WO2023109785A1 - 一种用于检测血清中sTNFR2的抗体及试剂盒 - Google Patents

一种用于检测血清中sTNFR2的抗体及试剂盒 Download PDF

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WO2023109785A1
WO2023109785A1 PCT/CN2022/138525 CN2022138525W WO2023109785A1 WO 2023109785 A1 WO2023109785 A1 WO 2023109785A1 CN 2022138525 W CN2022138525 W CN 2022138525W WO 2023109785 A1 WO2023109785 A1 WO 2023109785A1
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antibody
antigen
antibodies
binding fragment
detection
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PCT/CN2022/138525
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English (en)
French (fr)
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赵晓峰
高珍娜
曹卓晓
唐任宏
任晋生
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山东先声生物制药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention relates to the fields of biotechnology and immunology, in particular to an anti-TNFR2 antibody for detecting serum sTNFR2, a kit and applications thereof.
  • Tumor necrosis factor TNF is an important immune-related cytokine, which is closely related to the programmed death of cells and the occurrence and development of various tumors, inflammation and autoimmune diseases.
  • the biological effect of TNF is realized through the mediation of TNFR receptor.
  • Studies have shown that when TNF binds to TNFR receptors on the cell membrane, TNFR1 and TNFR2 on the cell membrane fall off from the cell membrane under the action of TNF- ⁇ converting enzyme (TACE), forming soluble forms (sTNFR1 and sTNFR2) (Ermert M et al. Cytokine. 2003 May).
  • TACE TNF- ⁇ converting enzyme
  • sTNFR1 and sTNFR2 forming soluble forms
  • ELISA detection has the characteristics of simple sampling, good patient compliance, high sensitivity, and easy operation. It is convenient for multi-time point detection of the concentration of sTNFR2 in the sample and continuous monitoring of its level.
  • the present invention provides an antibody and a kit for detecting sTNFR2 in serum.
  • the invention prepares and obtains the anti-TNFR2 antibody and the kit thereof for detecting serum sTNFR2, and verifies the application of the TNFR2 detection antibody and the kit thereof.
  • the present invention includes an antibody or antigen-binding fragment that specifically binds TNFR2, wherein the antibody or antigen-binding fragment comprises a combination of heavy chain CDRs and light chain CDRs:
  • the heavy chain CDRs combination includes: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the following or are combined with the sequence Compared to sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:
  • the light chain CDRs combination includes: CDR1-VL, CDR2-VL and CDR3-VL, and the CDR1-VL, CDR2-VL and CDR3-VL have any sequence combination selected from the following or match the sequence combination Combinations of sequences with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions than:
  • Each of CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is encoded according to the prevailing analysis method of KABAT; preferably, the substitutions are conservative amino acid substitutions.
  • the antibody or antigen-binding fragment of the invention comprises a combination of heavy chain CDRs and light chain CDRs selected from the group consisting of: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5 , VH6+VL6, VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, or VH14+VL14, and combinations of said heavy and light chain CDRs Combinations of CDRs having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequences.
  • said antibody or antigen-binding fragment comprises:
  • the heavy chain variable region has a sequence shown in SEQ ID NO: 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98; the light chain variable region has The sequence shown in SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 or 112;
  • the framework region of the antibody or antigen-binding fragment has at least 90% identity with the framework region of the amino acid sequence shown in (1) above, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the dissociation constant (KD) of the antibody or antigen-binding fragment binding to human TNFR2 is no greater than 2 ⁇ 10 -9 M.
  • the antibody or antigen-binding fragment is: (1) chimeric antibody or fragment thereof; (2) humanized antibody or fragment thereof; (3) fully human antibody or fragment thereof; preferably Yes, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), monovalent antibodies, multivalent antibodies, Full-length antibody, antibody fragment, naked antibody, conjugated antibody, humanized antibody, fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody or single domain antibody .
  • the antibody comprises a sequence derived from any one of the constant regions of human, mouse, rat, goat or rabbit antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; It preferably comprises sequences derived from the constant regions of human, mouse, rat, goat or rabbit antibodies IgGl, IgG2, IgG3 or IgG4.
  • the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, cytotoxic agents or immunomodulators, and the tracer
  • the agent is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from alkaloids, methotrexate (methotrexate), anthracyclines (doxorubicin), taxanes (taxanes) or toxin compounds.
  • the present invention includes a detection reagent comprising the antibody or antigen-binding fragment of the first aspect above.
  • the detection reagent comprises a coating molecule that specifically binds to soluble TNFR2 (sTNFR2) protein and a detection molecule.
  • sTNFR2 soluble TNFR2
  • the coating molecule and the detection molecule respectively bind to different, non-overlapping epitopes on the sTNFR2 protein.
  • the coating molecule comprises the antibody or antigen-binding fragment described in the first aspect above; and/or the detection molecule comprises the antibody or antigen-binding fragment described in the first aspect above. Antigen-binding fragments.
  • the Fc fragment contained in the coating molecule and the Fc fragment contained in the detection molecule are from different species, preferably from human, mouse, rat, sheep or rabbit.
  • the present invention includes a detection kit, which comprises the antibody or antigen-binding fragment described in the first aspect above or the detection reagent described in the second aspect above.
  • the detection kit further includes an antibody labeled with a labeling substance.
  • the labeling substance is an enzyme; preferably, the enzyme is selected from horseradish peroxidase, alkaline phosphatase and ⁇ -galactosidase.
  • the present invention includes a method for detecting sTNFR2 protein concentration in vitro, which comprises the antibody or antigen-binding fragment described in the first aspect above, the detection reagent described in the second aspect above, or the third aspect described above
  • the detection kit described in the aspect detects the sample to be tested.
  • the antibody pairs used in the sTNFR2 detection kit in the present disclosure are all anti-TNFR2 monoclonal antibodies, which can ensure the stability of antibody batches.
  • the antibody pairs in this kit have been screened and can be used together/concomitantly with anti-TNFR2 monoclonal antibody drugs targeting different epitopes.
  • the blood samples against the test drug can accurately quantify the sTNFR2 concentration, and can be used for subsequent detection of serum sTNFR2 in clinical patients before and after receiving anti-TNFR2 antibody drug treatment.
  • Fig. 1A shows the comparison result of the serum soluble TNFR2 (sTNFR2) level between the administration group and the control group mice;
  • Fig. 1 B shows the association analysis of the serum soluble TNFR2 level of administration group mice and mouse CT26 tumor size
  • Figure 1C shows the correlation analysis between the serum soluble TNFR2 level of control group mice and the size of mouse CT26 tumors
  • Fig. 2 shows the anti-TNFR2 serum titer detection result of people's TNFR2 (His tag) or TNFR2 (mFc) protein immune Balb/c or SJL mice;
  • Figure 3 shows a schematic diagram of the detection principle of the human soluble TNFR2 ELISA kit
  • Figure 4 shows the standard curve fitting diagram of 7 concentration points of antibody pairing mode (1)#234(hFc)-#327(mFc);
  • Figure 5 shows the standard curve fitting diagram of 7 concentration points of antibody pairing mode (2)#489(hFc)-#327(mFc).
  • the invention discloses an antibody and a kit for detecting sTNFR2 in serum. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
  • the raw materials and reagents used in the antibody for detecting sTNFR2 in serum and the kit provided by the present invention can be purchased from the market.
  • TNFR2 refers to Tumor Necrosis Factor Receptor 2, also known as Tumor Necrosis Factor Receptor Superfamily Member 1B (TNFRSF1B) or CD120b, a membrane-binding tumor necrosis factor- ⁇ (TNF ⁇ ) receptor.
  • TNFRSF1B Tumor Necrosis Factor Receptor Superfamily Member 1B
  • CD120b a membrane-binding tumor necrosis factor- ⁇ receptor.
  • Said TNFR2 is preferably human TNFR2.
