WO2022117079A1 - 结合胸腺基质淋巴细胞生成素的抗体及其应用 - Google Patents

结合胸腺基质淋巴细胞生成素的抗体及其应用 Download PDF

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WO2022117079A1
WO2022117079A1 PCT/CN2021/135410 CN2021135410W WO2022117079A1 WO 2022117079 A1 WO2022117079 A1 WO 2022117079A1 CN 2021135410 W CN2021135410 W CN 2021135410W WO 2022117079 A1 WO2022117079 A1 WO 2022117079A1
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seq
antibody
tslp
amino acid
heavy chain
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French (fr)
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应华
石金平
胡齐悦
黎婷婷
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

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  • the present disclosure relates to the field of antibody drugs, in particular, the present disclosure relates to anti-TSLP antibody drugs and applications thereof.
  • Asthma is a serious chronic inflammatory disease of the airways, and there are approximately 334 million asthma patients worldwide. As the environment deteriorates and air pollution increases, more people may suffer from this disease, which seriously endangers human life and health.
  • TSLP is an interleukin-7 (IL-7)-like cytokine first discovered in the conditioned medium of thymic stromal cells in mice.
  • TSLP is mainly expressed in lung, skin and intestinal epithelial cells.
  • TSLP is composed of four ⁇ -helices and two loops of AB and CD. There are three pairs of disulfide bonds composed of six cysteines in the molecule, two N glycosylation sites, and the molecular weight is about 15-20kD.
  • the receptor of TSLP is a complex, including two parts, one part is TSLPR and the other part is IL7R ⁇ . TSLP first binds to TSLPR with relatively low affinity, and then recruits IL7R ⁇ binding with high affinity, and finally activates signaling pathways such as stat5, leading to the maturation of DCs and the differentiation of T cells.
  • Bone marrow-derived dendritic cells are the main effector cells of TSLP.
  • TSLP acts on immature mDCs, and mDCs secrete cytokines IL-8, eotaxin-2, TARC and MDC, and at the same time highly express OX40L.
  • OX40L binds to natural CD4 + T cells to differentiate into Th2 cells, which in turn secrete Th2 cells such as IL-5, IL-4, IL-9, IL-13 and TNF Factors that induce the body's Th2 inflammatory response.
  • TSLP can also induce DC cells to produce the cytokine IL-8, which in turn recruits neutrophils, leading to neutrophil innate immune inflammation.
  • TSLP can also induce DC to produce eotaxin-2, eotaxin-2 recruits eosinophils, and works together with IL5 to rapidly enter the inflammatory state of eosinophil infiltration.
  • TSLP also acts on mast cells and natural killer cells, and mediates natural inflammation by inducing the production of IL-4, IL-6, and IgE.
  • TSLP can cause both natural inflammation and Th2 inflammation, which in turn increases tissue mucus, airway remodeling leads to tracheal stenosis, and severe cellular fibrosis, which gradually evolves into three major allergic diseases, including asthma, allergic dermatitis, and allergic rhinitis. disease. Therefore, blocking TSLP is a potentially effective strategy for the treatment of diseases such as asthma and atopic dermatitis.
  • WO2008155365, WO2009035577, WO2011056772, WO2016142426, WO2017004149 disclose anti-TSLP antibodies, but there is no corresponding antibody on the market. Therefore, it is necessary to continue to develop effective drugs for the treatment of TSLP-related diseases.
  • the present disclosure provides an anti-TSLP antibody.
  • the anti-TSLP antibody as previously described comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region amino acid sequences are HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 19, respectively LCDR1, LCDR2 and LCDR3 shown in NO:20, SEQ ID NO:21 and SEQ ID NO:22;
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 24, respectively LCDR1, LCDR2 and LCDR3 shown in ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22;
  • variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID NO: 25, respectively, and the variable region of the light chain comprises the amino acid sequences shown in SEQ ID NO: 25, respectively LCDR1, LCDR2 and LCDR3 shown in NO:20, SEQ ID NO:21 and SEQ ID NO:22.
  • the anti-TSLP antibody is as previously described, wherein the anti-TSLP antibody is a murine, chimeric, or humanized antibody.
  • the anti-TSLP antibody as previously described comprises a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14, or with SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14
  • the sequences shown have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and the amino acid sequence of the light chain variable region is as SEQ ID NO: 6, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:6.
  • the anti-TSLP antibody as previously described comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; and an amino acid sequence as set forth in SEQ ID NO: 14; The light chain variable region shown in ID NO:6.
  • the anti-TSLP antibody as previously described comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:13; and a light chain variable region having an amino acid sequence set forth in SEQ ID NO:6 .
  • an anti-TSLP antibody as previously described, wherein the antibody has a light chain variable region and heavy chain variable region combination as follows:
  • the anti-TSLP antibody is as previously described, wherein the antibody further comprises an antibody constant region.
  • the anti-TSLP antibody as previously described has a heavy chain constant region selected from human IgGl, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, and a light chain constant region of the antibody selected from Human antibody kappa and lambda chain constant regions and conventional variants thereof.
  • the anti-TSLP antibody as previously described, wherein the anti-TSLP antibody comprises a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO:34; and/or an amino acid sequence as set forth in SEQ ID NO:35 light chain constant region shown.
  • the anti-TSLP antibody as previously described, wherein the anti-TSLP antibody comprises a heavy chain and a light chain, wherein:
  • the heavy chain amino acid sequence is as set forth in SEQ ID NO:36, SEQ ID NO:38 or SEQ ID NO:39, or has at least the same sequence as set forth in SEQ ID NO:36, SEQ ID NO:38 or SEQ ID NO:39 85% sequence identity;
  • the light chain amino acid sequence is set forth in SEQ ID NO:37, or has at least 85% sequence identity to SEQ ID NO:37.
  • Said at least 85% sequence identity includes but is not limited to 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% % sequence identity.
  • the anti-TSLP antibody as previously described has one or more of the following properties:
  • a) binds to human TSLP with a KD value of less than 9 pM
  • the anti-TSLP antibody as previously described binds to human TSLP with a KD value of less than 9 pM, less than 5 pM, or less than 3 pM.
  • the anti-TSLP antibody as previously described binds cynomolgus monkey TSLP with a KD value of less than 9 pM, less than 5 pM, less than 3 pM, or less than 2 pM.
  • the KD value can be detected using the Biacore method. In some embodiments, wherein the KD value can be measured with reference to the method described in Test Example 1.
  • the anti-TSLP antibody as previously described blocks the binding of TSLP to TSLPR with an IC50 value of less than 0.6 nM, less than 4 nM, or less than 2 nM.
  • the IC50 value can be measured by an ELISA assay. In some embodiments, the IC50 value can also be detected with reference to Test Example 2 of the present disclosure.
  • the anti-TSLP antibody as previously described can inhibit TSLP-induced differentiation of naive CD4 + T cells into Th2 cells.
