Antibodies that bind to tereprenil monoclonal antibodies and uses thereof

Abstract

The invention provides an antibody or an antigen binding part of the antibody, a nucleic acid molecule for coding the antibody or the antigen binding part of the antibody, a carrier containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the carrier, and a detection reagent or kit containing the antibody or the antigen binding part of the antibody. And their use.

Description

Technical Field

[0001] The present application relates to the field of immunology, and more particularly, to antibodies binding to Teplizumab and uses thereof. Background Art

[0002] Tumor immunotherapy fights tumors by activating the human immune system, with PD-1 antibody drugs performing prominently. PD-1 is a 55 kDa transmembrane protein expressed on the surface of immune cells such as T cells, and its ligands PD-L1 / PD-L2 regulate immune responses under normal physiological conditions. However, many tumors (e.g., colorectal cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, etc.) highly express PD-L1 and can achieve immune escape by binding to PD-1 on T cells. Blocking the PD-1 / PD-L1 pathway has become an important treatment strategy, of which toripalimab is 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 antibodies (ADA) will help optimize its clinical application. Summary of the Invention

[0004] In a first aspect, the present application provides an antibody or an antigen-binding portion 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 the HCDR1 comprises the sequence shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 comprises the sequence shown in SEQ ID NO: 4, the amino acid sequence of the HCDR3 comprises the sequence shown in SEQ ID NO: 6, the amino acid sequence of the LCDR1 comprises the sequence shown in SEQ ID NO: 10, the amino acid sequence of the LCDR2 comprises the sequence shown in SEQ ID NO: 12, and the amino acid sequence of the LCDR3 comprises the sequence shown in SEQ ID NO: 14.

[0005] In a second aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof described in the first aspect.

[0006] In a third aspect, the present application provides a vector comprising the nucleic acid molecule described in the second aspect.

[0007] In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule described in the second aspect or the vector described in the third aspect.

[0008] In a fifth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof described in the first aspect.

[0009] In a sixth aspect, the present application provides use of the antibody or antigen-binding portion thereof described in the first aspect in detecting the concentration of toripalimab or its anti-drug antibody in a biological sample of an individual. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0011] Figure 2 This is the SDS-PAGE detection result of the purified exemplary antibody (9A6) of the present application that binds to toripalimab, wherein N represents the antibody under non-reducing conditions, and R represents the antibody under reducing conditions.

[0012] Figure 3 Methodologies for isotyping of antibodies are described.

[0013] Figure 4 The ELISA test results shown in the figure illustrate the binding of the purified exemplary antibodies binding to toripalimab to toripalimab and human IgG, respectively. The antibodies at different concentrations all showed significant binding to toripalimab and essentially no binding to human IgG.

[0014] Figure 5 The ELISA test shown verifies the neutralizing activity of the exemplary antibody (9A6) binding to Teplizumab of the present application at different concentrations. As the concentration of the antibody increases, its blocking rate of Teplizumab binding to PD-1 also increases.

[0015] Sequence Description

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

[0017] DETAILED DESCRIPTION

[0018] The following definitions and methods are provided to better define the present application and to guide those skilled in the art in practicing the present application. Unless otherwise specified, the terms of the present application are understood according to conventional usage by those skilled in the relevant art.

[0019] definition

[0020] The practice of the present application employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of 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 and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art.The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0023] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" recorded should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges terminating with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0024] In a broad sense, the term "antibody" refers to an immunoglobulin molecule that is capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus encompasses intact antibodies / full-length antibodies, single antibody chains, or any antigen-binding portion of an antibody (also referred to as an "antigen-binding fragment"), such as Fab, Fab', F(ab')2, Fv, scFv, Fd fragments, single domain antibodies, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen-recognition site of desired specificity, including glycosylation 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 intact body relative to the "antigen-binding portion / antigen-binding fragment", and the two together correspond to the broad concept of an antibody.

[0025] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the 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). A full-length antibody can be any class of antibody, such as IgD, IgE, IgG, IgA or IgM (or subclasses of the above), but antibodies do not need to belong to any particular class. Based on the antibody amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to different classes. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided 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 portion" or "antigen-binding fragment" are used interchangeably and refer to a portion or region of an intact antibody molecule that is responsible for binding to an antigen. The antigen-binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementary determining regions (CDR1, CDR2, and CDR3).

[0027] For a general antibody, examples of its antigen-binding portion include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv), which can be a single polypeptide chain composed of a VH domain and a VL domain via a peptide connector; and (5) (scFv)2, which can comprise two VH domains connected by a peptide connector and two VL domains, wherein the two VL domains are combined with the two VH domains via a disulfide bridge.

[0028] In some specific embodiments of the present application, the “antigen binding portion” includes but is not limited to a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment, a scFv fragment, a Fd fragment and a single domain antibody.

