.

[0126] Unknown samples: The %CV (concentration) of the replicates of the sample must not exceed 20.0%. If it does, the sample needs to be reanalyzed.

[0127] The backcount concentration of the sample (here, backcount concentration refers to the original backcount concentration, that is, the concentration before multiplying by a dilution factor other than MRD) is determined by the average value of the replicates and must satisfy: LLOQ ≤ backcount concentration ≤ ULOQ.

[0128] If the concentration of one or two replicates is greater than ULOQ, the sample will be diluted with a larger dilution factor and reanalyzed.

[0129] If both replicates of a sample have concentrations below the limit of quantitation (LOQ) and the dilution factor is greater than the minimum dilution factor (MRD), the sample will be diluted with a smaller dilution factor (but still greater than the MRD) and reanalyzed. If the dilution factor is equal to the MRD, the sample result will be reported as "less than the LQ". However, if the LQ was increased due to a failure of the original LQ, the sample will be reanalyzed.

[0130] If the backcount concentration of one well of a sample is less than LLOQ and the backcount concentration of another well is greater than or equal to LLOQ, and if the backcount concentration of the well greater than or equal to LLOQ is less than or equal to 125% of the LLOQ concentration and the dilution factor is the minimum dilution factor, then the sample does not need to be reanalyzed, and the value within the quantitation range should be reported. Otherwise, the sample needs to be reanalyzed.

[0131] The statistical formula involved in this invention is as follows: Average (the average of two or more values): Average value = total number / number of items.

[0132] Standard deviation (SD): , Where N = number of samples, Xi = Single sample signal value, = The average value of the signal.

[0133] Coefficient of variation (%CV):

[0134] Bias (%Bias):

[0135] Total error (%Total error):

[0136] Example 3: Validation of an ELISA method for detecting total antibody concentration in human serum The reagents, instruments, and methods used in this embodiment are as follows: Matrix: A mixture of human serum prepared from at least 10 different batches of drug-free individuals was used as the matrix; Standard curve and quality control sample preparation: Standard: Antibody-drug conjugate A solution (20 mg / mL); Standard curve samples: Dilute antibody-drug conjugate A solution with mixed human serum to the following concentrations: 3200,000, 1600,000, 800,000, 400,000, 200,000, 100,000, and 50,000 ng / mL, where 50,000 ng / mL is the anchor point.

[0137] Quality control samples: Dilute with mixed human serum to the following concentrations: 3200.000 (ULOQ), 2400.000 (HQC), 600.000 (MQC), 240.000 (LQC), 100.000 ng / mL (LLOQ). Sample dilution: All samples were diluted 5-fold with analytical buffer (MRD) before analysis, and additionally diluted with mixed human serum if necessary.

[0138] The analytical instruments used in this embodiment are as follows: Name: Molecular Devices Microplate Reader; Model: SpectraMax plus 384; Supplier: Molecular Devices.

[0139] The data analysis application software used in this embodiment is as follows: The raw data will be acquired using SoftMax Pro GxP v7.0.3. Subsequent processing and data analysis will be performed using Watson LIMS, version 7.5 SP1. For data that Watson LIMS cannot handle, Microsoft Excel (Microsoft Excel 2016) will be used.

[0140] (1) Establishment of the standard curve The above standard curve samples were subjected to enzyme-linked immunosorbent assay (ELISA) under the determined optimal experimental conditions according to Example 2. The total antibody concentration of human tissue factor detected was plotted on the x-axis, and OD was used as the metric. 450 Plot a four-parameter curve with the value as the ordinate, such as... Figure 1 As shown.

[0141] The curve equation is y=(0.0681807-3.22078) / (1+(x / 663.572)^1.17259)+ 3.22078), and the weighting coefficient is 1 / Y^2.

[0142] (2) Accuracy and precision assessment To evaluate the inter-batch and intra-batch precision and accuracy of the method, at least two analysts complete six acceptable analytical batches over at least two analytical days.

[0143] Each analytical batch includes one standard curve, a blank control point, and three sets of quality control samples (QCs). Each QC set contains five concentration levels (ULOQ, HQC, MQC, LQC, LLOQ). To simulate real samples, frozen quality control samples are preferred. It is recommended to freeze the prepared quality control samples for at least 24 hours before use.