  • the present disclosure also provides nucleic acid molecules encoding the anti-TSLP antibodies as described above.
  • the present disclosure also provides expression vectors comprising the nucleic acid molecules as previously described.
  • the present disclosure also provides a host cell comprising the nucleic acid molecule as described above or the expression vector as described above; preferably, wherein the host cell is a bacterium, a fungal cell, an insect animal cell or a mammalian cell .
  • the mammal includes, but is not limited to, 293, CHO cells; in some embodiments, wherein the mammalian cell is not a human embryonic cell.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of an anti-TSLP antibody as described above, or a nucleic acid molecule as described above, or a host cell as described above, and a One or more pharmaceutically acceptable carriers, diluents, buffers or excipients; preferably, the therapeutically effective amount is a unit dose of the composition containing 0.1-3000 mg or 1-1000 mg as previously described Anti-TSLP antibody.
  • the present disclosure provides a method of making a TSLP antibody as previously described.
  • the present disclosure provides a method for in vitro or ex vivo immunodetection or determination of TSLP, the method comprising the step of using an anti-TSLP antibody as previously described.
  • the present disclosure provides the use of an anti-TSLP antibody as previously described in the preparation of a reagent for the immunodetection of human TSLP.
  • the present disclosure provides an anti-TSLP antibody as previously described for use in the immunodetection or determination of TSLP.
  • the present disclosure provides a kit comprising an anti-TSLP antibody as previously described.
  • the present disclosure provides an anti-TSLP antibody as described above, or a nucleic acid molecule as described above, or a host cell as described above, or a pharmaceutical composition as described above, for use in the preparation of :
  • the aforementioned TSLP-related inflammatory conditions include, but are not limited to, allergy, dermatitis, asthma, allergic conjunctivitis, allergic rhinitis, and allergic sinusitis.
  • the aforementioned TSLP-related fibrotic disorders include, but are not limited to, scleroderma, interstitial lung disease, idiopathic pulmonary fibrosis, fibrosis due to chronic hepatitis B or C, radiation-induced fibrosis and wound healing-induced fibrosis.
  • the present disclosure provides an anti-TSLP antibody as described above, or a nucleic acid molecule as described above, or a host cell as described above, or a pharmaceutical composition as described above, prepared for use in therapy Use in the medicament of an inflammatory disease or a fibrotic disease.
  • the present disclosure provides a method of treating an inflammatory disease or a fibrotic disease, the method comprising administering to a subject a therapeutically effective amount of an anti-TSLP antibody as hereinbefore described, or a nucleic acid as hereinbefore described A molecule, or a host cell as described above or a pharmaceutical composition as described above.
  • the present disclosure provides an anti-TSLP antibody as described above, or a nucleic acid molecule as described above, or a host cell as described above, for use as a medicament. In some embodiments, wherein the medicament is used to treat an inflammatory disease or a fibrotic disease.
  • the aforementioned inflammatory diseases include, but are not limited to, allergy, dermatitis, asthma, allergic conjunctivitis, allergic rhinitis, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungi Sinusitis, chronic eosinophilic pneumonia, eosinophilic bronchitis, inflammatory bowel disease, and allergic sinusitis.
  • the aforementioned fibrotic diseases include, but are not limited to, scleroderma, interstitial lung disease, idiopathic pulmonary fibrosis, fibrosis due to chronic hepatitis B or C, radiation-induced fibrosis, and wounds Healing-induced fibrosis.
  • the aforementioned inflammatory disease or fibrotic disease is associated with TSLP.
  • Figure 1 shows the activity of the antibody to inhibit the production of Th2-related cytokine IL-13;
  • Figure 1B shows the activity of the antibody to inhibit the production of Th2-related cytokine IL-4;
  • Figure 1C shows the antibody to inhibit the production of Th2-related cytokine IL-5 The activity produced;
  • FIG. 1D is the activity of the antibody to inhibit the production of the Th2-related cytokine TNF- ⁇ .
  • TSLP thymic stromal lymphopoietin
  • IL-7 interleukin-7
  • DC dendritic cells
  • TSLP includes variants, isoforms, homologs, orthologs and paralogs of TSLP.
  • antibody is used herein in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full-length antibodies, and antibody fragments ( or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • “Native antibody” refers to naturally-occurring immunoglobulin molecules with different structures.
  • native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains joined by disulfide bonds.
  • VH variable domain
  • CH2 heavy chain variable domain
  • CH3 constant domains
  • VL variable region
  • CL constant light domain
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen.
  • VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • complementarity determining region refer to the region within the variable domain that mainly contributes to antigen binding
  • framework or "FR” refers to the variable domain residues other than CDR residues.
  • VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3
  • VL contains 3 CDR regions: LCDR1, LCDR2, and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer specificity for binding an antigen.
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: the "Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health , Bethesda, MD), "Chothia” numbering scheme, “AbM” numbering scheme, "contact” numbering scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering Rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278) et al. The relationship between these numbering conventions is well known to those skilled in the art, as shown in Table 2 below.
  • the "class" of an antibody refers to the type of constant region possessed by its heavy chain.
  • Antibody light chains include two types, kappa ( ⁇ ) and lambda ( ⁇ ), according to the constant region amino acid sequence.
  • antibodies can be divided into five categories, or antibody isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chains respectively , delta chains, gamma chains, alpha chains, and epsilon chains.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region mentioned in the present disclosure refer to the human-derived heavy chain constant regions disclosed in the prior art that do not alter the structure and function of the antibody variable region or variants of the light chain constant region
  • exemplary variants include IgG1, IgG2, IgG3, IgG4 heavy chain constant region variants with site-directed engineering and amino acid substitutions in the heavy chain constant region.
  • the substitution is a YTE mutation, a L234A and/or L235A mutation, an S228P mutation, and/or a mutation that obtains a knob-into-hole structure. These mutations have been shown to confer new properties to the antibody without altering the function of the variable region of the antibody.
  • the antibody is of the IgGl isotype with P329G, L234A, and L235A mutations in the hinge region to reduce effector function. In some embodiments, the antibody is of the IgG2 isotype. In some embodiments, the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibodies, diabodies, linear antibodies, single chain antibody molecules; and multispecific antibodies formed from antibody fragments Sexual antibodies.
  • antibody framework or "FR region” refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • Fc region or “fragment crystallizable region” are used to define the C-terminal region of an antibody heavy chain, including native sequence Fc regions and variant Fc regions.
  • the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus (according to the EU numbering system).
  • the boundaries of the Fc region of an antibody heavy chain may also vary, for example deletion of the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region or deletion of the C-terminal glycine and lysine (according to the EU numbering system) of the Fc region. residues 446 and 447).
  • a composition of intact antibodies may include a population of antibodies with all K447 residues and/or G446+K447 residues removed.
  • a composition of intact antibodies may include a population of antibodies without removal of K447 residues and/or G446+K447 residues.