  • anti-tumor necrosis factor receptor 2 antibody refers to any immunoglobulin molecule comprising at least a part (such as but not limited to at least one complementarity determining region (CDR) of a heavy chain or a light chain or a ligand-binding portion thereof, a heavy chain or light chain variable region, heavy or light chain constant region, framework region or any portion thereof) protein or peptide-containing molecule.
  • CDR complementarity determining region
  • TNFR2 antibodies also include antibody-like protein scaffolds such as the tenth fibronectin type III domain (10Fn3), which contain BC, DE and FG structural loops similar in structure and solvent accessibility to antibody CDRs.
  • the tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and by replacing the residues of the BC, DE and FG loops of 10Fn3 with CDR-H1, CDR-H2 or CDR from TNFR2 monoclonal antibody - Residue replacement in the H3 region, those skilled in the art can graft the CDR of the TNFR2 monoclonal antibody onto the fibronectin scaffold.
  • kit comprises a kit comprising one or more components as defined herein, and the invention also provides kits particularly suitable for carrying out any of the methods defined herein.
  • antibody refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g. bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), antibody conjugates) As well as antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation by the method by which the antibody was produced.
  • the terms "antigen-binding fragment” and “antibody fragment” are interchangeable and refer to one or more antibody fragments that retain the ability to specifically bind a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • the antibody fragment may be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, Nanobody, aptamer or domain antibody.
  • binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment comprising two Fab fragments connected at the hinge region by disulfide bonds; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and VH domains consisting of a single arm of the antibody (V) dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546,1989); (vii) consisting of VH or VL (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which CDRs may optionally be joined by a synthetic linker.
  • a Fab fragment a mono
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be joined using recombinant methods through a linker that enables them to be made in which the VL and VH regions pair to form A single protein chain of a monovalent molecule (termed a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 ,1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as whole antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • CDRs complementarity determining regions
  • FR framework regions
  • amino acid positions representing the hypervariable regions of an antibody may vary according to the context and various definitions known in the art. Some positions within variable domains can be considered heterozygous hypervariable positions because these positions can be considered to be within a hypervariable region under one set of criteria (such as IMGT or KABAT) but under a different set Outside the hypervariable region under the standard (such as KABAT or IMGT). One or more of these positions may also be found in extended hypervariable regions.
  • variable domains of the native heavy and light chains each comprise four framework regions predominantly in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets , and in some cases form part of the lamellar structure.
  • CDR1, CDR2, and CDR3 CDR1, CDR2, and CDR3 that form loops connecting the sheets , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are held tightly together by the FR regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference).
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VH CDR combination) of the chain (or its variable region);
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and CDR of the light chain variable region (VL), respectively.
  • the third CDR, these three CDRs constitute the CDR combination (VL CDR combination) of the light chain (or its variable region).
  • Kabat numbering system or "KABAT” generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • IMGT numbering system or “IMGT” herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, e.g., Chothia & Lesk (1987) J . Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • VH refers to the variable region of an immunoglobulin heavy chain of an antibody, including that of an Fv, scFv or Fab.
  • VL refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv or Fab).
  • the term “heavy chain constant region” refers to the carboxy-terminal portion of an antibody's heavy chain, which is not directly involved in binding the antibody to antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to The variable domains of antibodies have more conserved amino acid sequences.
  • the "heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment”, the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • a typical "heavy chain constant region segment" can be selected from CH1, Fc or CH3 domains.
  • the term "light chain constant region” refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the carboxy-terminal portion of an antibody obtained by papain hydrolysis of an intact antibody, typically comprising the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the EUKabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, thus the Fc region can comprise or excluding Lys447.
  • percent (%) sequence identity and “percent (%) sequence identity” are interchangeable and refer to the sequence after aligning sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity ( For example, for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and non-homologous sequences may be ignored for comparison purposes), after the amino acid (or nucleotide) of the candidate sequence ) residues are identical to the amino acid (or nucleotide) residues of the reference sequence.
  • alignment can be achieved in a number of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAi) software.
  • a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% to 100% sequence identity.
  • the length of a candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence .
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, such as those detected by antibodies or their antigens Binding fragment-specific recognition.
  • An antibody or antigen-binding fragment thereof that specifically binds an antigen will bind the antigen with a KD of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds an antigen will bind the antigen with a KD of up to 100 nM (eg, between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a K for that particular antigen or epitope thereof of greater than 100 nM (e.g., greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM).
  • Antibodies that are specifically immunoreactive with a particular protein or carbohydrate can be selected using a variety of immunoassay formats. For example, solid-phase ELISA immunoassays are routinely used to select for antibodies that are specifically immunoreactive with proteins or carbohydrates.
  • multispecific antibody refers to an antibody having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different antigen. binding to different epitopes.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • the term “bispecific antibody” refers to an antibody, usually a human or humanized antibody, that has monoclonal binding specificities for at least two different antigens.
  • one of the binding specificities can be detected against an epitope of TNFR2 and the other can be detected against another epitope of TNFR2 or any other antigen, for example against a cell surface protein, receptor, receptor subunit , tissue-specific antigens, viral-derived proteins, viral-encoded envelope proteins, bacterial-derived proteins or bacterial surface proteins, etc.
  • chimeric antibody refers to an antibody that has variable sequences derived from immunoglobulins of one source organism (such as a rat or mouse) and immunoglobulins derived from a different organism (such as a human). The constant region of an immunoglobulin.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; incorporated by reference above This article.
  • antibody conjugate refers to a couple/conjugate formed by chemically bonding an antibody molecule to another molecule either directly or through a linker.
  • Said another molecule may be a therapeutic agent or a tracer; preferably said therapeutic agent is selected from radioisotopes, chemotherapeutics or immunomodulators, said tracer is selected from radiological contrast agents, paramagnetic ions, metal , fluorescent markers, chemiluminescent markers, ultrasound contrast agents or photosensitizers.
  • An “antibody conjugate” is for example an antibody-drug conjugate (ADC), wherein the drug molecule is said other molecule.
  • ADC antibody-drug conjugate
  • antigen chimeric receptor herein refers to an artificial immune effector cell surface receptor engineered to be expressed on an immune effector cell and specifically binds an antigen, comprising at least (1) an extracellular antigen binding domain, Examples include variable heavy or light chains of antibodies, (2) transmembrane domains that anchor CARs into immune effector cells, and (3) intracellular signaling domains. CARs are able to redirect T cells and other immune effector cells to a target of choice, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen-binding domain.
  • vector includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses or other suitable replicons (eg, viral vectors).
  • DNA vectors eg, plasmids
  • RNA vectors eg, viruses or other suitable replicons
  • viral vectors eg, viral vectors.
  • a variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
  • Expression vectors of the invention contain polynucleotide sequences together with additional sequence elements, eg, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells.
  • vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids that contain regulatory sequences, such as promoter and enhancer regions, that direct transcription of the gene.
  • Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA resulting from transcription of the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosomal entry sites (IRES), and polyadenylation signal sites to direct the efficient transcription of genes carried on the expression vector.
  • the expression vector of the present invention may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourthricin.
  • the terms "subject”, “subject” and “patient” refer to an organism receiving treatment for a particular disease or condition, such as cancer or an infectious disease, as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the bovid family (such as domestic cattle, bison, buffalo, elk, and yaks), cattle, sheep, horses, and bison.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology in the subject being treated, such as a cell proliferative disorder (such as cancer or infectious disease).
  • a cell proliferative disorder such as cancer or infectious disease.
  • Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • the term "diagnosis" is an in vitro method, preferably the individual to be tested is a human.
  • a diagnostic kit is provided, which refers to accurately quantifying the patient's blood sample by detecting the TNFR2 monoclonal antibody test drug that does not have an epitope competition relationship with the monoclonal antibody of the present disclosure. Serum sTNFR2 concentrations before and after receiving treatment.
  • mice were divided into two groups: the control group was given 10mpk isotype control antibody (Biolegend, catalog number 400967), and the experimental group was given 10mpk anti-mouse TNFR2 monoclonal antibody (BioXcell, catalog number BE0247).
