Antibodies binding to teriparatide and uses thereof

Abstract

The present application provides antibodies or antigen-binding portions thereof that bind to tezepelumab, nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or the vectors, detection reagents or kits comprising the antibodies or antigen-binding portions thereof, and uses thereof.

Description

Technical Field

[0001] This application relates to the field of immunology, and more specifically, to antibodies that bind to toripalimab and their uses. Background Technology

[0002] Tumor immunotherapy works by activating the body's immune system to fight tumors, with PD-1 antibody drugs showing particularly strong performance. PD-1 is a 55 kDa transmembrane protein expressed on the surface of immune cells such as T cells. Its ligands, PD-L1 / PD-L2, regulate the immune response under normal physiological conditions. However, many tumors (e.g., colorectal cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, etc.) highly express PD-L1, enabling them to evade the immune system by binding to PD-1 on T cells. Blocking the PD-1 / PD-L1 pathway has become an important therapeutic strategy, with toripalimab being one of the representative drugs.

[0003] Therefore, developing an antibody that can monitor the blood concentration of toripalimab and the production of its anti-drug antibody (ADA) will help optimize clinical application. Summary of the Invention

[0004] In a first aspect, this application provides an antibody or antigen-binding moiety thereof that binds to toripalimab, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein, according to the IMGT definition, the amino acid sequence of HCDR1 comprises the sequence shown in SEQ ID NO:2, the amino acid sequence of HCDR2 comprises the sequence shown in SEQ ID NO:4, the amino acid sequence of HCDR3 comprises the sequence shown in SEQ ID NO:6, the amino acid sequence of LCDR1 comprises the sequence shown in SEQ ID NO:10, the amino acid sequence of LCDR2 comprises the sequence shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 comprises the sequence shown in SEQ ID NO:14.

[0005] Secondly, this application provides a nucleic acid molecule that encodes the antibody or its antigen-binding portion as described in the first aspect.

[0006] Thirdly, this application provides a vector containing the nucleic acid molecules described in the second aspect.

[0007] Fourthly, this application provides a host cell that contains the nucleic acid molecules described in the second aspect or the vector described in the third aspect.

[0008] Fifthly, this application provides a detection reagent or kit containing the antibody or its antigen-binding portion as described in the first aspect.

[0009] Sixthly, this application provides the use of the antibody or its antigen-binding portion described in the first aspect in detecting the concentration of toripalimab or its anti-antibody in a biological sample of an individual. Attached Figure Description

[0010] Figure 1 The preparation of the toripalimab immunogen is described.

[0011] Figure 2 The results are SDS-PAGE assays of the purified exemplary antibody (9A6) of this application bound to toripalimab, where N represents the antibody under non-reducing conditions and R represents the antibody under reducing conditions.

[0012] Figure 3 The methodology for antibody subtype identification is described.

[0013] Figure 4 The ELISA results shown illustrate the binding of the purified exemplary toripalimab-binding antibody of this application to both toripalimab and human IgG. Different concentrations of the antibody showed significant binding to toripalimab and virtually no binding to human IgG.

[0014] Figure 5 The ELISA assay shown validated the neutralizing activity of different concentrations of the exemplary antibody (9A6) binding to toripalimab of this application. As the concentration of the antibody increased, its blocking rate against the binding of toripalimab to PD-1 also increased.

[0015] Sequence Description

[0016] The sequencing results of the antibody (9A6) that binds to toripalimab provided in this application are shown in the table below.

[0017] Detailed Implementation

[0018] The following definitions and methods are provided to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terminology used in this application shall be understood in accordance with the conventional usage of those skilled in the art.

[0019] definition

[0020] Unless otherwise specified, this application is implemented using conventional molecular biology, microbiology, cell biology, biochemistry and immunology techniques in the art.

[0021] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0022] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0023] Although the numerical ranges and parameter approximations shown in the broad scope of this application are intended to be as accurate as possible in the specific embodiments, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the stated range “1 to 10” should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (inclusive); that is, all subranges beginning with a minimum value of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any references marked “incorporated herein” should be understood to be incorporated herein in their entirety.

[0024] In a broad sense, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Therefore, it encompasses complete antibodies / full-length antibodies, single-chain antibodies, or any antigen-binding portion of an antibody (also known as an "antigen-binding fragment"), such as Fab, Fab', F(ab')2, Fv, scFv, Fd fragments, single-domain antibodies, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site of desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. When "antibody" and "antigen-binding portion / antigen-binding fragment" appear in the same context, "antibody" can be understood as the complete entity relative to the "antigen-binding portion / antigen-binding fragment," both corresponding to the broad concept of antibody.

[0025] Typically, a full-length or complete antibody consists of two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be assigned to different classes based on the antibody's amino acid sequence of the heavy chain constant domain. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are called... , , , ,as well as The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0026] As used herein, the terms "antigen-binding region" or "antigen-binding fragment" are used interchangeably and refer to a portion or region of the complete antibody molecule responsible for binding the antigen. An antigen-binding domain may contain a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0027] Examples of antigen-binding portions of a general antibody include, but are not limited to: (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) an F(ab')2 fragment, which may be a divalent fragment having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; (3) an Fv fragment having a single arm of the antibody with VL and VH domains; (4) a single-chain Fv (scFv), which may be a single polypeptide chain consisting of VH and VL domains connected by peptide linkers; and (5) (scFv)2, which may contain two VH domains and two VL domains connected by peptide linkers, the two VL domains being combined with the two VH domains via disulfide bridges.