  • the composition of intact antibodies has a population of antibodies with and without a mixture of antibodies of K447 residues and/or G446+K447 residues.
  • Suitable native sequence Fc regions for the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service , National Institutes of Health, Bethesda, MD, 1991.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, and the remainder of the heavy and/or light chain is derived from a different source or species.
  • humanized antibody is an antibody that retains the antigen-binding activity of a non-human antibody, while being less immunogenic in humans. This can be accomplished, for example, by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (i.e., the constant regions and framework portions of the variable regions).
  • the antibodies provided herein are chimeric antibodies.
  • a chimeric antibody comprises non-human variable regions (eg, variable regions derived from mouse, rat, hamster, rabbit, or non-human primate, such as monkey) and human constant regions.
  • a chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • the chimeric antibody is a humanized antibody.
  • non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable regions in which the CDRs, or portions thereof, are derived from non-human antibodies, and the FRs, or portions thereof, are derived from human antibodies.
  • the humanized antibody will also comprise human constant regions.
  • some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody providing the CDR sequences) to restore or improve antibody specificity or affinity.
  • antibodies of the present disclosure can be isolated by screening combinatorial libraries for antibodies having the desired activity or activities. Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al., Nature Reviews 16:498-508 (2016). In some phage display methods, repertoires of VH and VL genes are separately cloned by PCR and randomly recombined in a phage library, which can then be used to screen for antigen-binding phage, as described in Winter et al., Annual Review of Immunology 12 : 433-455 (1994). Phages typically display antibody fragments as scFv fragments or as Fab fragments, and immunized libraries can provide high-affinity antibodies to the immunogen without the need to construct hybridomas.
  • unimmunized repertoires can be cloned to provide antibodies against a wide range of non-self and self-antigens without any immunization, as described by Griffiths et al., EMBO Journal 12:725-734 (1993) of.
  • non-immunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, amplifying the highly variable CDR3-encoding regions using PCR primers containing random sequences, and achieving rearrangement in vitro, as described by As described by Hoogenboom and Winter, Journal of Molecular Biology 227:381-388 (1992).
  • full-length antibody intact antibody
  • complete antibody complete antibody
  • whole antibody used interchangeably herein to refer to an antibody in substantially intact form, as distinguished from antigen-binding fragments as defined herein.
  • the term specifically refers to antibodies whose light and heavy chains comprise constant regions.
  • single-chain antibody single-chain Fv or “scFv” is meant to comprise an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) joined by a linker molecule.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Numerous linkers suitable for linking antibody VH and VL have been disclosed in the prior art, for example consisting of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl . Acad. Sci.
  • antigen refers to a molecule or molecular portion capable of being bound by a selective binding agent such as an antigen binding protein (including, for example, an antibody), and capable of being used in an animal to generate an antibody capable of binding the antigen.
  • Antigens can have one or more epitopes capable of interacting with different antigen binding proteins (eg, antibodies).
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to an internal binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • KD dissociation constant
  • antibodies are prepared at about 1 x 10-7 M or less (eg, about 1 x 10-8 M or less, about 1 x 10-9 M or less, about 1 x 10-10 M or less, about 1 x 10-10 M or less, about An equilibrium dissociation constant (KD) of 1 ⁇ 10 ⁇ 11 M or less, or about 1 ⁇ 10 ⁇ 12 M or less) binds the antigen or an epitope thereof.
  • KD equilibrium dissociation constant
  • the KD of an antibody for binding to an antigen is 10%, or 1%, of the KD of the antibody for binding to a non-specific antigen (eg, BSA, casein). KD can be measured using standard procedures, such as by measured by surface plasmon resonance assay.
  • a non-specific antigen eg, BSA, casein
  • antibodies that specifically bind to an antigen or an epitope thereof may be cross-reactive to other relevant antigens, for example, to those from other species (homologous) such as humans or monkeys, eg, Macaca fascicularis (cynomolgus , cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmosets (Callithrix jacchus) (common marmoset, marmoset) corresponding antigens are cross-reactive.
  • homologous such as humans or monkeys, eg, Macaca fascicularis (cynomolgus , cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmosets (Callithrix jacchus) (common marmoset, marmoset) corresponding antigens are cross-reactive.
  • KD refers to the dissociation constant, which is obtained from the ratio of kd to ka (ie kd/ka) and expressed as molar concentration (M).
  • M molar concentration
  • the KD value of an antibody can be determined using methods well known in the art. Methods for determining antibody KD include measuring surface plasmon resonance using biosensing systems such as systems, or measuring affinity in solution by solution equilibrium titration (SET).
  • nucleic acid is used interchangeably herein with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, synthetic, naturally occurring or non-naturally occurring, that have binding properties similar to the reference nucleic acid, and Metabolized in a manner similar to the reference nucleotide.
  • nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • isolated nucleic acid encoding an anti-TSLP antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such one or more nucleic acids in a single vector or in separate vectors molecules, and such one or more nucleic acid molecules present in one or more locations in a host cell.
  • nucleic acid sequence encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as those explicitly indicated.
  • degenerate codon substitutions can be obtained by generating sequences in which one or more selected (or all) codons are substituted at the third position by mixed bases and/or deoxy Inosine residue substitution (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).
  • amino acid sequence identity means that when aligning amino acid sequences, gaps are introduced where necessary to achieve maximum percent sequence identity, and no conservative substitutions are considered part of the sequence identity, between the first sequence and the second sequence.
  • the percentage of amino acid residues that are identical to amino acid residues. Determination of percent amino acid sequence identity can be accomplished by a variety of means of aligning sequences that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome.
  • AAV or AAV2 adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), 293 cells and NSO cells.
  • the antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind human TSLP. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-containing broth can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody was eluted by pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product usually needs to be refrigerated, or frozen, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, eg, a composition comprising any of the binding compounds of the present disclosure, to a patient having one or more disease symptoms for which the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a patient or population to be treated in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms or to inhibit progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to relieve symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of each target disease, the method of The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • H test Kruskal-Wallis test
  • Jonckheere-Terpstra test Jonckheere-Terpstra test
  • Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are set forth in the table "Exemplary Conservative Amino Acid Substitutions" below.
  • an “effective amount” or “effective dose” refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction in severity, or delay in onset of disorders, including biochemical, tissue academic and/or behavioral symptoms.
  • beneficial or desired outcomes include clinical outcomes, such as reducing the incidence or amelioration of one or more symptoms of the various target antigen-related disorders of the present disclosure, reducing the amount of other agents required to treat the disorder dose, enhances the efficacy of another agent, and/or delays the progression of a disorder associated with the target antigen of the present disclosure in a patient.
  • Exogenous refers to a substance produced outside an organism, cell, or human body, as the case may be.
  • Endogenous refers to a substance produced in a cell, organism, or human body as the case may be.
  • Homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position . The percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100.