  • the mouse serum was collected, and the soluble TNFR2 (mouse sTNFR2) in the mouse serum was detected with an ELISA kit (Abcam, Cat. No. ab202412).
  • Embodiment 2 human TNFR2 protein animal immunization
  • mice Female SJL mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) or Balb/c mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) aged 6-8 weeks were used for immunization.
  • 25 ⁇ g of human TNFR2 (his tag) purchased from Sino Biological, catalog number: 10417-H08H) or human TNFR2 (mFc tag) (purchased from Novoprotein, product number: C830) recombinant protein
  • the adjuvants for the last three immunization injections were incomplete freund's adjuvant (IFA, purchased from SIGMA, product number: F5506) and CpG ( Synthesized from Shanghai Sangon Biotechnology, product number: ODN1826), mixed with 25 ⁇ g of the same protein.
  • IFA incomplete freund's adjuvant
  • the foot pads and the back, and the third and fourth immunizations were injected subcutaneously and on the back of the tail to obtain high-titer, high-affinity, high-specificity antisera and specific immune cells.
  • human TNFR2 his tag
  • human TNFR2 mFc tag
  • anti-TNFR2 serum with high titer and high specificity was obtained.
  • the mice were euthanized and the spleen was aseptically removed, and the mouse spleen lymphocytes (Splenocytes) were aseptically isolated and frozen in liquid nitrogen.
  • the cells Resuscitate the cells when they need to be used later, sort the TNFR2-specific single B cells in the spleen or lymph nodes of the immunized mice into a 96-well plate with a BDAriaIII flow sorter, and reverse-transcribe the mRNA of the single cells into cDNA. Nested PCR was then performed using cDNA as a template to amplify the heavy and light chains of the antibody, respectively. The heavy chain variable region and the light chain variable region of the antibody are amplified, and cloned into the heavy chain expression vector and the light chain expression vector by homologous recombination method respectively. The constant regions of the heavy chain expression vector and the light chain expression vector were derived from human IgG1 and mouse IgG1, respectively.
  • the complete heavy chain expression sequence is signal peptide-VH-CH1-hinge region-CH2-CH3, and the complete light chain expression sequence is signal peptide-V ⁇ -CK.
  • the cloning and expression of the single B cell antibody described above are all performed in a 96-well plate in a high-throughput manner to achieve rapid identification and discovery of antibodies.
  • a total of 14 candidate antibody molecules were obtained after Octet screening for the affinity of the heavy and light chains of the cloned and expressed antibodies.
  • the CDRs of their sequences were analyzed by KABAT software.
  • the VH and VL sequences of the candidate antibody molecules, Table 2 shows the KABAT analysis results of the candidate antibody molecules.
  • Example 3 BIAcore detects the specificity of anti-TNFR2 antibody binding to human TNFR2-his protein
  • the ProteinA chip was used, and the time required for the chip to capture the diluted antibody was determined by manual operation (manual run), so that the saturated binding antigen Rmax was 50RU.
  • the human TNFR2 (his tag) protein was serially diluted to 32, 16, 8, 4 and 2nM.
  • the affinity of the antibody to the antigen was measured using multi-cycle kinetics. In each cycle, after the injection of the antibody, a gradient concentration of TNFR2 (his tag) protein is injected to cause the binding and dissociation process of the antigen and the antibody. ProteinA chips were regenerated with Glycine-HCl pH 1.5 after each cycle.
  • BIAcore T200 analysis software was used to fit the affinity KD value of antibody antigen. The BIAcore detection results are shown in Table 3. There is specific binding between the 14 anti-TNFR2 antibodies screened in Example 2 and the human TNFR2 (his tag) protein, and the affinity level is relatively high.
  • Embodiment 4 Octet detects the epitope binding of 14 anti-TNFR2 antibodies on human TNFR2 protein
  • Octet was used to detect the epitope binding of 14 anti-TNFR2 antibodies on human TNFR2 protein.
  • the In-tandem method was adopted, and the principle is as follows: the antigen human TNFR2 (his tag) was immobilized on the HIS1K sensor (Fortebio, catalog number: 18-5120) by capture, and then combined with the first antibody (Ab1) ( The combination of Ab1 and the sensor needs to be saturated), and then combined with the second antibody (Ab2). Whether Ab1 and Ab2 recognize the same epitope is determined by detecting the binding signal of Ab2.
  • HIS1K sensor was used to explore the loading concentration of 14 antibodies to reach saturation in the kinetic detection mode.
  • Experimental conditions for epitope binding After HIS1K sensor activation, equilibrate for 30s, immerse in 100nM human TNFR2 protein (his tag) solution, and immobilize human TNFR2 protein (his tag) antigen on it by capturing; equilibrate for 30s , combined with the first antibody (the saturation concentration explored in the pre-experiment, 200s); after 30s of equilibrium, combined with the second antibody (the saturation concentration explored in the pre-experiment, 200s), and finally the sensor was regenerated, and a new one could be performed. wheel cycle.
  • the ForteBio DataAnalysis 11.0 software was used to process the data and generate an array diagram. According to the percentage value, the epitope competition relationship between antibodies can be known, as shown in Table 4. It can be seen from Table 4 that the #234 antibody does not conflict with the remaining 13 antibodies on the epitope, and the #327 antibody does not conflict with the remaining 13 antibodies on the epitope. #489 and #631 overlap each other's epitopes, but do not conflict with the remaining 12 antibodies on epitopes. Antibodies with non-conflicting epitopes can be used for pairing, so there are a variety of antibody formats that can be paired.
  • Example 5 Octet detects the epitope competition relationship between 14 anti-TNFR2 antibodies and test drug antibody A and test drug antibody B on human TNFR2 protein
  • Test drug antibody A and test drug antibody B are both anti-TNFR2 antibodies.
  • the kit described in the present disclosure is intended to be used to detect the level of soluble TNFR2 in human serum samples containing test drug antibody A and test drug antibody B. Epitope competition relationship between A and test drug antibody B.
  • the experimental method is the same as in Example 4, and the results are shown in Table 5.
  • the ELISA antibodies that can be used to detect the ELISA containing test drug antibody A and test drug antibody B are #489, #234, #327, and #631, wherein #489 and #631 has epitope competition, and the KD value of #489 is better than #631, so #489, #234 and #3273 antibodies were selected for subsequent experiments.
  • the Fc of the above three antibodies are all human IgG1, which cannot be paired with sandwich ELISA antibodies.
  • the hFc of antibody #327 was transformed into mouse Fc. Combined with the epitope binding results of Example 5, the pairable forms of the above antibodies are shown in Table 6.
  • Test Drug Antibody A 5.2 8.8 4.8 116.5 99.5 8.5 103.1 12.3 102.2 7.2 98.9 117.9 88.4 71.0 Test Drug Antibody B 2.9 8.8 2.8 119.0 20.3 5.0 84.1 10.8 57.5 7.7 98.8 120.8 24.8 6.3
  • Example 6 3 antibodies were screened, and the Elisa detection kit was prepared by two antibody pairing methods, method (1): antibody #234 (hFc) was used as the coating antibody, and antibody #327 (mFc) was used as the detection antibody ; Mode (2): Antibody #489 (hFc) was used as the coating antibody, and antibody #327 (mFc) was used as the detection antibody.
  • Antibody pairing method (1) Antibody #234 (hFc) was used as the coating antibody, and antibody #327 (mFc) was used as the detection antibody.
  • the schematic diagram of the experiment principle is shown in Figure 3. The experimental steps are as follows: Dilute antibody #234 (hFc) with 50mM sodium carbonate-sodium bicarbonate buffer (pH9.5 ⁇ 9.6) to 1 ⁇ g/mL, add 100 ⁇ L/well to the microtiter plate (Hangzhou Shengyou, Cat. No. 1000096H) , overnight at 4°C. After washing the plate twice with washing solution (PBS-0.05% Tween20), add blocking solution (3% BSA-PBS) at 200 ⁇ L/well, and block at 37° C. for 1.0 hour.