[0028] In some specific embodiments of this application, the "antigen-binding portion" includes, but is not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, Fd fragments, and single-domain antibodies.

[0029] As used in this article, "single-chain antibody (scFv, single-chain fragment variable)" refers to an antibody with a single-chain structure, typically constructed using genetic engineering techniques. It is a polypeptide chain containing a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is usually designed between the heavy and light chain variable regions so that they can fold into the correct conformation to bind the antigen.

[0030] As used herein, the terms “Fab (fragment antigen binding) fragment,” “Fab portion,” or similar terms refer to antibody fragments that bind to antigens produced by treating an intact antibody with papain, including the intact light chain (VL-CL), the heavy chain variable region, and the CH1 fragment (VH-CH1).

[0031] As used herein, the terms “Fd fragment,” “Fd portion,” or similar terms refer to the heavy chain portion of the Fab fragment of an antibody, including the heavy chain variable region and the CH1 fragment (VH-CH1).

[0032] As used herein, the terms “Fc fragment,” “Fc domain,” and “Fc portion” are used interchangeably to refer to a portion of the antibody heavy chain constant region, including the hinge region, the CH2 and CH3 fragments of the heavy chain constant region, and are determined with reference to the EU numbering of human IgG1 antibodies.

[0033] As used herein, the term "single-domain antibody," also known as a VHH antibody or nanobody, can be defined as an amino acid sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Here, FR1-FR4 refer to framework regions 1-4, and CDR1-CDR3 refer to complementarity-determining regions 1-3. "VHH" refers to a variable antigen-binding domain from heavy chain antibodies derived from camels (camels, dromedaries, llamas, alpacas, etc.).

[0034] As is known to those skilled in the art, complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. There are several common definitions for the CDR amino acid sequence of VH or VL, including IMGT, Kabat, AbM, and Chothia definitions. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA86:9268-9272 (1989). In the embodiments of this application, the IMGT definition is used to determine the CDR amino acid sequence in the VH and VL amino acid sequences of the antibody.

[0035] For a given antibody's variable region amino acid sequence, the CDR amino acid sequence can be analyzed in various ways, such as using the online software Abysis (http: / / www.abysis.org / ).

[0036] As used in this article, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0037] As used in this article, “monoclonal antibody” or “monoclonal antibody” refers to an antibody obtained from a population of essentially homogeneous antibodies, that is, the individual antibodies that make up the population are identical except for the possibility of naturally occurring mutations in a small number of individuals.

[0038] As used in this article, "Toripalimab" refers to a PD-1 inhibitor, with the English name Toripalimab and the brand name Tuoyi. It is mainly used for indications such as melanoma, nasopharyngeal carcinoma, and urothelial carcinoma.

[0039] The term "identity / homology / consistency" in relation to amino acid or nucleic acid sequences is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of identity if desired. The methods and computer programs used for alignment are well known in the art.

[0040] Immunotherapy has become an important treatment for advanced malignant tumors. Instead of directly attacking cancer cells, immunotherapy works by activating the body's own immune system to fight tumors, exhibiting good safety and tolerability. Programmed cell death receptor 1 (PD-1) and its ligand-associated antibody drugs, as representative drugs in immunotherapy, have achieved great success in the treatment of advanced malignant tumors.

[0041] PD-1 is a type I transmembrane glycoprotein of approximately 55 kDa, belonging to the CD28 superfamily of receptors, and is mainly expressed on the surface of T cells, B lymphocytes, and activated macrophages. PD-1 has two ligands: PD-L1 and PD-L2. Under normal physiological conditions, the binding of PD-1 to PD-L1 / PD-L2 inhibits T cell activation, thereby protecting the body from attack by the autoimmune system. However, various solid tumors and some hematologic malignancies, such as melanoma, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, lymphoma, and leukemia, also express large amounts of PD-L1 in their cells. PD-L1 on the tumor cell membrane binds to PD-1 on T cells, inhibiting T cell activation, thus successfully evading recognition and attack by the body's immune system, achieving immune escape for tumor cells. Related studies have found that PD-L1 expression on tumor cells is associated with poor prognosis in several tumor types. Therefore, blocking the binding of PD-1 to PD-L1 is a reasonable approach for tumor immunotherapy.

[0042] Toripalimab is a PD-1 inhibitor, classified as a Class I drug, primarily used for indications such as melanoma, nasopharyngeal carcinoma, and urothelial carcinoma. With the increasing clinical application of toripalimab, the development of an antibody that specifically recognizes and binds to it, in order to detect the drug's concentration in the blood and the production of its antidrug antibody (ADA), is crucial for guiding rational clinical drug use.

[0043] Based on the exploration of the above problems and through continuous experimentation, this application provides the following implementation plan:

[0044] In a first aspect, this application provides an antibody or antigen-binding moiety thereof that binds to toripalimab, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein, according to the IMGT definition, the amino acid sequence of HCDR1 comprises the sequence shown in SEQ ID NO:2, the amino acid sequence of HCDR2 comprises the sequence shown in SEQ ID NO:4, the amino acid sequence of HCDR3 comprises the sequence shown in SEQ ID NO:6, the amino acid sequence of LCDR1 comprises the sequence shown in SEQ ID NO:10, the amino acid sequence of LCDR2 comprises the sequence shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 comprises the sequence shown in SEQ ID NO:14.