[0029] As used herein, the term "single-chain antibody (scFv)" refers to an antibody with a single-chain structure, typically constructed using genetic engineering techniques, comprising a single polypeptide chain consisting of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is typically designed between the heavy and light chain variable regions to facilitate folding of the heavy and light chain variable regions into the correct conformation for antigen binding.

[0030] As used herein, the term "Fab (fragment antigen binding) fragment", "Fab portion" or similar terms refers to an antibody fragment capable of binding to an antigen produced by treating an intact antibody with papain, comprising a complete light chain (VL-CL), a heavy chain variable region and a CH1 fragment (VH-CH1).

[0031] As used herein, the term "Fd fragment", "Fd portion" or similar terms refers 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 and refer to a portion of the constant region of an antibody heavy chain, including the hinge region, the CH2 fragment, and the CH3 fragment of the heavy chain constant region, and are determined with reference to the EU numbering of a human IgG1 antibody.

[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. FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity determining regions 1-3, respectively. "VHH" refers to the variable antigen-binding domain of a heavy chain antibody from the Camelidae family (camel, dromedary, llama, alpaca, etc.).

[0034] It is well known to those skilled in the art that the complementarity determining regions (CDRs, generally CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of an antibody. There are several common definitions of the CDR amino acid sequences of VH or VL, including the IMGT definition, the Kabat definition, the AbM definition, and the Chothia definition. 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. USA 86:9268-9272 (1989). In embodiments of the present application, the IMGT definition is used to determine the CDR amino acid sequences in the antibody VH and VL amino acid sequences.

[0035] For a given antibody variable region amino acid sequence, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0036] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0037] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor individuals.

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

[0039] The term "identity / homology / identity" with respect to amino acid or nucleic acid sequences is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment and introduction of gaps, if necessary, to achieve the maximum percentage identity. Methods and computer programs for alignment are well known in the art.

[0040] Tumor immunotherapy has become an important treatment for advanced malignancies. Tumor immunotherapy does not directly attack cancer cells, but instead activates the body's own immune system to fight tumors, resulting in a well-established safety and tolerability profile. Antibodies targeting programmed cell death receptor 1 (PD-1) and its ligands, as representative agents of tumor immunotherapy, have achieved significant success in treating advanced malignancies.

[0041] PD-1 is a type I transmembrane glycoprotein of approximately 55 kDa, belonging to the CD28 superfamily of receptors. It is primarily expressed on the surfaces of T cells, B lymphocytes, and activated macrophages. The PD-1 protein 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 autoimmune attack. However, PD-L1 is also expressed abundantly on tumor cells related to various solid tumors and some hematological malignancies, such as melanoma, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, lymphoma, and leukemia. PD-L1 on the tumor cell membrane binds to PD-1 on T cells, inhibiting T cell activation and thereby evading recognition and attack by the immune system, achieving immune escape. Related studies have found that PD-L1 expression on tumor cells is associated with poor prognosis in multiple tumor types. Therefore, blocking the binding of PD-1 to PD-L1 is a reasonable approach for tumor immunotherapy.

[0042] Toripalimab is a Class I PD-1 inhibitor primarily used in melanoma, nasopharyngeal carcinoma, and urothelial carcinoma. As Toripalimab's clinical application expands, developing an antibody that specifically recognizes and binds to Toripalimab to detect its blood levels and the production of anti-drug antibodies (ADAs) is crucial for guiding rational clinical medication use.

[0043] Based on the exploration of the above problems, after continuous exploration, this application provides the following implementation plan: In a first aspect, the present application provides an antibody or an antigen-binding portion 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 the HCDR1 comprises the sequence shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 comprises the sequence shown in SEQ ID NO: 4, the amino acid sequence of the HCDR3 comprises the sequence shown in SEQ ID NO: 6, the amino acid sequence of the LCDR1 comprises the sequence shown in SEQ ID NO: 10, the amino acid sequence of the LCDR2 comprises the sequence shown in SEQ ID NO: 12, and the amino acid sequence of the LCDR3 comprises the sequence shown in SEQ ID NO: 14.

[0044] In some embodiments of the first aspect, the antibody or antigen-binding portion thereof specifically binds to toripalimab or a fragment 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 antigen-binding portion thereof is an anti-idiotypic antibody that binds toripalimab. In more specific embodiments, the antibody or antigen-binding portion thereof can at least partially block the binding of toripalimab to PD-1.

[0045] 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 more sequence identity to SEQ ID NO: 8. Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8.

[0046] 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 more sequence identity to SEQ ID NO: 16. Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16.

[0047] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region may differ from the amino acid sequence shown in SEQ ID NO:8 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0048] 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 can 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 maintaining similar function of the heavy chain variable region.