  • Human TNFR2 protein (His tag) (purchased from Sino Biological, product number: 10417-H08H) was diluted with diluent (0.5% BSA-PBS-0.05% Tween20) to 4ng/mL, followed by 2.5-fold serial dilution, a total of 7 concentration points , and a zero concentration point;
  • Antibody #327 (mFc) was diluted to 2 ⁇ g/mL with diluent. After the blocked ELISA plate was washed twice with washing solution, 50 ⁇ L/well was added with gradiently diluted human TNFR2 protein (His tag), and then 2 ⁇ g/mL antibody #327 (mFc) was added at 50 ⁇ L/well. Incubate for 2.0 hours.
  • Fig. 4 is a standard curve fitting diagram of 7 concentration points. According to the Guideline on bioanalytical method validation, the accuracy range of each concentration point of the standard curve should be within the range of 85% to 115%, and the highest point and the lowest point can be appropriately relaxed.
  • the data in Table 7 shows that antibody #234 (hFc) is used as the coating antibody, antibody #327 (mFc) is used as the detection antibody, and the accurate quantitative range is 20.48-2000 pg/mL.
  • OD, optical density, light absorption value is the value given by the instrument after detection by the microplate reader.
  • Antibody pairing method (2) Antibody #489 (hFc) is used as the coating antibody, and antibody #327 (mFc) is used as the detection antibody.
  • the experimental method is the same as the above method (1).
  • Fig. 5 is a standard curve fitting diagram of 7 concentration points.
  • the data in Table 8 shows that antibody #489 (hFc) is used as the coating antibody, antibody #327 (mFc) is used as the detection antibody, and the accurate quantitative range is 20.48-2000 pg/ml.
  • OD, optical density, light absorption value is the value given by the instrument after detection by the microplate reader.
  • Detection value in the original sample detection mean value ⁇ dilution factor
  • the kit in 6..1 of Example 6 is intended to be used to detect the level of soluble TNFR2 in human serum containing anti-TNFR2 antibody drug, so it is necessary to investigate the influence of TNFR2 antibody in serum on the detection effect of the kit.
  • Estimation of anti-TNFR2 antibody drug level in serum Assuming that the administration dose is 10mg/kg, the body weight is calculated as 60kg, the blood volume is about 4.8L, and the residual amount of anti-TNFR2 antibody drug in blood sampling is 10%.
  • the concentration of TNFR2 antibody drug is roughly 12.5ug/mL (10mg/kg ⁇ 60kg ⁇ 10% ⁇ 4.8L).
  • the sTNFR2 concentration is 800pg/mL (the midpoint value of the kit’s quantitative range)
  • the addition recovery rate of 12.5ug/mL test drug antibody A and test drug antibody B was investigated.

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Abstract

提供一种用于检测血清中sTNFR2浓度的试剂盒。具体而言，公开了用于检测病人血清中sTNFR2浓度的抗TNFR2 抗体、检测试剂及试剂盒，具有灵敏度高、操作简便等特点，对肿瘤病程发展及病人愈后有重要的指示意义。

Description

一种用于检测血清中sTNFR2的抗体及试剂盒
本申请要求于2021年12月14日提交中国专利局、申请号为202111524331.1、发明名称为“一种用于检测血清中sTNFR2的抗体及试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术和免疫学领域，特别涉及检测血清sTNFR2的抗TNFR2抗体、试剂盒及它们的用途。
背景技术
肿瘤坏死因子TNF是重要的免疫相关细胞因子，与细胞的程序性死亡及多种肿瘤，炎症和自身免疫性疾病的发生发展密切相关。TNF的生物学作用通过TNFR受体的介导来实现。研究显示，当TNF与细胞膜上的TNFR受体结合后，在TNF-α转换酶(TACE)的作用下，细胞膜上的TNFR1和TNFR2从细胞膜上脱落，形成可溶性的形式(sTNFR1和sTNFR2)(Ermert M et al.Cytokine.2003May)。ELISA检测具有采样简单，患者依从性好，灵敏度高，操作简便等特点，便于对样本中sTNFR2的浓度展开多时间点检测并对其水平进行持续的监测。
发明内容
有鉴于此，本发明提供一种用于检测血清中sTNFR2的抗体及试剂盒。
为了实现上述发明目的，本发明提供以下技术方案：
本发明制备获得了检测血清sTNFR2的抗TNFR2抗体及其试剂盒，以及验证了所述TNFR2检测抗体及其试剂盒的用途。
在第一个方面，本发明包括一种特异性结合TNFR2的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链CDRs组合和轻链CDRs组合：
(1)所述重链CDRs组合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
Figure PCTCN2022138525-appb-000001
Figure PCTCN2022138525-appb-000002
和，
(2)所述轻链CDRs组合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
Figure PCTCN2022138525-appb-000003
各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为根据KABAT的通行分析方法编码；优选地，所述替换为保守氨基酸的替换。
在某些实施方案中，本发明所述的抗体或抗原结合片段包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、或VH14+VL14，以及与所述重链和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
在一个具体的实施方案中，所述的抗体或抗原结合片段包含：
(1)重链可变区具有SEQ ID NO:85、86、87、88、89、90、91、92、93、94、95、96、97或98所示序列；轻链可变区具有SEQ ID NO:99、100、101、102、103、104、105、106、107、108、109、110、111或112所示序列；
(2)与上述(1)所示序列具有至少90％同一性的氨基酸序列，优选为至少91％、92％、93％、94％、95％、96％、97％、98％、99％同一性；或，
(3)所述抗体或抗原结合片段的框架区与上述(1)所示氨基酸序列的框架区具有至少90％同一性，优选为至少91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