[0045] In some embodiments of the first aspect, the antibody or its antigen-binding portion specifically binds to toripalimab or fragments of toripalimab, such as the heavy chain, light chain, heavy chain variable region, light chain variable region, and / or constant region of toripalimab. For example, the antibody or its antigen-binding portion is an anti-idiotype antibody that binds toripalimab. In a more specific embodiment, the antibody or its antigen-binding portion can at least partially block the binding of toripalimab to PD-1.

[0046] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO:8. Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:8.

[0047] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO:16. Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16.

[0048] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region may be a substitution, deletion, and / or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from the amino acid sequence shown in SEQ ID NO:8.

[0049] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:8 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functionality to the heavy chain variable region.

[0050] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:8, and the resulting amino acid sequence still retains a similar function to the heavy chain variable region.

[0051] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:8, as long as the altered amino acid sequence substantially maintains a similar function to the heavy chain variable region.

[0052] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region may be substituted, deleted, and / or added by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids different from the amino acid sequence shown in SEQ ID NO:16.

[0053] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:16 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functionality to the light chain variable region.

[0054] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:16, and the resulting amino acid sequence still retains a similar function to the variable region of the light chain.

[0055] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:16, as long as the altered amino acid sequence substantially maintains a similar function to the light chain variable region.

[0056] In some embodiments of the first aspect, the antigen-binding portion is selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, Fd fragments, or single-domain antibodies.

[0057] In some embodiments of the first aspect, the antibody is selected from murine antibodies, human antibodies, and humanized antibodies. Preferably, the antibody is a murine antibody.

[0058] In some embodiments of the first aspect, the antibody may be a monoclonal antibody.

[0059] In some embodiments of the first aspect, the heavy chain of the antibody is the IgG1 subtype, and the light chain of the antibody is the κ chain.

[0060] In a second aspect, this application provides a nucleic acid molecule that encodes the antibody or its antigen-binding portion described in the first aspect.

[0061] In a preferred embodiment, the nucleic acid described herein may be a codon-optimized nucleic acid suitable for expression in a host cell. For example, it may still encode the same protein based on codon degeneracy. Methods for codon optimization based on the host cell used are well known to those skilled in the art.

[0062] In some embodiments of the second aspect, the nucleic acid molecule may include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0063] In some embodiments of the second aspect, the nucleic acid molecule is operatively linked to a regulatory nucleotide sequence for expression in a host cell. In some embodiments, the nucleic acid molecule is operatively linked to a regulatory nucleotide sequence contained in a vector (e.g., a plasmid) for expression in a host cell. The host cell may be derived from mammals, such as mice, rats, dogs, cats, sheep, cattle, monkeys, camels, llamas, humans, etc. The host cell may be cultured in a culture medium to express the antibody or its antigen-binding moiety, and subsequently recovered to harvest the antibody or its antigen-binding moiety.

[0064] This application provides combinations of polynucleotides, the combinations comprising a polynucleotide encoding a light chain of an antibody of the present application or an antigen-binding moiety thereof and a polynucleotide encoding a heavy chain of an antibody of the present application or an antigen-binding moiety thereof.

[0065] In a third aspect, this application provides a vector comprising the nucleic acid molecules described in the second aspect.

[0066] In some embodiments of the third aspect, the vector is an expression vector. The vector (e.g., an expression vector) may comprise a combination of nucleic acid molecules or polynucleotides as described in the second aspect. In some embodiments, the expression vector of this application comprises a nucleic acid molecule or a combination of polynucleotides as described in this application, wherein the nucleic acid molecule or combination of polynucleotides is efficiently linked to a regulatory sequence that allows the polypeptide it encodes to be expressed in a host cell or cell-free expression system. The choice of expression vector depends on the choice of host cell and can be selected to achieve the desired expression and regulatory characteristics in the selected host cell.

[0067] An "expression vector" is a vector that includes one or more expression control sequences. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0068] The nucleic acid in the vector can be operatively linked to one or more expression control sequences. As used herein, "operatively linked" means incorporated into the genetic construct such that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence consisting of a region of the DNA molecule typically located within 100 nucleotides upstream of the transcription start site (usually near the start site of RNA polymerase II). For the coding sequence to be under the control of the promoter, the translation start site of the polypeptide translation reading frame must be located between 1 and 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function at different distances from the transcription start site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, and then the mRNA can be translated into the protein encoded by the coding sequence, the coding sequence is "operatively linked" to and "under the control" of the expression control sequence in the cell.

[0069] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, tobacco mosaic virus, herpesviruses, cytomegaloviruses, retroviruses, vaccinia virus, adenoviruses, and adeno-associated viruses. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0070] Expression vectors may include tag sequences. Tag sequences are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted at any position within the polypeptide, including the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, Flag™ tags (Kodak, NewHaven, CT), maltose E-binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the antibody of this application is contained in a vector of nucleic acid containing one or more domains encoding the constant region of the Ig heavy chain, said domains being, for example, amino acid sequences corresponding to the hinge region, CH2 region, and CH3 region of the human immunoglobulin Cγ1 chain (Fc fragments).