[0049] 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 can 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 similar function of 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 or deleted in the region 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 similar function of the heavy chain variable region.

[0051] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region may differ from the amino acid sequence shown in SEQ ID NO: 16 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and / or additions.

[0052] 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 can 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 maintaining similar function of the light chain variable region.

[0053] 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 can 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 similar function of 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 or deleted in the region 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 the similar function of the light chain variable region.

[0055] In some embodiments of the first aspect, the antigen binding portion is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, or a single domain antibody.

[0056] In some embodiments of the first aspect, the antibody is selected from a murine antibody, a human antibody, and a humanized antibody. Preferably, the antibody is a murine antibody.

[0057] In some embodiments of the first aspect, the antibody may be a monoclonal antibody.

[0058] In some embodiments of the first aspect, the heavy chain of the antibody is of the IgG1 subtype, and the light chain of the antibody is a kappa chain.

[0059] In a second aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof according to the first aspect.

[0060] In preferred embodiments, nucleic acids as described herein can be codon-optimized nucleic acids suitable for expression in host cells. For example, based on the degeneracy of codons, they still encode the same protein. Methods for codon optimization according to the host cell used are well known to those skilled in the art.

[0061] In some embodiments of the second aspect, the nucleic acid molecule may include a DNA molecule and an RNA molecule. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0062] In some embodiments of the second aspect, the nucleic acid molecule is operably connected to a regulatory nucleotide sequence for expression in a host cell. In some embodiments, the nucleic acid molecule is operably connected to a regulatory nucleotide sequence, and the regulatory nucleotide sequence is contained in a vector (e.g., a plasmid) for expression in a host cell. The host cell can be from a mammal, such as a mouse, rat, dog, cat, sheep, cattle, monkey, camel, llama, mankind, etc. The host cell can be cultivated in a culture medium to express the antibody or its antigen-binding portion thereof, and then recovered to harvest the antibody or its antigen-binding portion thereof.

[0063] The present application provides a combination of polynucleotides, comprising a polynucleotide encoding the light chain of the antibody or antigen-binding portion thereof of the present application and a polynucleotide encoding the heavy chain of the antibody or antigen-binding portion thereof of the present application.

[0064] In a third aspect, the present application provides a vector comprising the nucleic acid molecule described in the second aspect.

[0065] In some embodiments of the third aspect, the vector is an expression vector. The vector (e.g., expression vector) may comprise the nucleic acid molecules or polynucleotide combinations described in the second aspect. In some embodiments, the expression vector of the present application comprises a combination of nucleic acid molecules or polynucleotides described herein, and the combination of nucleic acid molecules or polynucleotides is effectively connected to a regulatory sequence that allows the encoded polypeptide to be expressed in a host cell or a cell-free expression system. The selection of the expression vector depends on the selection of the host cell, and can be selected so as to have desired expression and control characteristics in the selected host cell.

[0066] An "expression vector" is a vector that includes one or more expression control sequences, which are DNA sequences that control and regulate the transcription and / or translation of another DNA sequence.

[0067] The nucleic acid in the vector can be operably connected to one or more expression control sequences. As used herein, "operably connected" means incorporated into a genetic construct so 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 composed of a region of a DNA molecule generally within 100 nucleotides upstream of the transcription start point (generally near the start site of RNA polymerase II). In order to place the coding sequence under the control of the promoter, the translation start site of the polypeptide translation reading frame must be positioned between 1 and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, position, and level. Unlike promoters, enhancers can work when located at different distances from the transcription 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 "operably connected" to the expression control sequence in the cell and is "under the control" of the expression control sequence.

[0068] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. 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).

[0069] The expression vector may include a tag sequence. The tag sequence is generally expressed as a fusion with the encoded polypeptide. Such tags can be inserted into 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, FlagTM tag (Kodak, New Haven, CT), maltose E binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the antibody of the present application is present in a vector containing a nucleic acid encoding one or more domains of the Ig heavy chain constant region, such as the amino acid sequence corresponding to the hinge region, CH2 region, and CH3 region of the human immunoglobulin Cγ1 chain (Fc fragment).

[0070] In a fourth aspect, the present application provides a host cell containing the nucleic acid molecule described in the second aspect or the vector described in the third aspect. In certain embodiments of the present application, the host cell can be a prokaryotic host cell, a eukaryotic host cell or a bacteriophage. The prokaryotic host cell can be Escherichia coli (e.g., Escherichia coli cells well known in the art such as DH5α, BL21), Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic host cell can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, mammalian cells such as HEK293 cells, BHK cells, CHO cells, COS cells, myeloma cells. In some embodiments, the host cell described in the present application is preferably a mammalian cell, more preferably HEK293 cells, BHK cells, CHO cells, NSO cells or COS cells.