  • sequences when sequences are optimally aligned, two sequences are 60% homologous if 6 matches or homology at 10 positions in the two sequences; if 95 matches at 100 positions in the two sequences or homologous, then the two sequences are 95% homologous.
  • comparisons are made when aligning two sequences to give the greatest percent homology.
  • the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are chosen to give the maximum match between the respective sequences over the entire length of the respective reference sequences.
  • the following references refer to BLAST algorithms frequently used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W.
  • progeny As used herein, the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny. Thus, “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. Where a different name is meant, it is clear from the context.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as a physiologically/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in formulations for delivery of antibodies or antigen-binding fragments.
  • the carrier can be an antiadherent, binder, coating, disintegrant, filler or diluent, preservative (eg, antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent agents, emulsifiers, buffers, etc.
  • suitable pharmaceutically acceptable carriers include water, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (eg, olive oil), saline, buffers, buffered saline, and the like Osmotic agents such as sugars, polyols, sorbitol and sodium chloride.
  • the present disclosure includes an agent for the treatment of a disease associated with TSLP comprising the anti-TSLP antibody of the present disclosure as an active ingredient.
  • the disease associated with TSLP in the present disclosure is not limited as long as it is a disease associated with TSLP, for example, a therapeutic response induced by the molecules of the present disclosure can block TSLP by binding to human TSLP and then blocking the binding of TSLP to its receptor. related signaling pathways that inhibit disease progression.
  • TSLP-related diseases can be diagnosed by detecting or assaying TSLP-expressing cells with the antibodies of the present disclosure.
  • the present disclosure relates to methods for immunodetection or assay of a target antigen (eg TSLP), reagents for immunodetection or assay of a target antigen (eg TSLP), for immunodetection or assay of cells expressing a target antigen (eg TSLP)
  • a target antigen eg, TSLP
  • a diagnostic agent for diagnosing a disease associated with a target antigen (eg, TSLP) positive cell comprising the present disclosure that specifically recognizes the target antigen (eg, human TSLP) and binds to the amino acid sequence of the extracellular region or its three-dimensional structure the antibody or antibody fragment as the active ingredient.
  • the method for detecting or determining the amount of the target antigen can be any known method.
  • it includes immunodetection or assay methods.
  • An immunodetection or assay method is a method for detecting or measuring the amount of antibody or antigen using labeled antigen or antibody.
  • immunodetection or assay methods include radioactive substance-labeled immunoantibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescence immunoassays, Western blotting, physicochemical methods Wait.
  • the biological sample used to detect or measure the target antigen eg TSLP
  • the target antigen eg TSLP
  • cells expressing the target antigen eg TSLP
  • tissue cells blood, plasma , serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.
  • Reagents for performing antigen-antibody reactions include buffers, salts, and the like.
  • Reagents for detection include those commonly used in immunodetection or assay methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and a substrate corresponding to the label, and the like.
  • HEK293E cells were inoculated into Freestyle expression medium (containing 1% FBS) at 0.8 ⁇ 10 6 /mL the day before, and placed in a constant temperature shaker (120 rpm) at 37° C. for 24 hours.
  • the transfection plasmid and transfection reagent PEI were sterilized with a 0.22 ⁇ m filter, and then the transfection plasmid was adjusted to 100 ⁇ g/100 mL cells, and the mass ratio of PEI (1 mg/mL) and plasmid was 3:1, with Take the transfection of 200mL HEK293E cells as an example, mix 10mL Opti-MEM and 200 ⁇ g plasmid, and let stand for 5 minutes (min); take another 10mL Opti-MEM and 600 ⁇ g PEI, mix well, and let stand for 5 minutes. The plasmid and PEI were mixed and allowed to stand for 15 min.
  • the plasmid and PEI mixture was slowly added to 200 mL of HEK293E cells, and cultured in a shaker at 8% CO 2 , 120 rpm, and 37°C. On day 3 of transfection, feed medium was supplemented with 10% volume. On the 6th day of transfection, the cell supernatant was collected by centrifugation at 4500 rpm for 10 min, filtered, and the recombinant TSLP and TSLP receptor protein supernatant was purified by Example 2.
  • the purified protein can be used in the experiments of the following examples. The relevant sequence looks like this:
  • Fc-tagged human TSLP receptor extracellular domain (human-TSLPR-Fc-ECD) amino acid sequence:
  • the underlined part is the extracellular region of human-TSLPR, and the italicized part is the linker-human Fc-tag.
  • the cell expression supernatant was centrifuged at high speed to remove impurities, filtered, the nickel column was equilibrated with PBS solution, and washed 10 times the column volume. The filtered supernatant was applied to the column. The column was rinsed with 30 mM imidazole in PBS until the A280 reading dropped to baseline. The target protein was eluted with a PBS solution containing 300 mM imidazole, and the elution peaks were collected. PBS was concentrated and exchanged, and LC-MS was identified as correct and then used. His-tagged human TSLP and cynomolgus TSLP were obtained.
  • the cell expression supernatant was centrifuged at high speed to remove impurities, and the recombinant antibody expression supernatant was purified with Protein A column.
  • the column was rinsed with PBS until the A280 reading dropped to baseline.
  • the target protein was eluted with 100 mM acetic acid, pH 3.5, and neutralized with 1 M Tris-HCl, pH 8.0. Concentrate and change the medium, the obtained protein was identified by electrophoresis and identified as correct by LC-MS and then used.
  • CHO-K1 and BaF3 cell lines expressing both human TSLP receptor and human IL7R ⁇ were constructed.
  • the target gene TSLPR/IL7R ⁇ was packaged by lentivirus and cloned into cells to form a stable high-expression cell line.
  • human TSLPR and human IL7R ⁇ genes were cloned into pCDH-CMV-MCS-EF1-puro and pCDH-CMV-MCS-EF1-Neo (SBI, CD500B-1) plasmids, respectively, and then human TSLPR was infected by lentivirus.
  • the cells were cloned into CHO-K1 and BaF3 cell lines, and selected and cultured for three weeks under the selection pressure of 10 ⁇ g/mL puromycin (Gibco, US). On this basis, a second round of infection was carried out, and the human IL7R ⁇ gene was cloned in and screened with 1 mg/mL G418 (Gibco, US) and 10 ⁇ g/mL puromycin for two to three weeks. Finally, by flow sorting, CHO-K1 and BaF3 monoclonal cell lines with high expression of TSLPR and IL7R ⁇ were screened.
  • Antibodies in the present disclosure can be obtained by techniques such as hybridoma screening, phage display, affinity maturation, and the like.
  • the inventors of the present disclosure have obtained a series of high-affinity TSLP antibodies through extensive experiments.
  • the sequences of anti-TSLP antibodies are as follows:
  • variable region sequence of anti-TSLP is as follows:
  • the underlined part is the complementarity determining region sequence.