[0071] In a fourth aspect, this application provides a host cell containing the nucleic acid molecules described in the second aspect or the vector described in the third aspect. In some embodiments of this application, the host cell may be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell may be *Escherichia coli* (e.g., *E. coli* cells well-known in the art such as DH5α and BL21), *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The eukaryotic host cell may be a fungus such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharizoa*, *Trichoderma*, etc.; an insect cell such as *Ardisia crenata*; a plant cell such as tobacco; or a mammalian cell such as HEK293 cells, BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the host cell described in this application is preferably a mammalian cell, more preferably HEK293 cells, BHK cells, CHO cells, NSO cells, or COS cells.

[0072] In some embodiments, this application also provides a method for preparing the antibody or its antigen-binding portion as described in the first aspect, comprising:

[0073] a) Culturing the host cells described in the fourth aspect; and

[0074] b) Recover the antibody or its antigen-binding fraction from the host cell or the culture supernatant of the host cell.

[0075] In some specific embodiments, this application also discloses a method for preparing antibodies bound to toripalimab, the method comprising: culturing host cells under expression conditions to express antibodies bound to toripalimab; and isolating and purifying the expressed antibodies bound to toripalimab. Using the above method, crude antibodies can be obtained. These crude antibodies are then purified using methods including affinity purification based on toripalimab, non-denaturing gel purification, HPLC, or RP. HPLC, size exclusion, purification on a protein G column, or any combination of these techniques will purify the antibody bound to toripalimab into a substantially homogeneous substance, such as on SDS-PAGE. On PAGE electrophoresis, it appears as a single band.

[0076] Fifthly, this application provides a detection reagent or kit containing the antibody or its antigen-binding portion as described in the first aspect.

[0077] In some embodiments of the fifth aspect, the detection reagent or kit is based on antibody detection techniques conventional in the art, including but not limited to ELISA, Western blotting, or electrochemiluminescence techniques.

[0078] In some embodiments, the detection reagent or kit is based on ELISA technology to detect toripalimab or its anti-antibody. For example, in addition to the antibody or its antigen-binding portion described in the first aspect, the detection reagent or kit may also include a solid-phase carrier, a secondary antibody, and / or a chromogenic substrate. In some specific embodiments, the antibody or its antigen-binding portion described in the first aspect may, for example, be coated onto the solid-phase carrier as a primary antibody. After the primary antibody binds to toripalimab, the binding is determined by the secondary antibody and the chromogenic substrate. In other specific embodiments, the antibody or its antigen-binding portion described in the first aspect may, for example, be used as a positive control to determine the presence or concentration of toripalimab anti-antibody.

[0079] In some embodiments, the detection reagent or kit is based on Western blotting technology to detect toripalimab or its anti-antibody. For example, in addition to the antibody or its antigen-binding moiety described in the first aspect, the detection reagent or kit may also include SDS-PAGE electrophoresis reagents, membranes (on which proteins are transferred), and other reagents and equipment.

[0080] In some embodiments, the detection reagent or kit is based on electrochemiluminescence technology to detect toripalimab or its anti-antibody. For example, in addition to the antibody or its antigen-binding moiety described in the first aspect, the detection reagent or kit may also include reagents and equipment required for electrochemiluminescence technology.

[0081] The antibodies or their antigen-binding portions described herein may bind to detectable portions. Exemplary detectable portions include, but are not limited to, radioactive isotopes such as iodine-125, iodine... 131. Cesium 137, Iridium-192 and Cobalt-60, horseradish peroxidase, fluorescein isothiocyanate, biotin, alkaline phosphatase, chemiluminescent agents such as luminol, etc. Those skilled in the art can select suitable detectable portions to bind with the antibody or its antigen-binding portion as needed to achieve different detection purposes.

[0082] In a sixth aspect, this application provides the use of the antibody or its antigen-binding portion described in the first aspect in detecting the concentration of toripalimab or its anti-antibody in a biological sample of an individual.

[0083] As used herein, the term "individual" refers to a mammal, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0084] In some implementations of the sixth aspect, "biological sample" means any sample taken from an individual (e.g., a human or other animal), such as a person with cancer or suspected of having cancer, and that may contain toripalimab or an anti-antibody thereof. Biological samples can be bodily fluids, such as blood, plasma, serum, urine, vaginal fluid, fluid from the scrotum (e.g., ascites from the testes), vaginal douches, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, fluid expelled from the nipple, fluid aspirated from different parts of the body (e.g., thyroid, breast), intraocular fluid (e.g., aqueous humor), etc.

[0085] In this specification and claims, the words “comprising,” “including,” and “containing” mean “including but not limited to” and are not intended to exclude other parts, additives, components, or steps.

[0086] It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of this application may be applied to any other aspect, embodiment or example described herein, unless there is any contradiction.

[0087] The foregoing disclosure generally describes this application. The embodiments are further illustrative of this application and should not be construed as limiting it. The embodiments do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, for example, see Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press.