[0144] Unless there is clear evidence of technical error, all data generated from passing accuracy and precision analysis (standard curve meets acceptance criteria) will be included in the statistical analysis. For outliers, data including and excluding outliers should be analyzed and compared to scientifically and accurately calculate intra- and inter-plate accuracy and precision.

[0145] For each analytical batch, intra-batch precision and accuracy must be calculated for quality control samples at each concentration level. Inter-batch precision and accuracy are assessed using all measurements at each concentration level from all accepted analytical batches.

[0146] The precision of the method is expressed as the coefficient of variation (%CV); the accuracy is expressed as the bias (%Bias). The accuracy of the kit was analyzed using these parameters. The results are shown in Table 5.

[0147] Table 5

[0148] Table 5 shows that the intra-batch coefficient of variation (%CV) for the five concentration levels was ≤14.5%, the intra-batch %Bias range for HQC, MQC, and LQC was -15.6% to 6.4%, and the intra-batch %Bias range for ULOQ and LLOQ was -22.9% to 6.4%; the inter-batch %Bias range for ULOQ and LLOQ was -10.6% to -5.7%, the inter-batch %CV range for ULOQ and LLOQ was 8.2% to 12.9%, the inter-batch %Bias range for HQC, MQC, and LQC was -6.6% to -5.6%, and the inter-batch %CV range for HQC, MQC, and LQC was 8.1% to 9.9%. The above results indicate that the quantitative detection of total human tissue factor antibodies in samples using the human tissue factor total antibody detection kit provided by this invention can meet the requirements of intra-assay coefficient of variation and bias ≤20% (for LLOQ and ULOQ, the intra-assay / inter-assay %Bias acceptance standard is within ±25.0%), demonstrating that the detection method provided in this application has good accuracy.

[0149] (3) Investigation of selectivity / matrix effect The selectivity of the method should be verified using serum samples from at least 10 healthy individuals. Each selective assay plate should include a selective control sample prepared from a mixture of human serum samples used to prepare the quality control sample.

[0150] In addition, at least one hemolyzed serum (prepared by adding 2% lysed blood cells to mixed human serum) and one high-lipid serum (prepared by adding synthetic lipid compounds to mixed human serum to achieve a lipid concentration ≥300 mg / dL) were tested to assess their matrix effects.

[0151] Standards were added to individual serum, hemolyzed serum, hyperlipidemic serum, and mixed human serum at final concentrations of 100,000 ng / mL (LLOQ level) and 2,400,000 ng / mL (HQC level). For individual serum, samples with and without standards were analyzed in duplicate. For hemolyzed and hyperlipidemic serum, samples with and without standards were analyzed in triplicate, with each analysis performed in duplicate. For mixed human serum, selective control samples prepared to LLOQ and HQC levels were analyzed in triplicate on each analytical plate, with each analysis performed in duplicate.

[0152] The matrix selectivity results for healthy individuals are shown in Table 6, and the matrix selectivity results for hyperlipidemic hemolytic individuals are shown in Figure 7. "*" indicates that the acceptance criteria were not met and the individuals were not included in the inter-batch calculation.

[0153] Acceptance criteria for selective control samples: 1) The average concentration %Bias of the duplicate wells is within ±20.0% (the average concentration %Bias at the LLOQ level is within ±25.0%); 2) Polypore concentration %CV ≤ 20.0% (LLOQ level polypore concentration %CV ≤ 25.0%); 3) At least 2 / 3 of the samples at each level must meet the above acceptance criteria; otherwise, the samples on the analysis plate need to be reanalyzed.

[0154] Selective sample acceptance criteria for matrices from healthy individuals: 1) The average concentration %Bias of the duplicate wells is within ±20.0% (the average concentration %Bias at the LLOQ level is within ±25.0%). 2) Polypore concentration %CV ≤ 20.0% (LLOQ level polypore concentration %CV ≤ 25.0%); 3) For samples with added standards, at least 80% of each level must meet the above acceptance criteria; 4) For individual matrices without added standards, at least 80% of the assay concentrations must be below LLOQ.

[0155] If the selectivity of individual matrices does not meet the above criteria, 10 additional individual matrices may be selected for evaluation. The results of these 10 matrices will be evaluated together with those from the first test. The results of all 20 individual matrices should meet the above acceptance criteria. If more than four individual matrices fail to meet the criteria in the first test, it is not necessary to select additional individual matrices for testing. A second analysis will be performed using the same healthy individual serum for confirmation. If the result is satisfactory, a third analysis will be performed for confirmation. If two out of the three analyses are satisfactory, the selectivity is considered satisfactory.