在另一个具体的实施方案中，所述的抗体或抗原结合片段与人TNFR2结合的解离常数(KD)不大于2×10 -9M。
在一个具体的实施方案中，所述抗体或抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；(3)全人源抗体或其片段；优选的，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人抗体、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
在一个具体的实施方案中，所述抗体包含来源于人、小鼠、大鼠、羊或兔抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含来源于人、小鼠、大鼠、羊或兔抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
在一个具体的实施方案中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、细胞毒性剂或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂；更优选地，所述细胞毒性剂选自生物碱类(alkaloids)、甲氨蝶呤(methotrexate)、蒽环类抗生素(doxorubicin)、紫杉烷类(taxanes)或毒素化合物。
在第二个方面，本发明包括一种检测试剂，所述的检测试剂包含上述第一个方面所述的抗体或抗原结合片段。
在某些实施方案中，所述的检测试剂包含能特异性结合可溶性TNFR2(sTNFR2)蛋白的包被分子和检测分子。
在一个具体的实施方案中，所述的包被分子和检测分子分别结合sTNFR2蛋白上不同的、非重叠的表位。
在一个具体的实施方案中，所述的包被分子为包含上述第一个方面所述的抗体或抗原结合片段；和/或所述的检测分子为包含上述第一个方面所述的抗体或抗原结合片段。
在一个具体的实施方案中，所述的包被分子包含的Fc片段与检测分子包含的Fc片段来自于不同种属，优选来源于人、小鼠、大鼠、羊或兔。
在第三个方面，本发明包含一种检测试剂盒，所述的检测试剂盒包含上述第一个方面所述的抗体或抗原结合片段或上述第二个方面所述的检测试剂。
在某些实施方案中，所述的检测试剂盒还包括用标记物质标记的抗体。
在一个具体的实施方案中，所述的标记物质是酶；优选的，其中所述的酶选自辣根过氧化物酶、碱性磷酸酶和β-半乳糖苷酶。
在第四个方面，本发明包含一种体外检测sTNFR2蛋白浓度的方法，其包含上述第一个方面所述的抗体或抗原结合片段、上述第二个方面所述的检测试剂或上述第三个方面所述的检测试剂盒对待测样品进行检测。
有益效果：本公开中的sTNFR2检测试剂盒所采用的抗体对均为抗TNFR2单抗，可保证抗体批间的稳定性。另外，本试剂盒中的抗体对已经过筛选，可与靶向抗原表位不同的抗TNFR2单抗药物共同/伴随使用，对于含有与本公开中单抗不存在表位竞争关系的抗TNFR2单抗受试药物的血液样本，均能准确地定量sTNFR2浓度，可以用于后续临床病人在接受抗TNFR2抗体药物治疗前后的血清sTNFR2检测。
附图说明
图1A示给药组与对照组小鼠的血清可溶性TNFR2(sTNFR2)水平对比结果；
图1B示给药组小鼠的血清可溶性TNFR2水平与小鼠CT26肿瘤大小的关联性分 析；
图1C示对照组小鼠的血清可溶性TNFR2水平与小鼠CT26肿瘤大小的关联性分析；
图2示人TNFR2(His tag)或TNFR2(mFc)蛋白免疫Balb/c或SJL小鼠的抗TNFR2血清滴度检测结果；
图3示人可溶性TNFR2ELISA试剂盒检测原理示意图；
图4示抗体配对方式(1)#234(hFc)-#327(mFc)7个浓度点的标准曲线拟合图；
图5示抗体配对方式(2)#489(hFc)-#327(mFc)7个浓度点的标准曲线拟合图。
具体实施方式
本发明公开了一种用于检测血清中sTNFR2的抗体及试剂盒，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的一种用于检测血清中sTNFR2的抗体及试剂盒中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
术语解释:
除非另外说明，本文所用术语具有所属技术领域普通技术人员通常理解的含义。对于本文中明确定义的术语，则该术语的含义以所述定义为准。
如本文所用，术语“TNFR2”是指肿瘤坏死因子受体2，也称为肿瘤坏死因子受体超家族成员1B(TNFRSF1B)或CD120b，这是一种结合肿瘤坏死因子-α(TNFα)的膜受体。所述TNFR2优选地是人TNFR2。
如本文所用，术语“抗-肿瘤坏死因子受体2抗体”、“肿瘤坏死因子受体2抗体”、“抗TNFR2抗体”、“TNFR2抗体”、“抗-TNFR2抗体部分”和/或“抗-TNFR2抗体片段”等是指任何包含能够特异性结合TNFR2的免疫球蛋白分子的至少一部分(例如但不限于重链或轻链的至少一个互补决定区(CDR)或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分)的含蛋白质或肽的分子。TNFR2抗体还包括抗体样蛋白支架(如第十纤连蛋白III型结构域(10Fn3))，其含有与抗体CDR在结构和溶剂可及性上相似的BC、DE和FG结构环。10Fn3结构域的三级结构类似于IgG重链可变区的三级结构，并且通过将10Fn3的BC、DE和FG环的残基用来自TNFR2单克隆抗体的CDR-H1、CDR-H2或CDR-H3区的残基替换，本领域技术人员可以将例如TNFR2单克隆抗体的CDR接枝到纤连蛋白支架上。
如本文所用，术语“试剂盒”包含本文所限定的一种或更多种组分的试剂盒，本发明还提供了特别地适于进行本文所限定的任何方法的试剂盒。
如本文所用，术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体)，抗体缀合物)以及抗体的抗原结 合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容援引加入本文)。
如本文所用，术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
如本文所用，术语“抗原结合片段”和“抗体片段”可互换，是指保留特异性结合靶抗原的能力的一个或更多个抗体片段。抗体的抗原结合功能可以由全长抗体的片段执行。抗体片段可以是Fab、F(ab’)2、scFv、SMIP、双抗体、三抗体、亲和体(affibody)、纳米抗体、适体或结构域抗体。涵盖术语抗体的“抗原结合片段”的结合片段的实例包括但不限于：(i)Fab片段，一种由VL、VH、CL和CHl结构域组成的单价片段；(ii)F(ab)2片段，一种包含由二硫键在铰链区连接的两个Fab片段的双价片段；(iii)由VH和CHl结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)包含VH和VL结构域的dAb；(vi)由VH结构域组成的dAb片段(Ward等人，Nature 341:544-546,1989)；(vii)由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)；以及(ix)两个或更多个分离的CDR的组合，所述CDR可以任选地由合成接头连接。此外，虽然Fv片段的两个结构域VL和VH是通过独立的基因编码的，但是这两个结构域可以使用重组方法通过接头接合，该接头能够使其制成其中VL和VH区配对以形成单价分子的单蛋白质链(称为单链Fv(scFv)；参见例如，Bird等人，Science 242:423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883,1988)。这些抗体片段可以使用本领域技术人员已知的常规技术获得，并且这些片段被筛选用于与完整抗体相同的方式使用。可以通过重组DNA技术、完整免疫球蛋白的酶促或化学裂解、或在一些实施方式中通过本领域已知的化学肽合成程序来产生抗原结合片段。
如本文所用，术语“互补决定区”(CDR)指在轻链和重链可变结构域中均发现的高变区。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的可高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的可高变区之外。这些位置中的一个或更多个也可以在延伸的可高变区中找到。本发明包括在这些杂合可高变的位置中包含修饰的抗体。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Proteins of Immunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分别是指重链可变区(VH)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了重链(或其可变区)的CDR组合(VH CDR组合)；CDR1-VL、CDR2-VL和CDR3-VL分别是指轻链可变区(VL)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构 成了轻链(或其可变区)的CDR组合(VL CDR组合)。
如本文所用，术语“Kabat编号系统”或“KABAT”通常是指由ElvinA.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“IMGT编号系统”或“IMGT”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
如本文所用，术语“VH”是指抗体的免疫球蛋白重链(包括Fv、scFv或Fab的重链)的可变区。术语“VL”是指免疫球蛋白轻链(包括Fv、scFv、dsFv或Fab的轻链)的可变区。