[0088] Example

[0089] The experimental examples described below are exemplary and are used only to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the experimental examples, they should be performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0090] Example 1. Preparation of toripalimab immunogen

[0091] refer to Figure 1 This document explains the preparation of the toripalimab immunogen (supplier: Jinan Maixiang Health Technology Co., Ltd.; brand: Junshi Biosciences). Specifically, this example uses Pierce... TM The F(ab')2 preparation kit (ThermoScientific, Cat:44988) uses pepsin immobilized on microbeads of agarose resin to selectively digest toripalimab, yielding the toripalimab F(ab')2 fragment for use as an immunogen. Because resin-immobilized pepsin is used, the digestion reaction can be easily stopped and enzyme-free products obtained simply by removing the resin from the solution. Then, NAb is used... TM Protein A centrifuge column binds the Fc fragment and undigested IgG. After centrifugation, a flow-through solution containing only the F(ab')2 fragment is obtained. Finally, Zeba can be used... TM The toripalimab F(ab')2 fragment was rapidly prepared using a desalting centrifuge column.

[0092] Example 2. Preparation of hybridoma cell lines against toripalimab

[0093] Hybridoma cell lines against toripalimab can be prepared according to the following steps:

[0094] 1. Emulsification of immunogens. Dilute the toripalimab F(ab')2 fragment to 0.1 mg / mL using physiological saline. Then, mix 3.5 mL of the diluted toripalimab F(ab')2 fragment with an equal volume of Freund's complete adjuvant (Sigma, F5881) / Freund's incomplete adjuvant (Sigma, F5505). Stir the mixture in an ice bath for 3–5 minutes using an adjuvant emulsifier (Bio-Long, BDYQ1001) to ensure complete emulsification of the immunogen. Store on ice for later use.

[0095] 2. Animal immunization. Prepare six 6-8 week old Balb / c mice and subcutaneously inject emulsified toripalimab F(ab')2 fragment immunogen at multiple sites on their backs. For example, administer 0.2 mL / site, 1 mL / mouse (approximately 50 μg / mouse). Immunize every 2-3 weeks for a total of 3-4 times until a positive antibody titer is detected in the serum.

[0096] 3. Cell fusion. Cell fusion can be performed according to the following steps:

[0097] 3.1. Take mice that have completed the immunization process and whose serum antibody titers meet the requirements, collect blood from the orbital venous plexus, let them stand at room temperature for 30 minutes, then centrifuge at 3000g for 15 minutes to separate the serum, and store the serum at -80℃ for later use as a positive control.

[0098] 3.2. Mice were euthanized and disinfected by immersion in 75% alcohol. The spleen was aseptically removed in a clean bench and lysed to release individual spleen cells. The resulting cells were washed with incomplete culture medium to prepare a single-cell suspension. After lysing red blood cells, the cell suspension was filtered through a 40 μm cell filter. The cells were centrifuged and washed twice with incomplete culture medium. A small number of cells were collected and counted using a CountStar cell counter.

[0099] 3.3. Remove the prepared mouse myeloma cells (Sp2 / 0 cells) in the logarithmic growth phase from the cell culture incubator, centrifuge to remove the old culture medium, then wash the Sp2 / 0 cells with incomplete culture medium and count them.

[0100] 3.4. Mix the spleen cells obtained in step 3.2 with the Sp2 / 0 cells obtained in step 3.3 at a ratio of 3:1 to 5:1 and fuse them using a PEG / electrofusion instrument.

[0101] 3.5. The fused cells were gently resuspended in conditioned medium containing HAT supplement (Gibco, Cat: 21060-017) and Hybridoma Feeder (Bio-Long, Cat: CM-2001) and evenly seeded into 96-well plates for culture.

[0102] 3.6. After 10-14 days, when the cell clones in the 96-well plate have grown to more than 1 / 10 of the plate area, the cell culture supernatant is identified by ELISA. Positive clones are screened and transferred to conditioned medium containing HT supplement (Gibco, Cat:11067-030) for further culture.

[0103] 3.7. Perform cloning 2-3 rounds until a positive monoclonal cell line is obtained.

[0104] Example 3. Selection of positive clones

[0105] 1. Wrapped in:

[0106] 1.1. Preparation of coating working solution. Toripalimab and human IgG were diluted with 1×PBS to a coating working solution with a concentration of approximately 1 μg / mL.

[0107] 1.2. Add the prepared coating working solution sequentially to the 96-well plate, 100 μL / well, and coat overnight at 2-8℃.

[0108] 2. Closed. Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper. Add 300 μL of blocking buffer (1×PBST containing 1% (w / v) BSA) to the wells of the ELISA plate and incubate at 37±5°C for 120-130 minutes. Wash the plate three times with 350 μL of 1×PBST washing buffer and pat dry on clean paper. Store any excess plate in a sealed container at 2-8°C for later use.

[0109] 3. Sample preparation:

[0110] 3.1. Dilution of mouse positive serum. The dilution was carried out in two steps: (1) 5 μL of mouse positive serum was mixed with 45 μL of diluent PBS to form 50 μL of solution A, which is 10-fold diluted mouse positive serum; (2) 40 μL of solution A was mixed with 3960 μL of diluent PBS to form solution B, which is 1000-fold diluted mouse positive serum.

[0111] 3.2. Preparation of hybridoma cell supernatant. Approximately 120 μL of supernatant was taken from the 96-well cell culture plate from which the clones had grown, and used as the supernatant sample for the hybridoma to be tested.