  • anti-TSLP antibodies and their corresponding variable region sequences in the present disclosure are as follows:
  • VH2 VL1 Ab-2 VH2 VL1 Ab-23 VH17 VL1 Ab-6 VH3 VL2 Ab-24 VH18 VL1 Ab-9 VH3 VL3 Ab-25 VH19 VL1 Ab-27 VH21 VL1
  • a full-length antibody is formed by linking the variable regions of the light and heavy chains of the antibody with the constant regions of the light and heavy chains of the antibody.
  • the heavy chain variable region of the Ab-1 antibody is VH1
  • the light chain variable region is VL1
  • other analogies for example, the IgG1 heavy chain constant region (such as: SEQ ID NO: 34) and the lambda light chain can be used.
  • a constant region eg, SEQ ID NO: 35 serves as the constant region of the antibody in the present disclosure.
  • the heavy chain constant region of the antibody can be selected from the constant regions of IgGl, IgG2, IgG4 and variants thereof.
  • IgG1 constant region is used in the present disclosure, the sequence of which is shown in SEQ ID NO:34.
  • the light chain constant region may be selected from light chain constant regions of human kappa, lambda chains or variants thereof.
  • the constant region of the human lambda chain is used in the present disclosure.
  • the heavy and light chain variable regions of the present disclosure are recombined with the above-mentioned constant regions to obtain the full-length sequences of the heavy and light chains.
  • sequences of the antibodies are shown below:
  • control antibody used in this disclosure is Amg157, and its antibody sequence is as follows: > Heavy chain sequence of Amg157
  • the antibodies of the present disclosure can be cloned, expressed and purified by conventional gene cloning and recombinant expression methods.
  • Test Example 1 Biacore Determination of Binding of Anti-TSLP Antibodies to Human and Cynomolgus TSLP
  • the affinity of the humanized TSLP antibody to human and cynomolgus monkey TSLP was determined using a Biacore T200 (GE) instrument.
  • Biosensor chip (Cat. #29127556, GE) to affinity capture the molecule to be tested, and then flow through the antigen (huTSLP-his, cynoTSLP-his, prepared in Example 1) on the surface of the chip, and use Biacore T200 instrument in real time Binding and dissociation curves were obtained by detecting reaction signals. After the dissociation in each experimental cycle, the biosensor chip was washed and regenerated with a glycine-hydrochloric acid regeneration solution (pH 1.5Cat.#BR-1003-54, GE). Using BIAevaluation version 4.1, GE software was used to fit the data with a (1:1) Langmuir model, and the affinity values were obtained, as shown in the following table.
  • the anti-TSLP antibody in the present disclosure has a high affinity for human and cynomolgus monkey TSLP, which is better than the control Amg157 antibody.
  • Test Example 2 Experiment of ELSA-based anti-TSLP antibody blocking TSLP binding to TSLP receptor
  • the TSLP receptor has two subunits, TSLPR and IL7R, where TSLPR is a TSLP-specific receptor and IL7R is a shared receptor for TSLP and IL7.
  • TSLPR is a TSLP-specific receptor
  • IL7R is a shared receptor for TSLP and IL7.
  • TSLP first binds to TSLPR and then to IL7R. This test case is used to identify whether TSLP antibody can block the binding of TSLP to the extracellular domain of recombinantly expressed TSLPR receptor protein.
  • Human-TSLPR-Fc-ECD (2 ⁇ g/mL, SEQ ID NO: 3) was coated on an ELISA plate and incubated at 4°C overnight. After discarding the liquid, 200 ⁇ L/well of 5% nonfat milk blocking solution diluted with PBS was added, 37 Incubate for 2 hours in a °C incubator for blocking.
  • the blocking solution was discarded, and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20), and the biotin-labeled huTSLP-Fc antigen was prepared to 3nM, and the antibody to be tested started gradient from 200nM Dilute, mix antigen and antibody 1:1 and place at 37°C for 15min, add 100 ⁇ L per well to the microtiter plate, place at 37°C for 1 hour (h), wash the plate with PBST 3 times, add 100 ⁇ L/well of sample diluent Streptavidin-Peroxidase Polymer diluted 1:4000 and incubated at 37°C for 1 hour.
  • PBST buffer pH 7.4 PBS containing 0.05% Tween-20
  • Antibody IC50(nM) Amg157 0.61 Ab-1 0.18 Ab-2 0.18 Ab-3 0.2 Ab-6 0.2 Ab-9 0.26 Ab-14 0.2 Ab-23 0.19 Ab-24 0.21 Ab-25 1.12 Ab-27 5.98
  • Test Example 3 Anti-TSLP antibody inhibits TSLP-induced proliferation of TSLPR/IL7R-overexpressing BaF3 cells
  • TSLP can bind to TSLPR/IL7R on the surface of BaF3, thereby promoting the proliferation of BaF3.
  • This test example is used to identify that the disclosed antibodies can block the BaF3 proliferation-inducing activity of TSLP.
  • BaF3 cells overexpressing TSLPR/IL7R were cultured in RPMI1640 medium with 10% FBS and 2ng/mL rhIL3 (Linktech, Catalog No. 96-AF-300-03-20) at 37°C, 5% CO 2 Culture in an incubator, and the cell density does not exceed 1 ⁇ 10 6 cells/mL.
  • rhIL3 Linktech, Catalog No. 96-AF-300-03-20
  • the cell density does not exceed 1 ⁇ 10 6 cells/mL.
  • cells in the logarithmic growth phase were washed three times with PBS and centrifuged at 800 rpm for 5 min. The cell density was adjusted to 8000 cells/well/90 ⁇ L with RPMI1640 (2% FBS, recombinant human TSLP-Fc: 40 ng/mL), and 10 ⁇ L was added.
  • Antibody IC50(nM) Ab-1 2.24 Ab-2 2.63 Ab-3 2.5 Ab-6 12.34 Ab-9 13 Ab-14 0.43 Ab-23 0.43 Ab-24 0.37
  • Test Example 4 Anti-TSLP Antibody Blocks TSLP-Induced Differentiation of Native CD4 + T Cells to Th2 Cells
  • TSLP can induce the maturation of primary myeloid mDC cells, and mature mDC cells highly express OX40 ligands.
  • OX40 ligands can bind to OX40 on the surface of natural CD4 + T cells, thereby making natural CD4 + T cells differentiate into Th2 cells and produce IL4/ IL5/IL13 and other immune response-related factors make the body produce Th2 inflammatory response.
  • This test example is used to detect that the antibodies in the present disclosure can block TSLP-induced differentiation of Th2 cells.
  • the primary myeloid DCs were isolated and purified from human peripheral blood mononuclear cells (PBMCs) using the method of magnetic bead sorting (CD1c(BDCA-1) + Dendritic Cell Isolation Kit Miltenyi Biotec), and the obtained mDCs were grown on 96 well inside the cell culture plate.