[0156] Selective sample acceptance criteria for hemolysis and high lipid content: 1) The average concentration %Bias of the duplicate wells is within ±20.0% (the average concentration %Bias at the LLOQ level is within ±25.0%). 2) Polypore concentration %CV ≤ 20.0% (LLOQ level polypore concentration %CV ≤ 25.0%); 3) Selective samples prepared with at least 2 / 3 of hemolyzed serum or hyperlipidemic serum at each level (with added standards) must meet the above acceptance criteria; 4) The concentration of at least 2 / 3 of the hemolyzed serum or hyperlipidemic serum without added standards is lower than that of LLOQ.

[0157] If a hemolyzed or high-lipid selective sample does not meet the above criteria, a second identical analysis is performed to confirm the results. If the second analysis passes, a third analysis is performed for confirmation. If two out of three analyses pass, the sample is considered to have no matrix effect. If the second analysis does not meet the above criteria, the sample is considered to have a matrix effect under those conditions.

[0158] Table 6

[0159]

[0160] Table 7

[0161] As shown in Tables 6 and 7, all 10 samples with added standards for measuring individual matrix LLOQ levels met the acceptance criteria. Eight of the 10 samples with added standards for measuring individual matrix HQC levels met the acceptance criteria. The results for all 10 individual matrix samples without added standards were lower than the LLOQ level. For hemolyzed matrix, three measurements with added LLOQ and three measurements with added HQC standards met the acceptance criteria. For high-fat matrix, three measurements with added LLOQ and three measurements with added HQC standards met the acceptance criteria. The concentrations of the three measurements for both hemolyzed and high-fat matrix samples without added standards were lower than the LLOQ level.

[0162] The above results indicate that no significant matrix effect was observed in healthy individual matrix, hemolyzed matrix, and high-lipid matrix using the method provided by this invention.

[0163] (4) Investigation of dilution linearity and hook effect An initial concentration sample with a final concentration of 800,000.000 ng / mL was prepared in mixed human serum. This sample was then diluted 10, 100, 400, 800, 3200, and 20000 times with the mixed human serum, resulting in concentrations of 80,000.000 ng / mL, 8,000.000 ng / mL, 2,000.000 ng / mL, 1,000.000 ng / mL, 250.000 ng / mL, and 40.000 ng / mL, respectively, to evaluate the dilution linearity and hook effect of the method. Three sets of measurements were performed for each sample to assess dilution linearity and hook effect.

[0164] The dilution linearity results are shown in Table 8. As can be seen from Table 8, for the three sets of samples tested, all dilution points with theoretical concentrations within the quantification range met the acceptance criteria; for points with theoretical concentrations outside the quantification range, the average signal values ​​were all lower than the average signal values ​​of the LLOQ. The method maintained good dilution linearity even at a dilution factor of 3200.

[0165] Acceptance criteria for dilution linear samples: For concentration points with theoretical concentrations within the quantitation range, the measured values ​​should meet the following criteria: at least two-thirds of the samples at each concentration level have a backcalculated concentration %Bias within ±20.0%, and the replicate concentration %CV ≤ 20.0%; the %CV of the backcalculated concentration after dilution correction for all samples meeting the above criteria within a dilution sequence should not exceed 20.0%. For points with theoretical concentrations below the quantitation range, the following criteria should be met: at least two-thirds of the samples at each concentration level have a signal value whose average value is lower than the average signal value of the LLOQ.

[0166] The results of the hook effect are shown in Table 9. The acceptance criteria for the hook effect are as follows: For the hook effect, the signal values ​​at points above the upper limit of quantitation will be used for evaluation. If at least two-thirds of the sample signal values ​​at concentrations above the upper limit of quantitation increase with increasing concentration or remain unchanged accordingly, then it can be concluded that no "hook" effect was detected at that concentration. As shown in Table 9, no hook effect was detected even at a sample concentration of 800,000.000 ng / mL.