如本文所用，术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
如本文所用，术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
如本文所用，术语“Fc”是指完整抗体经木瓜蛋白水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据EUKabat编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
如本文所用，术语“百分比(％)序列一致性”和“百分比(％)序列同一性”可互换，是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分 子在那个位置是相同的。
如本文所用，术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM(例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow&Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow&Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件。
如本文所用，术语“多特异性抗体”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。术语“双特异性抗体”是指对至少两种不同的抗原具有单克隆结合特异性的抗体，其通常是人或人源化的抗体。在本发明中，结合特异性之一可以针对TNFR2的抗原表位而被检测，另一个可以针对TNFR2的另一个抗原表位或任何其他抗原，例如针对细胞表面蛋白、受体、受体亚基、组织特异性抗原、病毒来源蛋白、病毒编码的包膜蛋白、细菌来源蛋白或细菌表面蛋白等而被检测。
如本文所用，术语“嵌合抗体”是指以下抗体，其具有源自一种来源生物(如大鼠或小鼠)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985J Immunol Methods 125:191-202；以上通过援引加入并入本文。
如本文所用，术语“抗体缀合物”是指抗体分子直接或者通过连接接头与另一个分子化学键合而形成的偶联体/缀合物。所述另一个分子可以是治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。“抗体缀合物”例如抗体-药物缀合物(ADC)，其中药物分子就是所述的另一个分子。
本文术语“抗原嵌合受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工免疫效应细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
如本文所用，术语“载体”包括核酸载体，例如DNA载体(如质粒)，RNA载体，病毒或其他适合的复制子(例如病毒载体)。已经开发了多种载体用于将编码外源蛋白质的多核苷酸递送到原核或真核细胞中。本发明的表达载体含有多核苷酸序列以及例如用于表达蛋白质和/或将这些多核苷酸序列整合到哺乳动物细胞基因组中的附加序列元件。可以用于表达本发明的抗体和抗体片段的某些载体包括含有指导基因 转录的调控序列(如启动子和增强子区域)的质粒。用于表达抗体和抗体片段的其他有用的载体含有多核苷酸序列，其增强这些基因的翻译速率或改善由基因转录产生的mRNA的稳定性或核输出。这些序列元件包括例如5’和3’非翻译区、内部核糖体进入位点(IRES)和聚腺苷酸化信号位点，以便指导表达载体上携带的基因的有效转录。本发明的表达载体还可以含有以下多核苷酸，该多核苷酸编码用于选择含有这种载体的细胞的标记。适合的标记的实例包括编码抗生素(如氨苄青霉素、氯霉素、卡那霉素或诺尔丝菌素)抗性的基因。
如本文所用，术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、牛、绵羊、马和野牛等。
如本文所用，术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
如本文所用，术语“诊断”是一种体外方法，优选的待测个体是人。在本发明的实施方案中，提供了一种诊断试剂盒，是指通过检测患者的血液样本中与本公开的单抗不存在表位竞争关系的TNFR2单抗受试药物，从而准确地定量患者在接受治疗前后的血清sTNFR2浓度。
下面结合实施例，进一步阐述本发明：
备注：本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1 CT26同源移植小鼠模型(syngeneic model)中小鼠血清可溶性TNFR2(sTNFR2)的检测
实验小鼠分为2组：对照组给予10mpk同型对照抗体(Biolegend，货号400967)，实验组给予10mpk抗小鼠TNFR2单克隆抗体(BioXcell，货号BE0247)。实验结束后，采集小鼠血清，用ELISA试剂盒(Abcam，货号ab202412)对小鼠血清中可溶性TNFR2(mouse sTNFR2)进行检测。检测结果如图1A，给药组小鼠sTNFR2的水平显著低于对照组(n＝10，p＝0.0034，unpairedt test)。将sTNFR2水平与小鼠肿瘤大小进行相关性分析，如图1B所示，发现在给予抗小鼠TNFR2抗体的小鼠血清sTNFR2水平与肿瘤大小显著正相关(R＝0.9933，p<0.0001)，而在对照抗体组中无相关性，如图1C所示，说明在此模型中，血清sTNFR2水平可以作为抗鼠TNFR2抗体治疗后的预后生物标志物。
实施例2人TNFR2蛋白动物免疫
采用6～8周龄的雌性SJL小鼠(购自北京维通利华实验动物技术有限公司)或者Balb/c小鼠(购自上海斯莱克实验动物有限公司)进行免疫。第一次免疫注射时，用弗氏完全佐剂(complete freund’s adjuvant，CFA，购自SIGMA，货号：F5881)混合25μg人TNFR2(his tag)(购自Sino Biological，货号：10417-H08H)或人TNFR2(mFc tag)(购自Novoprotein，货号：C830)重组蛋白，后三次免疫注射的佐剂则采用弗氏不完全佐剂(incomplete freund’s adjuvant，IFA，购自SIGMA，货号：F5506)和CpG(合成自上海生工生物，货号：ODN1826)，混合25μg同样蛋白。第一次和第二次免疫注射后足垫和背部，第三次和第四次免疫注射尾部皮下及背部，以获得高滴度高亲和力高特异性的抗血清及特异性的免疫细胞。如图2所示，经人TNFR2(his tag)/人TNFR2(mFc tag)蛋白免疫后获得了高滴度高特异性的抗TNFR2血清。在末次免疫(第四次免疫)后的第7天，安乐死小鼠并无菌取出脾脏，无菌分离提取小鼠脾脏淋巴细胞(Splenocyte)，分装冻存于液氮中。在后面需要使用时重新复苏细胞，将免疫后小鼠的脾脏或淋巴结中的TNFR2特异的单个B细胞用BDAriaIII流式分选仪分选至96孔板中，将单细胞的mRNA反转录成cDNA。然后以cDNA为模板进行巢式PCR，分别进行抗体重链和轻链扩增。扩增得到抗体重链可变区和轻链可变区，分别通过同源重组方法克隆到重链表达载体和轻链表达载体。重链表达载体和轻链表达载体的恒定区分别来源于人IgG1和小鼠IgG1。完整重链表达序列是信号肽-VH-CH1-铰链区-CH2-CH3，完整轻链表达序列是信号肽-Vκ-Cκ。以上所述单B细胞抗体克隆和表达皆在96孔板内以高通量方式达到抗体的快速鉴定和发现。经过Octet对克隆表达的抗体重链和轻链亲和力筛选后共获得14个候选抗体分子，其序列的CDRs分别用KABAT软件分析，对应的序列信息如下表1至2所示，其中表1示出候选抗体分子的VH和VL序列，表2示出候选抗体分子的KABAT分析结果。
表1.候选抗体分子的VH和VL序列
Figure PCTCN2022138525-appb-000004
Figure PCTCN2022138525-appb-000005
Figure PCTCN2022138525-appb-000006
表2.KABAT分析候选抗体CDR区的结果
Figure PCTCN2022138525-appb-000007
Figure PCTCN2022138525-appb-000008
实施例3 BIAcore检测抗TNFR2抗体与人TNFR2-his蛋白结合的特异性
用BIAcore(型号T200)对筛选出来的14株抗体的精确KD值进行确认。
该实验采用ProteinA芯片，通过手动操作(manual run)测定出芯片捕获稀释后的抗体所需要的时间，以使得能饱和结合抗原Rmax为50RU。将人TNFR2(his tag)蛋白梯度稀释至32、16、8、4和2nM。采用多循环动力学测得抗体与抗原的亲和力。 在每一个循环中，注射抗体后再注入梯度浓度的TNFR2(his tag)蛋白，使抗原与抗体发生结合与解离过程。每个循环后用pH1.5的Glycine-HCl进行ProteinA芯片的再生。应用BIAcore T200分析软件拟合抗体抗原的亲和力KD值。BIAcore检测结果见表3，实施例2筛选出的14个抗TNFR2抗体与人TNFR2(his tag)蛋白之间存在特异性结合，且亲和力水平较高。
表3.Anti-TNFR2抗体与人TNFR2(his tag)蛋白特异性结合的BIAcore结果
Figure PCTCN2022138525-appb-000009
实施例4 Octet检测14个抗TNFR2抗体在人TNFR2蛋白上的表位结合
利用Octet检测14个抗TNFR2抗体在人TNFR2蛋白上的表位结合。本实验采用In-tandem的方法，其原理如下：抗原human TNFR2(his tag)通过捕获的方式固化于HIS1K传感器(Fortebio,货号：18-5120)上，然后与第一个抗体(Ab1)结合(Ab1与sensor的结合需要达到饱和)，再与第二个抗体(Ab2)结合。通过检测Ab2的结合信号来判定Ab1和Ab2是否识别同一表位。