[0071] In some embodiments, the present application also provides a method for preparing the antibody or antigen-binding portion thereof according to the first aspect, comprising: a) culturing the host cell according to the fourth aspect; and b) recovering the antibody or antigen-binding portion thereof from the host cell or the culture supernatant of the host cell.

[0072] In some embodiments, the present application also discloses a method for preparing an antibody that binds to Teplizumab, which may include: culturing a host cell under expression conditions to express an antibody that binds to Teplizumab; and isolating and purifying the expressed antibody that binds to Teplizumab. Using the above method, a crude antibody can be obtained. Purification methods, including affinity purification based on Teplizumab, non-denaturing gel purification, HPLC, or RP-HPLC, can then be used. Purify the antibody that binds to toripalimab to a substantially homogeneous material by HPLC, size exclusion, purification on a protein G column, or any combination of these techniques, for example, in an SDS-PAGE column. It showed a single band on PAGE electrophoresis.

[0073] In a fifth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof described in the first aspect.

[0074] In some embodiments of the fifth aspect, the detection reagent or kit is based on conventional antibody detection technology in the art, including but not limited to ELISA, Western Blotting or electrochemiluminescence technology.

[0075] In some embodiments, the detection reagent or kit detects Teplizumab or its anti-drug antibodies based on ELISA technology. For example, in addition to the antibody or antigen-binding portion thereof described in the first aspect, the detection reagent or kit may further comprise a solid phase carrier, a secondary antibody and / or a chromogenic substrate. In some specific embodiments, the antibody or antigen-binding portion thereof described in the first aspect can be coated on the solid phase carrier, for example, as a primary antibody, and after the primary antibody binds to Teplizumab, the binding status is determined by a secondary antibody and a chromogenic substrate. In other specific embodiments, the antibody or antigen-binding portion thereof described in the first aspect can be used, for example, as a positive control to determine the presence or concentration of Teplizumab anti-drug antibodies.

[0076] In some embodiments, the detection reagent or kit is based on Western blotting technology to detect toripalimab or its anti-drug antibody. For example, in addition to the antibody or antigen-binding portion thereof 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.

[0077] In some embodiments, the detection reagent or kit detects toripalimab or its anti-drug antibody based on electrochemiluminescence technology. For example, in addition to the antibody or antigen-binding portion thereof described in the first aspect, the detection reagent or kit may further include reagents and equipment required for electrochemiluminescence technology.

[0078] The antibodies or antigen-binding portions thereof described herein may be conjugated to a detectable moiety. Exemplary detectable moieties 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 moieties to be combined with the antibodies or antigen-binding portions thereof of the present application as needed to achieve different detection purposes.

[0079] In a sixth aspect, the present application provides use of the antibody or antigen-binding portion thereof described in the first aspect in detecting the concentration of toripalimab or the concentration of its anti-drug antibody in a biological sample of an individual.

[0080] 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. As used herein, "individual," "patient," and "subject" are used interchangeably.

[0081] In some embodiments of the sixth aspect, a "biological sample" refers to any sample obtained from an individual (e.g., a human or other animal), such as a human with cancer or a human suspected of having cancer, and that may contain toripalimab or its anti-drug antibodies. The biological sample can be a body fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from the scrotum (e.g., testicular ascites), vaginal washes, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, nipple discharge, aspirated fluid from various parts of the body (e.g., thyroid, breast), intraocular fluid (e.g., aqueous humor), etc.

[0082] Throughout the specification and claims, the words “comprises,” “comprising,” and “including” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0083] It will be appreciated that features, characteristics, components, or steps described in conjunction with a particular aspect, embodiment, or example of the present application may be applicable to any other aspect, embodiment, or example described herein unless incompatible therewith.

[0084] The foregoing disclosure generally describes the present application. The examples are provided to further illustrate the present application and should not be construed as limiting the present application. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in numerous publications, for example, see Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press.

[0085] Example

[0086] The experimental examples described below are illustrative and intended only to explain this application and are not to be construed as limiting this application. Where specific techniques or conditions are not specified in the experimental examples, the experiments were performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. For reagents or instruments used without manufacturer indication, all are commercially available conventional products.

[0087] Example 1. Preparation of Teplizumab Immunogen

[0088] refer to Figure 1 Understand the preparation of Tepliizumab (supplier, Jinan Maixiang Health Technology Co., Ltd.; brand, Junshi Biosciences) immunogen. Specifically, this example uses Pierce TM The F(ab')2 preparation kit (ThermoScientific, Cat:44988) selectively cleaves Teplizumab with pepsin immobilized on microbeaded agarose resin to obtain the Teplizumab F(ab')2 fragment used as an immunogen. Since resin-immobilized pepsin is used, the enzymatic cleavage reaction can be easily stopped by simply removing the resin from the solution and obtaining an enzyme-free cleavage product. TM Protein A spin column binds Fc fragments and undigested IgG, and after centrifugation, the flow-through containing only F(ab')2 fragments is obtained. TM Desalting centrifugal column was used to quickly prepare the F(ab')2 fragment of toripalimab.