[0112] 4. Add sample. Add solution B and the supernatant sample of hybridoma cells to be tested, 50 μL / well, sequentially to an ELISA plate coated with toripalimab and human IgG. After sealing the plate, gently agitate and incubate at 37°C for 60-70 minutes.

[0113] 5. Preparation of working solution for test reagents. The detection reagent used in this embodiment is goat anti-mouse IgG (H+L) HRP. The preparation of the working solution is carried out in two steps: (1) 5 μL of goat anti-mouse IgG (H+L) HRP and 495 μL of diluent PBS are mixed to form 500 μL of solution C, that is, 100 times diluted goat anti-mouse IgG (H+L) HRP with a concentration of 8000 ng / mL; (2) 400 μL of solution C is mixed with 79600 μL of diluent PBS to form solution D, that is, 20000 times diluted goat anti-mouse IgG (H+L) HRP with a concentration of 40 ng / mL.

[0114] 6. Add the working solution for testing. Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper; add the corresponding detection working solution to the microplate at 100 μL / well and incubate at 37°C in the dark for 60-70 minutes.

[0115] 7. Color development. Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper. Add 100 μL of TMB substrate solution to each well of the microplate and incubate at room temperature in the dark for 15-20 minutes.

[0116] 8. Detect, analyze, and select positive clones. Add 1M H2SO4 (50 μL / well) to the ELISA plate, gently agitate to ensure no orange-yellow discoloration at the well edges, and place the plate in a plate reader within 5 minutes for detection at a wavelength of 450-630 nm. Clones with an OD450 response value greater than 1 in the toripalimab-coated plate and less than 0.3 in the human IgG-coated plate are considered candidate positive clones for further cloning and screening, or can be frozen for later use.

[0117] Example 4. Selection of positive monoclonal antibodies

[0118] 1. Adjust cell density. Positive candidate master clones were counted, and the cell density was adjusted to 1 × 10⁶ cells using culture medium. 3 ~1×10 5 / mL.

[0119] 2. Lay the boards.Approximately 130 positive master clones were added to 6.5 mL of complete culture medium, resulting in a cell density of approximately 20 cells / mL. After mixing, 100 μL / well was added to three wells (A, B, and C) of a 96-well plate, averaging 2 cells per well. 2.9 mL of complete culture medium was added to the remaining 2.9 mL of cell suspension, resulting in a cell density of approximately 10 cells / mL. 100 μL / well was added to three wells (D, E, and F), averaging 1 cell per well. 2.2 mL of complete culture medium was added to the remaining 2.2 mL of cell suspension, resulting in a cell density of approximately 5 cells / mL. 100 μL / well was added to two wells (G and H), averaging 0.5 cells per well.

[0120] 3. Cultivation. After 4-5 days of culture, small clones can be seen growing under an inverted microscope; on the 8th-9th day, clearly clustered clones can be seen growing under the microscope, some of which are even visible to the naked eye.

[0121] 4. Screen for positive monoclonal antibodies. ELISA testing is performed, and positive monoclonal antibodies are selected based on the results, which can then be used for subsequent validation.

[0122] Alternatively, 2-3 rounds of cloning can be performed until satisfactory positive monoclonal cells are selected.

[0123] Note: For newly cloned hybridoma cells, HT supplement (Gibco, Cat: 11067-030) needs to be added to the complete culture medium.

[0124] In this embodiment, positive monoclonal 9A6 was selected.

[0125] Example 5. Positive monoclonal amplification and antibody purification

[0126] After expanding the culture of positive monoclonal cells, 200 mL of cell culture supernatant was collected and centrifuged at 3000 rpm for 20 minutes to remove residual cells and debris. The supernatant was then filtered through 0.45 μm and 0.22 μm filters. The filtered cell supernatant was then loaded onto a pre-equilibrated Protein G affinity chromatography column, washed with phosphate buffer until equilibrated, and eluted with 50 mM citrate buffer (pH 3.0) at a flow rate of 5 mL / min. The complete elution peak was collected, and the eluent was adjusted to pH 7.4 with 1 M Tris-HCl buffer (pH 9.0). The purified antibody was quantified using a NanoDrop micro-spectrophotometer and then aliquoted for use.

[0127] Example 6. Validation of purified antibody using SDS-PAGE

[0128] 1. Sample preparation.Based on the antibody quantitative concentration, take 5-10 μg of sample and add it to PBS, with a total volume not exceeding 30 μL. Add the loading buffer according to the ratio and treat in a boiling water bath for 5 minutes.

[0129] 2. Loading the sample. The protein prepreg gel (GenScript, Cat: M00659) was fixed in the electrophoresis tank, electrophoresis buffer was added, and the processed samples were loaded in sequence.

[0130] 3. Electrophoresis. Connect the power supply and perform electrophoresis at 150V for 1 hour.

[0131] 4. Examination and dyeing. After electrophoresis, remove the gel, rinse it with deionized water, and then stain it in 0.1% Coomassie brilliant blue staining solution for 1 hour.

[0132] 5. Decolorization. After staining, transfer the gel to a destaining solution for destaining. Change the destaining solution every 30-60 minutes until the protein bands on the gel are clearly visible.