正式实验前，在动力学检测模式下用HIS1K传感器摸索14个抗体达到饱和的loading浓度。表位结合的实验条件：HIS1K传感器活化后，经30s平衡，浸入100nM人TNFR2蛋白(his tag)溶液中，通过捕获的方式，将人TNFR2蛋白(his tag)抗原固化于其上；经过30s平衡，与第一个抗体结合(预实验摸索的饱和浓度，200s)；再经过30s平衡，与第二个抗体(预实验摸索的饱和浓度，200s)结合，最后传感器进行再生，又可进行新一轮的循环。应用ForteBio DataAnalysis 11.0软件对数据进行处理，生成列阵图，根据百分比值，可知抗体之间的表位竞争关系，如表4所示。从表4可知，#234抗体与其余13个抗体在表位上不冲突，#327抗体与其余13个抗体在表位上不冲突。#489和#631分别与彼此表位有重叠，但与其余12个抗体在表位上不冲突。表位不存在冲突的抗体都可用于配对，因此，可配对的抗体形式有多种。
表4.Octet检测14个anti-TNFR2抗体在人TNFR2蛋白上的表位结合结果
AB# #047 #160 #213 #234 #300 #327 #338 #454 #489 #508 #558 #627 #631 #670
#047 11.7 13.2 31.8 85.6 81.5 103.7 38.0 25.5 102.5 21.9 28.3 23.4 106.1 21.2
#160 12.9 13.8 22.6 78.2 39.7 99.3 18.9 27.7 102.3 21.9 29.1 24.0 104.4 22.5
#213 13.2 6.5 6.5 89.1 7.7 100.7 3.0 15.6 106.9 15.3 16.1 14.2 110.8 13.6
#234 89.5 87.4 95.2 8.9 101.5 99.8 95.4 95.7 100.1 95.6 91.0 92.8 105.4 101.6
#300 46.9 26.8 6.3 95.7 8.4 99.5 1.8 16.4 105.0 15.4 15.1 18.0 108.0 13.0
#327 96.6 95.1 98.6 99.3 100.8 13.0 94.9 97.3 99.5 91.7 94.5 98.2 100.8 94.8
#338 34.6 20.7 20.3 98.1 21.3 99.8 11.0 24.2 100.5 21.1 27.0 23.2 103.3 19.4
#454 14.6 14.4 18.2 94.2 22.5 99.2 12.6 6.1 112.7 2.9 7.0 5.9 109.7 2.7
#489 95.3 99.9 100.0 102.4 101.2 101.8 98.6 99.6 7.6 92.4 97.0 96.8 11.5 98.4
#508 24.7 24.9 32.0 97.9 32.6 97.6 23.6 22.9 95.7 14.0 24.8 21.2 99.5 15.3
#558 12.2 13.9 15.9 96.0 16.4 96.6 9.6 6.4 99.3 4.7 7.3 6.7 103.0 3.6
#627 17.7 16.3 22.2 88.8 29.0 97.0 14.7 12.7 95.1 7.5 13.5 10.5 99.5 7.4
#631 93.4 95.8 97.0 99.7 98.2 99.0 96.5 98.2 3.2 90.3 94.7 95.3 7.0 96.5
#670 26.7 27.0 33.3 97.6 34.5 96.2 23.4 23.3 95.1 15.0 25.5 20.9 100.2 13.1
备注：
11.7 表格中自反应信号值低于20％，证明本次实验有效。
85.6 表格中抗体与其他抗体反应的数值在60％及以上，表示完全不竞争。
实施例5 Octet检测14个抗TNFR2抗体分别与受试药物抗体A和受试药物抗体B在人TNFR2蛋白上的表位竞争关系
受试药物抗体A和受试药物抗体B(两者表位不同，为内部所研抗体)均为抗TNFR2抗体。本公开所述的试剂盒，拟用于检测含有受试药物抗体A和受试药物抗体B的人的血清样本中可溶性TNFR2的水平，因此，需要考察本公开候选抗体对分别与受试药物抗体A和受试药物抗体B的表位竞争关系。实验方法同实施例4，结果见表5，可共用于检测含受试药物抗体A和受试药物抗体B的ELISA用抗体为#489、#234、#327、和#631，其中#489和#631在表位上有竞争，且#489的KD值优于#631，故选择#489、#234和#3273个抗体进行后续实验。上述3个抗体的Fc均为人IgG1，无法进行夹心ELISA抗体的配对。将其中#327抗体的hFc改造为小鼠Fc。结合实施例5的表位结合结果，上述抗体的可配对形式如表6。
表5.Octet检测受试药物抗体A、B与14个Elisa试剂盒候选抗体在人TNFR2蛋白上的表位结合结果
AB# #047 #454 #160 #489 #213 #508 #234 #558 #300 #627 #327 #631 #338 #670
受试药物抗体A 5.2 8.8 4.8 116.5 99.5 8.5 103.1 12.3 102.2 7.2 98.9 117.9 88.4 71.0
受试药物抗体B 2.9 8.8 2.8 119.0 20.3 5.0 84.1 10.8 57.5 7.7 98.8 120.8 24.8 6.3
备注：489#与631#表位上是有竞争的。
表6.可用于检测含受试药物抗体A和受试药物抗体B样本的抗体配对形式
Figure PCTCN2022138525-appb-000010
实施例6可溶性TNFR2ELISA检测试剂盒制备
在实施例6中筛选到3个抗体，通过两种抗体配对方式进行Elisa检测试剂盒的制备，方式(1)：抗体#234(hFc)作为包被抗体，抗体#327(mFc)作为检测抗体；方式(2)：抗体#489(hFc)作为包被抗体，抗体#327(mFc)作为检测抗体。
6.1 ELISA检测试剂盒的制备
抗体配对方式(1)：抗体#234(hFc)作为包被抗体，抗体#327(mFc)作为检测抗体。实验原理示意图如图3所示。实验步骤如下：将抗体#234(hFc)用50mM 碳酸钠-碳酸氢钠缓冲液(pH9.5～9.6)稀释至1μg/mL，按100μL/孔加入酶标板(杭州生友，货号1000096H)中，4℃包被过夜。用洗液(PBS-0.05％Tween20)洗板2次后，按200μL/孔加入封闭液(3％BSA-PBS)，37℃封闭1.0小时。人TNFR2蛋白(His tag)(购自Sino Biological，货号：10417-H08H)用稀释液稀释(0.5％BSA-PBS-0.05％Tween20)至4ng/mL，后续2.5倍梯度稀释，共7个浓度点，以及一个零浓度点；抗体#327(mFc)用稀释液稀释至2μg/mL。封闭后的酶标板，用洗液洗涤2次后，按50μL/孔加入梯度稀释的人TNFR2蛋白(His tag)，随后按50μL/孔加入2μg/mL抗体#327(mFc)，400rpm室温共孵育2.0小时。洗板3次后加入1:10000稀释的酶标羊抗鼠IgG Fc(goat anti mouse IgG Fc-HRP,Jackson Immuno，货号115-035-071)，400rpm室温孵育1.0小时。洗板3次后，加入HRP底物TMB(Thermo，货号34029)进行显色，最后按50μL/孔加入终止液(1M H 2SO 4)终止反应。之后用酶标仪读取各孔吸光值，以人TNFR2蛋白(His tag)浓度为横坐标，OD450-OD630作为纵坐标，进行四参数拟合。图4为7个浓度点的标准曲线拟合图。根据指导原则Guideline on bioanalytical method validation，标准曲线各浓度点的准确度范围应在85％～115％范围内，对于最高点和最低点可适当放宽。表7数据表明，抗体#234(hFc)作为包被抗体，抗体#327(mFc)作为检测抗体，可准确定量的范围为20.48～2000pg/mL。
表7.标准曲线拟合数据(抗体#234(hFc)包被、抗体#327(mFc)检测)
Figure PCTCN2022138525-appb-000011
备注：
1.OD，optical density，光吸收值，为酶标仪检测后仪器给出的数值。
2.回算浓度：酶标仪软件SoftMax Pro 7.0通过标准曲线的理论浓度和OD值进行四参数拟合，得到四参数方程；再将OD值带入四参数方程，回算出实测浓度值。
3.准确度％＝平均回算值÷理论浓度值×100％。
抗体配对方式(2)：抗体#489(hFc)作为包被抗体，抗体#327(mFc)作为检 测抗体。实验方法与上述方式(1)相同。图5为7个浓度点的标准曲线拟合图。表8数据表明，抗体#489(hFc)作为包被抗体，抗体#327(mFc)作为检测抗体，可准确定量的范围为20.48～2000pg/ml。
表8.标准曲线拟合数据(抗体#489(hFc)包被、抗体#327(mFc)检测)
Figure PCTCN2022138525-appb-000012
备注：
1.OD，optical density，光吸收值，为酶标仪检测后仪器给出的数值。
2.回算浓度：酶标仪软件SoftMax Pro 7.0通过标准曲线的理论浓度和OD值进行四参数拟合，得到四参数方程；再将OD值带入四参数方程，回算出实测浓度值。
3.准确度％＝平均回算值÷理论浓度值×100％。
6.2健康人个体血清中可溶性TNFR2本底值检测
由于健康个体血清中天然存在一定量的可溶性TNFR2，因此需对血清基质本身的可溶性TNFR2本底值进行测定，确定大致的一个范围，以衡量实施例6.1中的试剂盒可定量范围是否能满足后续要求。选取41个健康个体(上海妙通，货号HSER-10ML)的血清，用商品化的soluble TNFR2试剂盒(R&D，货号SRT200)按说明书进行检测，血清样本稀释10倍后进行测定，测定结果见表9。从表9结果可知，健康个体血清中存在较高的可溶性TNFR2水平，41个个体的均值为2415.3pg/mL，范围为1181.4～5067.5pg/mL，经适当稀释后均落在我们已开发的试剂盒的可准确定量的范围内。因此，实施例6.1中的2个ELISA试剂盒的定量范围能满足检测要求。