[0089] Example 2. Preparation of Teplizumab-resistant hybridoma cell lines

[0090] Hybridoma cell lines resistant to Teplizumab can be prepared according to the following steps: 1. Emulsification of the immunogen. Dilute the toripalimab F(ab')2 fragment to 0.1 mg / mL in normal 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) or Freund's incomplete adjuvant (Sigma, F5505). Stir the mixture in an ice bath using an adjuvant emulsifier (BioAolong, BDYQ1001) for 3-5 minutes to fully emulsify the immunogen. Keep on ice until ready to use.

[0091] 2. Animal immunization. Prepare six 6-8 week old Balb / c mice and subcutaneously inject the emulsified toripalimab F(ab')2 fragment immunogen at multiple sites on the back. For example, administer 0.2 mL / site and 1 mL / mouse (approximately 50 μg / mouse). Immunize every 2-3 weeks for a total of 3-4 times until the serum antibody titer is positive.

[0092] 3. Cell fusion. Cell fusion can be performed as follows: 3.1. From mice that have completed the immunization process and whose serum antibody titers meet the requirements, collect blood from the orbital venous plexus. After standing at room temperature for 30 minutes, centrifuge at 3000g for 15 minutes to separate the serum. Store the serum at -80°C until use as a positive control.

[0093] 3.2. Euthanize mice and disinfect by soaking in 75% alcohol. Aseptically remove the mouse spleen in a clean bench and grind to release single splenocytes. Rinse the resulting cells with incomplete culture medium to prepare a single-cell suspension. After lysing red blood cells, filter the cell suspension through a 40 μm cell strainer. Centrifuge and wash the cells twice with incomplete culture medium. Remove a small number of cells and count them using a CountStar cell counter.

[0094] 3.3. Remove the previously 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.

[0095] 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 3:1-5:1 and fuse using a PEG / electrofusion instrument.

[0096] 3.5. The fused cells were gently resuspended in conditioned medium containing HAT supplement (Gibco, Cat: 21060-017) and Hybridoma Feeder supplement (Biolong, Cat: CM-2001) and evenly plated in 96-well plates for culture.

[0097] After 10-14 days, when cell colonies have grown to more than 1 / 10 of the well area in the 96-well plate, identify the cell culture supernatant by ELISA. Positive clones are screened and transferred to conditioned medium containing HT supplement (Gibco, Cat: 11067-030) for further culture.

[0098] 3.7. Perform 2-3 rounds of cloning until a positive monoclonal cell line is obtained.

[0099] Example 3. Selection of positive clones

[0100] 1. Coating: 1.1. Preparation of coating reagent working solution: Dilute toripalimab and human IgG with 1× PBS to a coating working solution with a concentration of approximately 1 μg / mL.

[0101] 1.2. Add the prepared coating solution to a 96-well plate at 100 μL / well and coat overnight at 2-8°C.

[0102] 2. Closed. Wash the plate three times with 1× PBST plate washer at a volume of at least 300 μL / well and pat dry on a clean tissue paper. Add 300 μL / well of blocking solution (1× PBST containing 1% (w / v) BSA) to the ELISA plate wells and block in a 37±5°C incubator for 120-130 minutes. Wash the plate three times with 350 μL / well of 1× PBST plate washer and pat dry on a clean tissue paper. Store excess plate in a sealed container at 2-8°C until ready for use.

[0103] 3. Sample preparation: 3.1. Dilution of mouse positive serum. Dilution was performed 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, i.e., 10-fold diluted mouse positive serum; (2) 40 μL of solution A was mixed with 3960 μL of diluent PBS to form solution B, i.e., 1000-fold diluted mouse positive serum.

[0104] 3.2. Preparation of hybridoma cell supernatant. Remove approximately 120 μL of supernatant from the 96-well cell culture plate where the clones were grown and use it as the hybridoma supernatant sample to be tested.

[0105] 4. Add sample. To an ELISA plate coated with toripalimab and human IgG, add Solution B and the hybridoma cell supernatant sample to be tested, 50 μL / well. Seal the plate with gentle shaking and incubate at 37°C for 60-70 minutes.

[0106] 5. Preparation of detection reagent working solution. The detection reagent used in this example is goat anti-mouse IgG (H+L) HRP. The working solution is prepared in two steps: (1) 5 μL of goat anti-mouse IgG (H+L) HRP is mixed with 495 μL of diluent PBS to form 500 μL of solution C, which is a 100-fold dilution of 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, which is a 20000-fold dilution of goat anti-mouse IgG (H+L) HRP with a concentration of 40 ng / mL.