[0133] The results are as follows Figure 2 As shown, lane N represents the 9A6 antibody under non-reducing conditions, and R represents the 9A6 antibody under reducing conditions.

[0134] Example 7. Identification of antibody subtypes

[0135] Antibody subtypes were identified using the Pierce Rapid ELISA Mouse Monoclonal Antibody Genotyping Kit (Invitrogen, Cat: 37503).

[0136] 1. Prepare reagents:

[0137] Add 50 mL of 10×TBS to 450 mL of ultrapure water to dilute to 1×TBS for later use; add 30 mL of 30×wash buffer to 870 mL of ultrapure water to prepare 1×wash buffer for later use.

[0138] 2. Prepare the sample to be tested:

[0139] Each sample to be tested requires 450 μL for subtype identification.

[0140] Hybridoma cell supernatant: The recommended dilution range is 1:10-1:100; in this example, 1:50 is used. The dilution method is to add 20 μL of hybridoma cell supernatant to 980 μL of 1×TBS.

[0141] Ascites: The recommended dilution range is 1:50000-1:80000; in this example, 1:75000 is used. The dilution method is as follows: first, add 1 μL of ascites to 5 mL of 1×TBS, and then add 67 μL of the diluent obtained in the first step to 933 μL of 1×TBS.

[0142] Purified antibody: The recommended concentration range is 25 ng / mL to 2 μg / mL; in this example, the purified antibody was diluted to 250 ng / mL with 1×TBS.

[0143] 3. Antibody typing:

[0144] 3.1. Equilibrate the TMB substrate and the strips coated with capture antibodies at room temperature.

[0145] 3.2. For example Figure 3 As shown, for each sample, 50 μL of diluted sample was added to each well (i.e., 8 wells) in the corresponding AH row.

[0146] 3.3. For example Figure 3 As shown, add 50 μL of goat anti-mouse (IgG / IgA / IgM)-HRP to each of the eight wells in each column. Gently tap the strips to mix.

[0147] 3.4. After sealing with sealing film, incubate at room temperature for 1 hour.

[0148] 3.5. Discard the solution in the wells. Add 1× washing buffer to the wells, wash 3 times, and pat the strips dry on absorbent paper.

[0149] 3.6. Add 75 μL of TMB substrate equilibrated to room temperature to each well. Positive wells will turn blue after approximately 1 minute. The development time and color intensity vary depending on the antibody concentration and subtype.

[0150] 3.7. After 5-15 minutes, add 75 μL of stop solution to each well to terminate the reaction. The color of the plate wells will then change from blue to yellow. The light intensity at a wavelength of 450 nm can be measured using a spectrophotometer, or it can be determined directly by visual inspection.

[0151] 3.8. For example Figure 3 As shown, the subtype of the antibody to be tested is determined. In each sample, there should be exactly one positive well in the AF wells (heavy chain typing), and exactly one positive well in the GH wells (light chain typing). The results show that the heavy chain of the 9A6 antibody is the IgG1 subtype, and the light chain of the antibody is the κ chain.

[0152] Example 8. Comparison of the binding activity of purified antibody with toripalimab and human IgG.

[0153] 1. Wrapped in:

[0154] 1.1. Dilute toripalimab and human IgG with 1×PBS to prepare a coating working solution with a concentration of approximately 1 μg / mL.

[0155] 1.2. Add the prepared coating working solution sequentially to the 96-well plate, 100 μL / well, and coat overnight at 2-8℃.

[0156] 2. Closed. Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper. Add 300 μL of blocking buffer (1×PBST containing 1% (w / v) BSA) to the wells of the ELISA plate and incubate at 37±5°C for 120-130 minutes. Wash the plate three times with 350 μL of 1×PBST washing buffer and pat dry on clean paper. Store any excess plate sealed at 2-8°C for later use.

[0157] 3. Sample preparation:

[0158] For mouse positive serum, dilute it according to the following steps: (1) Mix 5 μL of mouse positive serum with 45 μL of diluent PBS to form 50 μL of solution E, which is 10 times diluted mouse positive serum; (2) Take 40 μL of solution E and mix it with 3960 μL of diluent PBS to form solution F, which is 1000 times diluted mouse positive serum.

[0159] For the purified antibody, dilute it sequentially with PBS at a ratio of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000, and mix well before use.

[0160] 4. Add sample. Add solution F and serially diluted purified antibody samples (100 μL / well) sequentially to an ELISA plate coated with toripalimab and human IgG. After sealing the plate, gently shake and incubate at 37°C for 60-70 minutes.

[0161] 5. Preparation of the working solution for the test reagent:

[0162] The detection reagent used in this embodiment is goat anti-mouse IgG (H+L) HRP. The preparation of the working solution is carried out in two steps: (1) 5 μL of goat anti-mouse IgG (H+L) HRP and 495 μL of diluent PBS are mixed to form 500 μL of solution G, which is 100 times diluted goat anti-mouse IgG (H+L) HRP with a concentration of 8000 ng / mL; (2) 400 μL of solution G is mixed with 79600 μL of diluent PBS to form solution H, which is 20000 times diluted goat anti-mouse IgG (H+L) HRP with a concentration of 40 ng / mL.

[0163] 6. Add the working solution for testing. Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper; add the corresponding detection working solution to the microplate at 100 μL / well and incubate at 37°C in the dark for 60-70 minutes.