[0167] Table 8

[0168] Table 9

[0169] (5) Target interference investigation The target substance human tissue factor-His present in the sample may interfere with the detection of total antibody-drug conjugate A. To investigate the effect of human tissue factor-His in the sample on the detection of total antibody-drug conjugate A, target interference samples were prepared by mixing equal volumes of antibody-drug conjugate A at final concentrations of 2×100.000 ng / mL and 2×3200.000 ng / mL, and human tissue factor-His at final concentrations of 2×0.500 ng / mL, 2×1.000 ng / mL, 2×2.000 ng / mL, 2×5.000 ng / mL, and 2×10.000 ng / mL in mixed serum. After preparation, the target interference samples needed to be pre-incubated at room temperature for at least 60 minutes (500 rpm) and then frozen at -70°C for at least 24 hours before subsequent experimental analysis. All samples needed to be MRD diluted before analysis. Three measurements need to be performed on the target interference sample.

[0170] The results are shown in Table 10, where "*" indicates that the acceptance criteria were not met. The acceptance criteria for target interference are as follows: if, in the analytical batch, for each antibody-drug conjugate A concentration, the target interference samples all satisfy the condition that the deviation of the backcalculated concentration of at least 2 / 3 of the sample replicates is within ±25.0%, and %CV ≤ 25.0%, then it can be concluded that no target interference was detected at human tissue factor-His concentrations up to this level. If target interference at a certain concentration fails to meet the acceptance criteria (%CV ≤ 25.0%), the target interference experiment at that concentration will be repeated.

[0171] Table 10

[0172] As shown in Table 10, the total antibody concentration of antibody-drug conjugate A was 3200.000 ng / mL, and tissue factor-His at 10.000 ng / mL did not interfere with the detection of the total antibody of antibody-drug conjugate A; the total antibody concentration of antibody-drug conjugate A was 100.000 ng / mL, and tissue factor-His at 10.000 ng / mL did not interfere with the detection of the total antibody of antibody-drug conjugate A.

[0173] (6) Method robustness test The robustness of the method requires examination of at least the longest and shortest incubation times for critical steps, which can be evaluated together with the accuracy and precision experiments. Acceptance criteria and intra-batch accuracy are the same as for precision experiments. Alternatively, the method can be evaluated separately, with the same procedures as acceptance criteria and intra-batch accuracy and precision experiments. All longest and shortest incubation conditions must be performed separately within the same analytical batch. The target shortest and longest incubation times are shown in Table 11 below.

[0174] Table 11

[0175] The results are shown in Table 5 for analysis batches 3 and 5. As can be seen from Table 5, all of the above meet the acceptance criteria.

[0176] (7) Stability test In the stability experiment, ultra-high concentration stable samples (SUHQC, 800,000.000 ng / mL, MRD (minimum dilution factor)) were prepared in a mixed matrix using standards and diluted 1,000 times with mixed human serum before stability assessment. High concentration stable samples (SHQC, 2,400.000 ng / mL) and low concentration stable samples (SLQC, 240.000 ng / mL) were also prepared. Stability was assessed by three determinations for each condition. Except for the stability samples tested on the day of preparation, the three stability samples should be from different aliquots. Each determination was performed in duplicate.

[0177] Short-term stability study: Evaluate stability for the following periods at room temperature and 2–8°C: Evaluate stability for at least 48 hours at room temperature. If stability does not meet acceptance criteria after 48 hours, evaluate stability for a shorter period. Evaluate stability for at least 72 hours at 2–8°C. If stability does not meet acceptance criteria after 72 hours, evaluate stability for a shorter period.

[0178] Freeze-thaw cycle stability assessment: Evaluate stability after 6 freeze-thaw cycles at -20°C and -70°C. The initial freezing time should be at least 24 hours. For each subsequent freeze-thaw cycle, the stability sample must be frozen for at least 12 hours, then thawed naturally at room temperature and left to stand for at least 2 hours, except on the day of testing. If instability is found after 6 freeze-thaw cycles, stability testing for 4 more freeze-thaw cycles is required.

[0179] Long-term stability: Stability was evaluated for 1 month, 3 months, 6 months, and 12 months under storage conditions of -20°C and -70°C. The stability study results are shown in Table 12, where "*" indicates that the percentage bias (%Bias) does not meet the acceptance criteria. The stability acceptance criteria are: the average percentage bias (%Bias) of the duplicate well concentration relative to the theoretical value is within ±20.0%; and the percentage CV (%CV) of the two duplicate wells is ≤20.0%. For each stability condition, at least two-thirds of the stable samples at each concentration level must meet the above criteria.