  • the serially diluted antibody and recombinantly expressed human TSLP (huTSLP-his, final concentration of 50ng/mL) were pre-incubated (37°C) for about 45 minutes, and then added to each cell culture well containing mDC, and incubated at 37°C for 24 hours. Stimulated mature mDCs were collected and washed twice with PBS.
  • CD4 + CD45RA + naive T cells were extracted from PBMCs by magnetic bead separation (Myltenyi, Biotec). The isolated naive T cells and mature mDCs were mixed and seeded in a 96-well cell culture plate at a ratio of 5:1, and co-cultured for 6 days. Cells were collected, seeded in 96-well plates pre-coated with anti-CD3 (10 ⁇ g/mL), and anti-CD28 (1 ⁇ g/mL) was added to stimulate differentiated T cells for 24 hours. Finally, the cell culture supernatant was collected. . Th2-related cytokines secreted by cells in the supernatant were detected by ELISA.
  • IL-4 and IL-5 cytokines were detected by R&D's ELISA kit, and TNF- ⁇ and IL-13 were detected by Xinbosheng's ELISA kit. The results are shown in Table 9 and Figures 1A-1D.
  • IL-13 (pg/mL) 1789.9 537.9 IL-4 (pg/mL) 50.1 38.7 IL-5 (pg/mL) 99.1 68.1 TNF- ⁇ 263.2 194.2
  • the results show that the antibody Ab-23 obtained in the present disclosure can significantly inhibit the production of Th2 cytokines IL4, IL5, IL13 and TNF- ⁇ , indicating that the antibody obtained in the present disclosure can block the differentiation of Th2 cells induced by TSLP.

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Abstract

涉及能结合胸腺基质淋巴细胞生成素的抗体及其应用。具体地，涉及抗TSLP抗体，其药物组合物，以及其作为治疗哮喘药物的用途。

Description

结合胸腺基质淋巴细胞生成素的抗体及其应用
本申请要求申请日为2020/12/3的中国专利申请202011412850.4的优先权。本申请引用上述中国专利申请的全文。
技术领域
本披露涉及抗体药物领域，具体地本披露涉及抗TSLP抗体药物以及其应用。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
哮喘是严重的气道慢性炎症疾病，全球约有3.34亿哮喘病人。随着环境恶化和空气污染加剧，可能会有更多人罹患此病，严重危害人类的生命健康。
TSLP是一种类白细胞介素7(IL-7)细胞因子，最早从小鼠的胸腺基质细胞条件培养基中发现。TSLP主要在肺、皮肤和肠道上皮细胞中表达。TSLP由4个α-螺旋以及AB和CD两个环组成，分子内有三对由六个半胱氨酸组成的二硫键，有两个N糖基化位点，分子量约为15-20kD。TSLP的受体是个复合物，包括两部分，一部分为TSLPR，另一部分为IL7Rα。TSLP先与TSLPR以相对较低的亲和力结合，然后以高亲和力招募IL7Rα的结合，最终激活stat5等信号通路，导致DC的成熟和T细胞的分化。
骨髓源性树突状细胞(myeloid dendritic cell，mDC)是TSLP最主要的效应细胞，TSLP作用于未成熟的mDC，mDC分泌细胞因子IL-8，eotaxin-2，TARC和MDC，同时高表达OX40L，在没有IL-12的前提下，OX40L与天然CD4 +T细胞结合，使其分化成Th2细胞，进而Th2细胞分泌IL-5、IL-4、IL-9，IL-13和TNF等Th2细胞因子，诱导机体Th2炎症反应。另外，TSLP还可以诱导DC细胞产生细胞因子IL-8，进而招募嗜中性粒细胞，导致嗜中性粒细胞天然免疫炎症。TSLP还可以诱导DC产生eotaxin-2，eotaxin-2招募嗜酸性粒细胞，和IL5一起作用，使机体迅速进入嗜酸性粒细胞浸润的炎症状态。TSLP也作用于肥大细胞、自然杀伤细胞，通过诱导产生IL-4、IL-6、IgE等介导天然炎症。综上，TSLP可同时导致天然炎症和Th2炎症，进而使组织黏液增多，气道重塑导致气管狭窄，细胞纤维化严重，进而逐步演变成哮喘、过敏性皮炎和过敏性鼻炎等三大过敏性疾病。因此，阻断TSLP对治疗哮喘、过敏性皮炎等疾病是一个潜在的有效策略。
目前，WO2008155365、WO2009035577、WO2011056772、WO2016142426、WO2017004149公开了抗TSLP的抗体，但并没有相应的抗体上市，因此，有必要继续开 发有效的治疗TSLP相关疾病的药物。
发明内容
本披露提供一种抗TSLP抗体。
在一些实施方案中，如前所述抗TSLP抗体，其包含重链可变区和轻链可变区，其中：
i)重链可变区氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示的HCDR1、HCDR2和HCDR3；且轻链可变区包含氨基酸序列分别如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的LCDR1、LCDR2和LCDR3；
或
ii)重链可变区包含氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；且轻链可变区包含氨基酸序列分别如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的LCDR1、LCDR2和LCDR3；
或
iii)重链可变区包含氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:23和SEQ ID NO:25所示的HCDR1、HCDR2和HCDR3，轻链可变区包含氨基酸序列分别如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如前所述抗TSLP抗体，其中所述抗TSLP抗体是鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，如前所述抗TSLP抗体，其包含重链可变区和轻链可变区，其中：