[0164] 7. Color development. Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper. Add 100 μL of TMB substrate solution to each well of the microplate and incubate at room temperature in the dark for 15-20 minutes.

[0165] 8. Detect, analyze data, and screen for active antibodies. Add 1 M H₂SO₄, 50 μL / well, to the microplate, gently shake to ensure no orange-yellow discoloration at the well edges, and place the microplate in a plate reader within 5 minutes for detection at a wavelength of 450-630 nm. Results are as follows. Figure 4 As shown.

[0166] Example 9. Blocking activity of purified antibody against toripalimab

[0167] 1. Wrapped

[0168] 1.1. Preparation of coating working solution: Dilute PD-1 (Bio-Techne, R&D SYSTEMS, Cat: 8986-PD-100) with 1×PBS to a coating working solution with a concentration of approximately 1 μg / mL.

[0169] 1.2. Add the prepared coating working solution sequentially to the 96-well plate, 100 μL / well, and coat overnight at 2-8℃.

[0170] 2. Closed.Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper. Add 300 μL of blocking buffer (1×PBST containing 1% (w / v) BSA) to the wells and incubate at 37±5℃ for 120-130 minutes. Wash the plate three times with 350 μL of 1×PBST washing buffer and pat dry on clean paper. Store any excess plate sealed at 2-8℃ for later use.

[0171] 3. Sample preparation:

[0172] Dilute toripalimab to 1 μg / mL using the following steps: (1) Mix 2 μL of toripalimab (5 mg / mL) with 98 μL of diluent PBS to form 100 μL of solution I, which is toripalimab diluted 50 times; (2) Mix 90 μL of solution I with 8910 μL of diluent PBS to form solution J, which is toripalimab diluted 5000 times (1 μg / mL).

[0173] The purified 9A6 antibody was diluted with PBS to concentrations of 60, 30, 15, 7.5, 3.75, and 1.875 μg / mL. 150 μL of each concentration was then mixed with 150 μL of toripalimab (1 μg / mL) for later use. Simultaneously, 150 μL of toripalimab (1 μg / mL) was mixed with 150 μL of PBS as a control. The mixture was incubated at 37°C for 60 minutes.

[0174] 4. Add sample. In a PD-1 coated microplate, add the pretreated sample to each well in duplicate, 100 μL / well. After sealing the plate, gently shake and incubate at 37°C for 60-70 minutes.

[0175] 5. Preparation of working solution for test reagents. The detection reagent used in this embodiment is goat anti-mouse IgG (H+L) HRP. The preparation of the working solution is carried out in two steps: (1) 5 μL of goat anti-mouse IgG (H+L) HRP and 495 μL of diluent PBS are mixed to form 500 μL of solution K, which is 100 times diluted goat anti-mouse IgG (H+L) HRP with a concentration of 8000 ng / mL; (2) 400 μL of solution K is mixed with 79600 μL of diluent PBS to form solution L, which is 20000 times diluted goat anti-mouse IgG (H+L) HRP with a concentration of 40 ng / mL.

[0176] 6. Add the working solution for testing.Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper; add the corresponding detection working solution to the microplate at 100 μL / well and incubate at 37°C in the dark for 60-70 minutes.

[0177] 7. Color development. Wash the plate three times with at least 300 μL of 1×PBST washing buffer and pat dry on clean paper. Add 100 μL of TMB substrate solution to each well of the microplate and incubate at room temperature in the dark for 15-20 minutes.

[0178] 8. Detection and analysis. Add 1 M H₂SO₄, 50 μL / well, to the microplate, gently shake to ensure no orange-yellow discoloration at the well edges, and place the microplate in a plate reader within 5 minutes for detection at a wavelength of 450-630 nm. Results are as follows. Figure 5 As shown.

Claims

1. An antibody or its antigen-binding moiety that binds to toripalimab, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and in, According to the IMGT definition, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 6, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 10, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

14.

2. The antibody or its antigen-binding portion according to claim 1, wherein, The amino acid sequence of the heavy chain variable region has at least 90% identity with the sequence shown in SEQ ID NO: 8; and / or The amino acid sequence of the light chain variable region has at least 90% identity with the sequence shown in SEQ ID NO:

16.

3. The antibody or its antigen-binding portion according to claim 1 or 2, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8; and / or The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

16.

4. The antibody or its antigen-binding portion according to claim 1, wherein the antigen-binding portion is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, or a scFv fragment.

5. The antibody or its antigen-binding portion according to claim 1, wherein the antibody is a murine antibody or a humanized antibody; and / or The heavy chain of the antibody is the IgG1 subtype, and the light chain of the antibody is the κ chain.

6. A nucleic acid molecule encoding an antibody or an antigen-binding portion thereof as described in any one of claims 1-5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. The carrier according to claim 7, wherein the carrier is an expression carrier.

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

10. A detection reagent or kit comprising the antibody or antigen-binding portion thereof as described in any one of claims 1-5.

11. The detection reagent or kit according to claim 10, wherein the detection reagent or kit is based on ELISA, Western blotting, or electrochemiluminescence technology.

12. The use of the antibody or its antigen-binding portion as described in any one of claims 1-5 for non-disease diagnostic purposes in detecting the concentration of toripalimab or its anti-antibody in a biological sample of an individual.

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