[0180] Table 12

[0181] Table 12 shows that the stable samples were stable after 48 hours of storage at room temperature; and stable samples were stable after 72 hours of storage at 2–8℃. At -20℃, the stable samples were stable after 6 freeze-thaw cycles; at -70℃, the stable samples were stable after 6 freeze-thaw cycles; at -20℃, the stable samples were stable after 95 days of storage; and at -70℃, the stable samples were stable after 95 days of storage.

[0182] In summary, the enzyme-linked immunosorbent assay (ELISA) method for quantitative detection of total anti-tissue factor antibodies in serum provided in this application involves coating an ELISA plate with an antibody to form a solid-phase carrier, which binds to the total anti-human tissue factor antibodies in the biological sample to be tested, and then passing a specific detection antibody to form a solid-phase coated antibody-antibody-detection antibody-ELISA complex. Because the antibody pairs obtained through screening (coating antibody and detection antibody) have high specificity, the method established in this application has high specificity, high sensitivity, and good precision and accuracy.

[0183] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. An antibody for detecting the total antibody concentration in blood, characterized in that, The antibodies include: (1) A first antibody comprising a heavy chain variable region VH1 and a light chain variable region VL1, wherein VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 41; and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 42; and / or, (2) A second antibody comprising a heavy chain variable region VH2 and a light chain variable region VL2, wherein the VH2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 43; and the VL2 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:

44.

2. The antibody according to claim 1, characterized in that, The complementarity-determining region of the antibody is defined based on the IMGT antibody coding system, wherein, The first antibody has HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; LCDR1 as shown in SEQ ID NO:4, LCDR2 with an LTS amino acid sequence, and LCDR3 as shown in SEQ ID NO:6; and / or, The second antibody has HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11, LCDR1 as shown in SEQ ID NO:12, LCDR2 with the amino acid sequence NAK, and LCDR3 as shown in SEQ ID NO:

14.

3. The antibody according to claim 1 or 2, characterized in that, The VH1 comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 41; and / or, the VL1 comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 42; And / or, the VH2 comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 43; and / or, the VL2 comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

44.

4. The antibody according to claim 3, characterized in that, The VH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 41; and / or, the VL1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 42; And / or, the VH2 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 43; and / or, the VL2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

44.

5. The antibody according to claim 1, characterized in that, The antibody is a full-length antibody, comprising a heavy chain and a light chain, wherein the heavy chain and light chain amino acid sequences of the first antibody and / or the second antibody include: (1) The heavy chain of the first antibody comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:7; the light chain comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:8; and / or, (2) The heavy chain of the second antibody contains an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:15; the light chain contains an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

16.

6. The antibody according to claim 5, characterized in that, The amino acid sequence of the heavy chain of the first antibody is shown in SEQ ID NO:7, and the amino acid sequence of the light chain is shown in SEQ ID NO:8; and / or, The amino acid sequence of the heavy chain of the second antibody is shown in SEQ ID NO:15, and the amino acid sequence of the light chain is shown in ID NO:

16.

7. An isolated polynucleotide, characterized in that, The polynucleotide encodes the antibody according to any one of claims 1 to 6.

8. A carrier, characterized in that, The carrier comprises the polynucleotide of claim 7.

9. A host cell, characterized in that, The host cell comprises the polynucleotide of claim 7 or the vector of claim 8.

10. A reagent kit, characterized in that, The kit includes the antibody as described in any one of claims 1 to 6.

11. The reagent kit according to claim 10, characterized in that, The kit includes a coating antibody and a detection antibody, wherein the coating antibody is a first antibody and the detection antibody is a second antibody; the detection antibody is labeled with a detectable marker, wherein the detectable marker is selected from one or more of biotin, fluorescein, and enzymes; And / or, the kit may further include one or more of the following: HRP-labeled streptavidin, coating buffer, washing buffer, analysis buffer, blocking buffer, chromogenic buffer, stop solution, or antibody-drug conjugate reconstituted solution or standard.

12. Use of the antibody according to any one of claims 1 to 6, or the kit according to claim 10 or 11, in the preparation of a product for detecting the total antibody concentration in blood.

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