[0107] 6. Add the assay working solution. Wash the plate three times with 1× PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the corresponding detection working solution to the ELISA plate and incubate in a dark oven at 37°C for 60-70 minutes.

[0108] 7. Color rendering. Wash the plate three times with 1× PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the chromogenic substrate TMB solution to the ELISA plate and incubate at room temperature in the dark for 15-20 minutes.

[0109] 8. Detect, analyze, and select positive clones. Add 1M H2SO4 (50 μL / well) to the ELISA plate and gently shake to ensure there is no orange-yellow coloration around the edges of the wells. Within 5 minutes, place the plate in a plate reader and analyze at a wavelength of 450-630 nm. Clones with an OD450 response greater than 1 in the toripalimab-coated plate and less than 0.3 in the human IgG-coated plate should be considered candidate positive clones for cloning and subsequent screening, or frozen for future use.

[0110] Example 4. Selection of positive monoclones

[0111] 1. Adjust cell density. The cells of the positive candidate main clone were counted and the cell density was adjusted to 1×10 3 ~1×10 5 / mL.

[0112] 2. Lay the boards. Approximately 130 positive primary clone cells were added to 6.5 mL of complete medium. At this point, the cell density was approximately 20 cells / mL. After mixing, 100 μL / well was added to the wells in rows A, B, and C of a 96-well plate, with an average of 2 cells per well. 2.9 mL of complete medium was added to the remaining 2.9 mL of cell suspension. At this point, the cell density was approximately 10 cells / mL. 100 μL / well was added to the wells in rows D, E, and F, with an average of 1 cell per well. 2.2 mL of complete medium was added to the remaining 2.2 mL of cell suspension. At this point, the cell density was approximately 5 cells / mL. 100 μL / well was added to the wells in rows G and H, with an average of 0.5 cells per well.

[0113] 3. Cultivation. After 4-5 days of culture, small clones can be seen growing under an inverted microscope; on the 8th-9th day, obvious clusters of clones can be seen growing under the microscope, and some can even be seen with the naked eye.

[0114] 4. Screening of positive monoclonal clones. Perform ELISA testing, select positive monoclonal clones based on the results, and then proceed with subsequent verification work.

[0115] Alternatively, 2-3 rounds of cloning can be performed until satisfactory positive monoclonal cells are screened.

[0116] Note: For the initial cloning of hybridoma cells, HT supplement (Gibco, Cat: 11067-030) needs to be added to the complete culture medium.

[0117] In this example, a positive monoclonal clone 9A6 was screened out.

[0118] Example 5. Positive monoclonal amplification and antibody purification

[0119] After expanding the 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 filtered through 0.45 μm and then 0.22 μm filters. The filtered cell supernatant was then applied to a pre-equilibrated Protein G affinity chromatography column. The column was washed with phosphate buffer until equilibrium, and then eluted with 50 mM citrate buffer (pH 3.0) at a flow rate of 5 mL / min. The entire elution peak was collected, and the eluate was finally 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 aliquoted for use.

[0120] Example 6. Verification of purified antibodies using SDS-PAGE

[0121] 1. Prepare samples. According to the quantitative concentration of the antibody, take out 5-10 μg of sample and add it to PBS. The total volume should not exceed 30 μL. Add loading buffer in proportion and treat in a boiling water bath for 5 minutes.

[0122] 2. Load the sample. Place the protein precast gel (GenScript, Cat: M00659) in the electrophoresis tank and fix it. Add electrophoresis buffer and load the processed samples in order.

[0123] 3. Electrophoresis. The power was turned on and electrophoresis was performed at a voltage of 150 V for 1 hour.

[0124] 4. Dyeing. After electrophoresis, the gel was removed, rinsed with deionized water, and then stained in 0.1% Coomassie Brilliant Blue solution for 1 hour.

[0125] 5. Discoloration. After staining, transfer the gel to a destaining solution for destaining. Change the destaining solution every 30 to 60 minutes until the protein bands on the gel are clearly visible.

[0126] The results are as follows Figure 2 As shown, lane N represents the 9A6 antibody under non-reducing conditions, and lane R represents the 9A6 antibody under reducing conditions.

[0127] Example 7. Identification of Antibody Subtypes

[0128] The Pierce Rapid ELISA Mouse Monoclonal Antibody Identification Kit (Invitrogen, Cat: 37503) was used to identify the subtype of the antibody.

[0129] 1. Prepare reagents: 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.

[0130] 2. Prepare samples for testing: For each sample to be tested, 450 μL should be prepared for subtype identification.