所述重链可变区的氨基酸序列如SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14所示，或与SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14所示的序列具有至少90％、92％、94％、95％、96％、97％、98％或99％的序列同一性，且所述轻链可变区的氨基酸序列如SEQ ID NO:6所示，或与SEQ ID NO:6具有至少90％、92％、94％、95％、96％、97％、98％或99％的序列同一性。
在一些实施方案中，如前所述抗TSLP抗体，其包含氨基酸序列如SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14所示的重链可变区；和氨基酸序列如SEQ ID NO:6所示的轻链可变区。
在一些实施方案中，如前所述抗TSLP抗体，其包含氨基酸序列如SEQ ID NO:13所示的重链可变区；和氨基酸序列如SEQ ID NO:6所示的轻链可变区。
在一些实施方案中，如前所述抗TSLP抗体，其中所述抗体的轻链可变区和重链可变区组合如下所示：
表1.抗体的轻链和重链可变区的组合
抗体 VH VL
Ab-1 SEQ ID NO:9 SEQ ID NO:6
Ab-2 SEQ ID NO:10 SEQ ID NO:6
Ab-6 SEQ ID NO:11 SEQ ID NO:7
Ab-9 SEQ ID NO:11 SEQ ID NO:8
Ab-25 SEQ ID NO:15 SEQ ID NO:6
Ab-27 SEQ ID NO:16 SEQ ID NO:6
Ab-14 SEQ ID NO:12 SEQ ID NO:6
Ab-23 SEQ ID NO:13 SEQ ID NO:6
Ab-24 SEQ ID NO:14 SEQ ID NO:6
在一些实施方案中，如前所述抗TSLP抗体，其中所述抗体进一步包含抗体恒定区。
在一些实施方案中，如前所述抗TSLP抗体，所述抗体的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述抗体的轻链恒定区选自人抗体κ和λ链恒定区及其常规变体。
在一些实施方案中，如前所述抗TSLP抗体，其中所述的抗TSLP抗体包含氨基酸序列如SEQ ID NO:34所示的重链恒定区；和/或氨基酸序列如SEQ ID NO:35所示的轻链恒定区。
在一些实施方案中，如前所述抗TSLP抗体，其中所述的抗TSLP抗体包含重链和轻链，其中：
重链氨基酸序列如SEQ ID NO:36、SEQ ID NO:38或SEQ ID NO:39所示，或与SEQ ID NO:36、SEQ ID NO:38或SEQ ID NO:39所示的序列具有至少85％的序列同一性；且
轻链氨基酸序列如SEQ ID NO:37所示，或与SEQ ID NO:37具有至少85％的序列同一性。
所述的至少85％序列同一性包括但不限于85％、86％、87％、88％、89％、90％、92％、94％、95％、96％、97％、98％或99％的序列同一性。
在一些实施方案中，如前所述抗TSLP抗体，其中所述的抗TSLP抗体包含：
如SEQ ID NO:36的重链和SEQ ID NO:37的轻链；或
如SEQ ID NO:38的重链和SEQ ID NO:37的轻链；或
如SEQ ID NO:39的重链和SEQ ID NO:37的轻链。
在一些实施方案中，如前所述的抗TSLP抗体，其具有以下特性中的一种或多种：
a)以小于9pM的KD值与人TSLP结合；
b)以小于0.6nM的IC50值阻断TSLP与TSLPR的结合；或
c)以小于9pM的KD值与食蟹猴TSLP结合。
在一些实施方案中，如前所述的抗TSLP抗体，其以小于9pM、小于5pM或小于3pM的KD值与人TSLP结合。
在一些实施方案中，如前所述的抗TSLP抗体，其以小于9pM、小于5pM、小于3pM或小于2pM的KD值与食蟹猴TSLP结合。
在一些实施方案中，其中所述KD值可使用Biacore方法检测获得。在一些实施方案中，其中所述KD值可参照测试例1中所述的方法测得。
在一些实施方案中，如前所述的抗TSLP抗体，其以小于0.6nM、小于4nM或小于2nM的IC50值阻断TSLP与TSLPR的结合。
在一些实施方案中，其中所述的IC50值可以通过ELISA实验测得。在一些实施方案中，其中所述的IC50值也可参照本披露的测试例2检测。
在一些实施方案中，如前所述的抗TSLP抗体，其可以抑制TSLP诱导的天然CD4 +T细胞往Th2细胞的分化。
本披露还提供核酸分子，其编码如前所述的抗TSLP抗体。
本披露还提供表达载体，其包含如前所述的核酸分子。
本披露还提供一种宿主细胞，其包含如如前所述的核酸分子或如前所述的表达载体；优选地，其中所述的宿主细胞为细菌、真菌细胞、昆虫动物细胞或哺乳动物细胞。在一些实施方案中，其中所述哺乳动物包括但不限于293、CHO细胞；在一些实施方案中，其中所述的哺乳动物细胞不是人类胚胎细胞。
在一些实施方案中，本披露提供一种药物组合物，其含有治疗有效量的如前所述的抗TSLP抗体，或如前所述的核酸分子，或如前所述的宿主细胞，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂；优选地，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg或1-1000mg如前所述的抗TSLP抗体。
在一些实施方案中，本披露提供一种制备如前所述的TSLP抗体的方法。
在一些实施方案中，本披露提供一种用于体外或离体免疫检测或测定TSLP的方法，所述方法包括使用如前所述的抗TSLP抗体的步骤。
在一些实施方案中，本披露提供如前所述的抗TSLP抗体在制备免疫检测人TSLP的试剂中的用途。
在一些实施方案中，本披露提供一种用于免疫检测或测定TSLP的如前所述的抗TSLP抗体。
在一些实施方案中，本披露提供一种试剂盒，其包含如前所述的抗TSLP抗体。
在一些实施方案中，本披露提供如前所述的抗TSLP抗体，或如前所述的核酸分子，或如前所述的宿主细胞，或如前所述的药物组合物，其用于制备：
a.治疗TSLP相关炎症的药物；或
b.治疗TSLP相关纤维化病症的药物。
在一些实施方案中，前述的TSLP相关炎症包括但不限于过敏症、皮炎、哮喘、过敏性结膜炎、过敏性鼻炎和过敏性鼻窦炎。
在一些实施方案中，前述的TSLP相关纤维化病症包括但不限于硬皮症、间质性肺病、特发性肺纤维化、B型或C型慢性肝炎引发的纤维化、辐射诱发的纤维化和伤口愈合引发的纤维化。
在一些实施方案中，本披露提供如前所述的抗TSLP抗体，或如前所述的核酸分子，或如前所述的宿主细胞，或如前所述的药物组合物在制备用于治疗炎症疾病或纤维化疾病的药物中的用途。
在一些实施方案中，本披露提供一种治疗炎症疾病或纤维化疾病的方法，所述方法包括向受试者施用治疗有效量的如前所述的抗TSLP抗体，或如前所述的核酸分子，或如前所述的宿主细胞或如前所述的药物组合物。
在一些实施方案中，本披露提供一种用作药物的如前所述的抗TSLP抗体，或如前所述的核酸分子，或如前所述的宿主细胞。在一些实施方案中，其中所述的药物用于治疗炎症疾病或纤维化疾病。
在一些实施方案中，前述的炎症疾病包括但不限于过敏症、皮炎、哮喘、过敏性结膜炎、变应性鼻炎、嗜酸性粒细胞性胃肠炎、过敏性支气管肺曲霉病、过敏性真菌鼻窦炎、慢性嗜酸性粒细胞性肺炎、嗜酸性粒细胞性支气管炎、炎性肠病和过敏性鼻窦炎。
在一些实施方案中，前述的纤维化疾病包括但不限于硬皮症、间质性肺病、特发性肺纤维化、B型或C型慢性肝炎引发的纤维化、辐射诱发的纤维化和伤口愈合引发的纤维化。
在一些实施方案中，前述的炎症疾病或纤维化疾病与TSLP相关。
附图说明
图1：图1A为抗体抑制Th2相关的细胞因子IL-13产生的活性；图1B为抗体抑制 Th2相关的细胞因子IL-4产生的活性；图1C为抗体抑制Th2相关的细胞因子IL-5产生的活性；图1D为抗体抑制Th2相关的细胞因子TNF-α产生的活性。
具体实施方式
术语
本文所用的术语只是为了描述实施方案的目的，并非旨在进行限制。除非另外定义，本文所用的全部技术术语和科学术语具有与本披露所属领域的普通技术人员通常所理解的意义。
说明书和权利要求书中所用的单数形式“一个”、“一种”和“所述”包括复数指代，除非上下文清楚表明并非如此。