表9. 41个健康个体血清中可溶性TNFR2本底值检测结果
Figure PCTCN2022138525-appb-000013
Figure PCTCN2022138525-appb-000014
备注：1.F代表Female,M代表Male。此样本中女性血清20个，男性血清21个。
2.原始样品中的检测值＝检测均值×稀释倍数
6.3血清样本中存在的抗TNFR2抗体对ELISA试剂盒检测的影响
实施例6的6..1中的试剂盒拟用于检测含抗TNFR2抗体药物的人的血清中可溶性TNFR2的水平，因此需考察血清中TNFR2抗体对试剂盒检测效果的影响。血清中抗TNFR2抗体药物水平的推算：假定给药剂量为10mg/kg，体重按60kg计算、其血液量约为4.8L,采血时抗TNFR2抗体药物的残留量为10％，此时血样中抗TNFR2抗体药物的浓度大致为12.5ug/mL(10mg/kg×60kg×10％÷4.8L)。实施例6.1中2种抗体配对方式，均在sTNFR2浓度为800pg/mL(试剂盒定量范围中点值)时考察12.5ug/mL受试药物抗体A和受试药物抗体B的添加回收率。
实验结果见表10和表11，在sTNFR2浓度为800pg/mL时，12.5ug/mL受试药物抗体A的存在，抗体配对方式1能获得较好的回收率，12.5ug/mL受试药物抗体A的存在对检测干扰较小(回收率越接近100％越好，即干扰越小)，在可接受范围内，能满足检测需求。12.5ug/mL受试药物抗体B存在时，方式1和2添加回收率均不理想；12.5ug/mL受试药物抗体B的存在对检测干扰较大。
表10.抗体配对方式(1)(234hFc-327mFc)添加回收率结果
Figure PCTCN2022138525-appb-000015
备注：1.添加回收率％＝实测值/添加理论值×100％。
2.#1和#2代表2次重复实验。
表11.抗体配对方式(2)(489hFc-327mFc)添加回收率结果
Figure PCTCN2022138525-appb-000016
Figure PCTCN2022138525-appb-000017
备注：1.添加回收率％＝实测值/添加理论值×100％。
2.#1和#2代表2次重复实验。
以上对本发明所提供的一种用于检测血清中sTNFR2浓度的试剂盒进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
序列表
Figure PCTCN2022138525-appb-000018
Figure PCTCN2022138525-appb-000019
Figure PCTCN2022138525-appb-000020
Figure PCTCN2022138525-appb-000021
Figure PCTCN2022138525-appb-000022
Figure PCTCN2022138525-appb-000023
Figure PCTCN2022138525-appb-000024
Figure PCTCN2022138525-appb-000025
Figure PCTCN2022138525-appb-000026
Figure PCTCN2022138525-appb-000027
Figure PCTCN2022138525-appb-000028
Figure PCTCN2022138525-appb-000029
Figure PCTCN2022138525-appb-000030
Figure PCTCN2022138525-appb-000031
Figure PCTCN2022138525-appb-000032
Figure PCTCN2022138525-appb-000033
Figure PCTCN2022138525-appb-000034
Figure PCTCN2022138525-appb-000035
Figure PCTCN2022138525-appb-000036
Figure PCTCN2022138525-appb-000037
Figure PCTCN2022138525-appb-000038
Figure PCTCN2022138525-appb-000039
Figure PCTCN2022138525-appb-000040

Claims (16)

  1. 特异性结合TNFR2的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链CDRs组合和轻链CDRs组合：
    (1)所述重链CDRs组合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    Figure PCTCN2022138525-appb-100001
    和，
    (2)所述轻链CDRs组合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    Figure PCTCN2022138525-appb-100002
    各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为根据KABAT的通行分析方法编码；
    优选地，所述替换为保守氨基酸的替换。
  2. 如权利要求1所述的抗体或抗原结合片段，其特征在于，其包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、或VH14+VL14，以及与所述重链和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
  3. 如权利要求1～2任一项所述的抗体或抗原结合片段，其特征在于，所述的抗体或抗原结合片段包含：
    (1)重链可变区具有SEQ ID NO:85、86、87、88、89、90、91、92、93、94、95、96、97或98所示序列；轻链可变区具有SEQ ID NO:99、100、101、102、103、104、105、106、107、108、109、110、111或112所示序列；或
    (2)与上述(1)所示序列具有至少90％同一性的氨基酸序列，优选为至少91％、 92％、93％、94％、95％、96％、97％、98％、99％同一性；或
    (3)所述抗体或抗原结合片段的框架区与上述(1)所示氨基酸序列的框架区具有至少90％同一性，优选为至少91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
  4. 如权利要求1～3任一项所述的抗体或抗原结合片段，其特征在于，其与人TNFR2结合的解离常数(KD)不大于2×10 -9M。
  5. 如权利要求1～4任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含：
    (1)嵌合抗体或其片段；或
    (2)人源化抗体或其片段；或
    (3)全人源抗体或其片段；
    优选的，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人抗体、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
  6. 如权利要求1～5任一项所述的抗体或抗原结合片段，其特征在于，所述抗体包含来源于人、小鼠、大鼠、羊或兔抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含来源于人、小鼠、大鼠、羊或兔抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
  7. 如权利要求1～6任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、细胞毒性剂或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂；更优选地，所述细胞毒性剂选自生物碱类(alkaloids)、甲氨蝶呤(methotrexate)、蒽环类抗生素(doxorubicin)、紫杉烷类(taxanes)或毒素化合物。
  8. 检测试剂，其特征在于，所述检测试剂包含如权利要求1～7任一项所述的抗体或抗原结合片段。
  9. 如权利要求8所述的检测试剂，其特征在于，所述的检测试剂包含能特异性结合可溶性TNFR2(sTNFR2)蛋白的包被分子和检测分子。
  10. 如权利要求9所述的检测试剂，其特征在于，所述的包被分子和检测分子分别结合sTNFR2蛋白上不同的、非重叠的表位。
  11. 如权利要求10的检测试剂，其特征在于，所述的包被分子为包含如权利要求1～7任一项所述的抗体或抗原结合片段；和/或所述的检测分子为包含如权利要求1～7任一项所述的抗体或抗原结合片段。
  12. 如权利要求11的检测试剂，其特征在于，所述的包被分子包含的Fc片段与检测分子包含的Fc片段来自于不同种属，优选来源于人、小鼠、大鼠、羊或兔。
  13. 检测试剂盒，其特征在于，所述的检测试剂盒包含如权利要求1～7任一项所述的抗体或抗原结合片段或如权利要求8～12任一项所述的检测试剂。
  14. 如权利要求13的检测试剂盒，其特征在于，所述的检测试剂盒还包括用标记物质标记的抗体。
  15. 如权利要求14的检测试剂盒，其特征在于，所述的标记物质是酶；
    优选的，其中所述的酶选自辣根过氧化物酶、碱性磷酸酶和β-半乳糖苷酶。
  16. 体外检测sTNFR2蛋白浓度的方法，其包括使用以下任意项对待测样品进行检测；
    I、如权利要求1～7任一项所述的抗体或抗原结合片段；或
    II、如权利要求8～12任一项所述的检测试剂；或
    III、如权利要求13～15任一项所述的检测试剂盒。
PCT/CN2022/138525 2021-12-14 2022-12-13 一种用于检测血清中sTNFR2的抗体及试剂盒 WO2023109785A1 (zh)

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