[0131] Hybridoma cell supernatant: The recommended dilution range is 1:10-1:100; in this example, 1:50 was used. To dilute, add 20 μL of hybridoma cell supernatant to 980 μL of 1× TBS.

[0132] Ascites: The recommended dilution range is 1:50,000-1:80,000; in this example, 1:75,000 was used. For dilution, first add 1 μL of ascites to 5 mL of 1× TBS, then add 67 μL of the dilution from the first step to 933 μL of 1× TBS.

[0133] Purified antibody: The recommended concentration range is 25 ng / mL-2 μg / mL; in this example, the purified antibody was diluted to 250 ng / mL using 1× TBS.

[0134] 3. Antibody typing: 3.1. Equilibrate the TMB substrate and capture antibody-coated strips to room temperature.

[0135] 3.2. Figure 3 As shown, for each sample, 50 μL of the diluted sample to be tested is added to each well (ie, 8 wells) in rows A-H of the corresponding column.

[0136] 3.3. Figure 3 Add 50 μL of goat anti-mouse (IgG / IgA / IgM)-HRP to each of the 8 wells in each column as shown. Gently tap the plate to mix.

[0137] 3.4. Seal the plate with sealing film and incubate at room temperature for 1 hour.

[0138] 3.5. Pour off the solution in the wells. Add 1× wash buffer to the wells, wash three times, and pat the wells dry on absorbent paper.

[0139] 3.6. Add 75 μL of TMB substrate equilibrated to room temperature to each well. Positive wells should turn blue after approximately 1 minute. The development time and color intensity vary depending on the antibody concentration and isotype.

[0140] 3.7. After 5-15 minutes, add 75 μL of stop solution to each well to terminate the reaction. The well color will immediately change from blue to yellow. The light intensity at a wavelength of 450 nm can be measured using a spectrophotometer or visually.

[0141] 3.8. Figure 3 As shown, the subtype of the antibody to be tested should be determined. For each sample, there should be one and only one positive well in wells AF (heavy chain typing), and one and only one positive well in wells GH (light chain typing). The results showed that the heavy chain of the 9A6 antibody was of the IgG1 subtype, and the light chain of the antibody was a kappa chain.

[0142] Example 8. Comparison of Binding Activity of Purified Antibodies to Toripalimab and Human IgG

[0143] 1. Coating: 1.1. Dilute Toripalimab and human IgG in 1× PBS to a coating working solution with a concentration of approximately 1 μg / mL.

[0144] 1.2. Add the prepared coating solution to a 96-well plate at 100 μL / well and coat overnight at 2-8°C.

[0145] 2. Closed. Wash the plate three times with 1× PBST plate washer at a volume of at least 300 μL / well and pat dry on a clean tissue paper. Add 300 μL / well of blocking solution (1× PBST containing 1% (w / v) BSA) to the ELISA plate wells and block in a 37±5°C incubator for 120-130 minutes. Wash the plate three times with 350 μL / well of 1× PBST plate washer and pat dry on a clean tissue paper. Store excess plates in sealed containers at 2-8°C until needed.

[0146] 3. Sample preparation: For mouse positive serum, dilute 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 a 10-fold 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 a 1000-fold diluted mouse positive serum.

[0147] For the purified antibodies, dilute them with PBS in a gradient of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000, mix well and set aside.

[0148] 4. Add sample. Solution F and serially diluted purified antibody samples were added to the ELISA plate coated with Teplizumab and human IgG, 100 μL / well. The plate was sealed with gentle shaking and incubated at 37°C for 60-70 minutes.

[0149] 5. Preparation of detection reagent working solution: The detection reagent used in this example is goat anti-mouse IgG (H+L) HRP. The working solution is prepared in two steps: (1) 5 μL of goat anti-mouse IgG (H+L) HRP is mixed with 495 μL of diluent PBS to form 500 μL of solution G, which is a 100-fold dilution of 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 a 20000-fold dilution of goat anti-mouse IgG (H+L) HRP with a concentration of 40 ng / mL.

[0150] 6. Add the assay working solution. Wash the plate three times with 1× PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the corresponding detection working solution to the ELISA plate and incubate in a dark oven at 37°C for 60-70 minutes.

[0151] 7. Color rendering. Wash the plate three times with 1× PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the chromogenic substrate TMB solution to the plate and incubate at room temperature in the dark for 15-20 minutes.

[0152] 8. Detect, analyze data, and screen for active antibodies. Add 1 M H2SO4 to the ELISA plate, 50 μL / well, and shake gently to ensure that there is no orange-yellow color at the edge of each well. Place the ELISA plate in a plate reader within 5 minutes for detection at a wavelength of 450-630 nm. The results are as follows: Figure 4 shown.

[0153] Example 9. Blocking activity of purified antibodies against toripalimab

[0154] 1. Coating

[0155] 1.1. Prepare coating reagent working solution: Dilute PD-1 (Bio-Techne, R&D SYSTEMS, Cat: 8986-PD-100) with 1× PBS to a concentration of approximately 1 μg / mL.

[0156] 1.2. Add the prepared coating solution to a 96-well plate at 100 μL / well and coat overnight at 2-8°C.

[0157] 2. Closed. Wash the plate three times with 1× PBST plate washer at a volume of at least 300 μL / well and pat dry on a clean tissue paper. Add blocking solution (1× PBST containing 1% (w / v) BSA) to the enzyme-labeled wells at a volume of 300 μL / well and block in a 37±5°C incubator for 120-130 minutes. Wash the plate three times with 1× PBST plate washer at a volume of 350 μL / well and pat dry on a clean tissue paper. Store excess plates in sealed containers at 2-8°C until use.

[0158] 3. Sample Preparation: Toripalimab was diluted to 1 μg / mL according to the following steps: (1) 2 μL of toripalimab (5 mg / mL) was mixed with 98 μL of diluent PBS to form 100 μL of solution I, i.e., 50-fold diluted toripalimab; (2) 90 μL of solution I was mixed with 8910 μL of diluent PBS to form solution J, i.e., 5000-fold diluted toripalimab (1 μg / mL).

[0159] For the purified 9A6 antibody, dilute it with PBS to 60, 30, 15, 7.5, 3.75, and 1.875 μg / mL, then remove 150 μL of each aliquot and mix it with 150 μL of toripalimab (1 μg / mL) for later use. At the same time, mix 150 μL of toripalimab (1 μg / mL) with 150 μL of PBS as a control. Incubate at 37°C for 60 minutes.

[0160] 4. Add sample. In the ELISA plate coated with PD-1, the pretreated samples were loaded into duplicate wells in sequence, 100 μL / well, sealed, shaken slightly, and incubated in a 37°C incubator for 60-70 minutes.

[0161] 5. Preparation of detection reagent working solution.The detection reagent used in this example is goat anti-mouse IgG (H+L) HRP. The working solution is prepared in two steps: (1) 5 μL of goat anti-mouse IgG (H+L) HRP is mixed with 495 μL of diluent PBS to form 500 μL of solution K, which is a 100-fold dilution of 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 a 20000-fold dilution of goat anti-mouse IgG (H+L) HRP with a concentration of 40 ng / mL.

[0162] 6. Add the assay working solution. Wash the plate three times with 1× PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the corresponding detection working solution to the ELISA plate and incubate in a dark oven at 37°C for 60-70 minutes.

[0163] 7. Color rendering. Wash the plate three times with 1× PBST at a volume of at least 300 μL / well and pat dry on a clean tissue. Add 100 μL / well of the chromogenic substrate TMB solution to the plate and incubate at room temperature in the dark for 15-20 minutes.

[0164] 8. Testing and Analysis. Add 1 M H2SO4 to the ELISA plate, 50 μL / well, and shake gently to ensure that there is no orange-yellow color at the edge of each well. Place the ELISA plate in a plate reader within 5 minutes for detection at a wavelength of 450-630 nm. The results are as follows: Figure 5 shown.

Claims

1. An antibody or antigen-binding portion 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 in, According to the IMGT definition, the amino acid sequence of the HCDR1 comprises the sequence shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 comprises the sequence shown in SEQ ID NO: 4, the amino acid sequence of the HCDR3 comprises the sequence shown in SEQ ID NO: 6, the amino acid sequence of the LCDR1 comprises the sequence shown in SEQ ID NO: 10, the amino acid sequence of the LCDR2 comprises the sequence shown in SEQ ID NO: 12, and the amino acid sequence of the LCDR3 comprises the sequence shown in SEQ ID NO:

14.

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

16.

3. The antibody or antigen-binding portion thereof 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 light chain variable region is shown in SEQ ID NO:

16.

4. The antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the antigen-binding portion is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment or a single domain antibody.

5. The antibody or antigen-binding portion thereof according to any one of claims 1 to 4, wherein the antibody is a murine antibody, a human antibody or a humanized antibody; and / or The heavy chain of the antibody is of IgG1 subtype, and the light chain of the antibody is a kappa chain.

6. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule according to claim 6, preferably an expression vector.

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

9. A detection reagent or kit comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 5.

10. The detection reagent or kit according to claim 9, which is based on ELISA, Western Blotting or electrochemiluminescence technology.

11. Use of the antibody or antigen-binding portion thereof according to any one of claims 1 to 5 for detecting the concentration of toripalimab or the concentration of its anti-drug antibody in a biological sample of an individual.

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