除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅限于”的意义。
术语“和/或”，例如“X和/或Y”应当理解为意指“X和Y”或“X或Y”并且应当被用来提供对两种含义或任一含义的明确支持。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“胸腺基质淋巴细胞生成素(Thymic Stromal Lymphopoietin，TSLP)”是四α-螺旋束I型细胞因子，也是响应促炎症刺激而产生的上皮细胞衍生的细胞因子，与白细胞介素-7(IL-7)密切相关，其通过刺激树突细胞(DC)起始***反应，是调节人体免疫反应的重要因子。术语“TSLP”包括TSLP的变体、同种型、同系物、直系同源物和旁系同源物。
本文中的术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。
“天然抗体”指具有不同结构的天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异四聚糖蛋白，由二硫键结合的两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域，或重链可变域，接着是三个恒定域(CH1、CH2和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域，或轻链可变域，接着是一个恒定轻域(CL)。
本披露的抗体包括鼠源抗体、嵌合抗体和人源化抗体。
术语“可变区”或“可变域”指抗体重链或轻链中涉及抗体结合抗原的域。VH和VL 各包含四个保守的框架区(FR)和三个互补决定区(CDR)。其中，术语“互补决定区”、“CDR”指可变结构域内主要促成与抗原结合的区域；“框架”或“FR”是指除CDR残基之外的可变结构域残基。VH包含3个CDR区：HCDR1、HCDR2和HCDR3；VL包含3个CDR区：LCDR1、LCDR2、和LCDR3。每个VH和VL由从氨基末端至羧基末端由按以下顺序排列的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。单个VH或VL可能足以赋予结合抗原的特异性。
可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则、“AbM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018Oct 16；9:2278)等。这些编号规则之间的关系是本领域技术人员熟知的，具体如下表2中所示。
表2.CDR编号***之间的关系
Figure PCTCN2021135410-appb-000001
除非另有说明，本披露实施例中的可变区和CDR序列均适用“Kabat”编号规则。
抗体的“类”指其重链拥有的恒定区的类型。根据恒定区氨基酸序列，抗体轻链包括两种类型，卡帕(κ)和拉姆达(λ)。根据抗体重链恒定区的氨基酸组成和排列顺序不同，可将抗体分为五类，或称为抗体同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。五类Ig中每类Ig都可以有κ链或λ链。
本披露中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术 已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3、IgG4重链恒定区变体。在一些实施方案中，所述替换是YTE突变，L234A和/或L235A突变，S228P突变，和/或获得杵臼(knob-into-hole)结构的突变。这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。在一些实施方案中，抗体是IgG1同种型的，具有铰链区中的P329G、L234A和L235A突变以降低效应子功能。在一些实施方案中，抗体是IgG2同种型。在一些实施方案中，抗体是IgG4同种型的，具有铰链区中的S228P突变以改善IgG4抗体的稳定性。
术语“抗体片段”指不同于完整抗体的分子，其包含完整抗体的部分，所述部分与完整抗体所结合的抗原相结合。抗体片段的实例包括但不限于Fv、Fab、Fab’、Fab’-SH、F(ab′)2、单域抗体、双抗体、线性抗体、单链抗体分子；以及由抗体片段形成的多特异性抗体。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“Fc区”或“片段可结晶区”用于定义抗体重链的C末端区域，包括天然序列Fc区和变体Fc区。在一些实施方式中，人IgG重链的Fc区定义为从Cys226位置处的氨基酸残基或从Pro230延伸至其羧基末端(根据EU编号***)。抗体重链的Fc区的边界还可以变化，例如缺失Fc区的C末端赖氨酸(根据EU编号***的残基447)或缺失Fc区的C末端甘氨酸和赖氨酸(根据EU编号***的残基446和447)。因此，在一些实施方式中，完整抗体的组合物可以包括去除了所有K447残基和/或G446+K447残基的抗体群体。在一些实施方式中，完整抗体的组合物可以包括没有去除K447残基和/或G446+K447残基的抗体群体。在一些实施方式中，完整抗体的组合物具有带有和不带有K447残基和/或G446+K447残基的抗体混合物的抗体群体。用于本文所述抗体的合适天然序列Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4。除非本文中另有规定，Fc区或恒定区中的氨基酸残基的编号方式依照EU编号***，又称作EU索引，如记载于Kabat等,Sequences of Proteins of Immunological Interest,第5版Public Health Service,National Institutes of Health,Bethesda,MD,1991。
术语“嵌合”抗体指其中的重和/或轻链的一部分自特定的来源或物种衍生，而重和/或轻链的剩余部分自不同来源或物种衍生的抗体。
术语“人源化”抗体是保留非人抗体的与抗原的结合活性，同时在人中具有较低免疫原性的抗体。例如，这可以通过保留非人CDR区并用其人对应物(即，恒定区以及可 变区的框架部分)替换抗体的其余部分来实现。
在某些实施方案中，本文中提供的抗体是嵌合抗体。在一个例子中，嵌合抗体包含非人可变区(例如自小鼠、大鼠、仓鼠、家兔、或非人灵长类，诸如猴衍生的可变区)和人恒定区。在又一个例子中，嵌合抗体是“类转换的”抗体，其中类或亚类已经自亲本抗体的类或亚类改变。嵌合抗体包括其抗原结合片段。
在某些实施方案中，嵌合抗体是人源化抗体。通常，将非人抗体人源化以降低对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。一般地，人源化抗体包含一个或多个可变区，其中CDR或其部分衍生自非人抗体，而FR或其部分衍生自人抗体。任选地，人源化抗体还会包含人恒定区。在一些实施方案中，将人源化抗体中的一些FR残基用来自非人抗体(例如提供CDR序列的抗体)的相应残基替代，以恢复或改善抗体特异性或亲和力。
人源化抗体及其生成方法综述于如Almagro and Fransson,Front.Biosci.13:1619-1633(2008)，并且进一步记载于如Riechmann等，Nature 332:323-329(1988)；Queen等，Proc.Nat'l Acad.Sci.USA 86:10029-10033(1989)。
此外，可以通过对组合文库筛选具有期望的一种或多种活性的抗体来分离本披露的抗体。用于筛选组合文库的方法综述于例如Lerner等，Nature Reviews 16:498-508(2016)。在某些噬菌体展示方法中，将VH和VL基因的全集分别通过PCR克隆，并在噬菌体文库中随机重组，然后可以用该噬菌体文库筛选抗原结合噬菌体，如记载于Winter等，Annual Review of Immunology 12:433-455(1994)的方法。噬菌体通常以scFv片段或以Fab片段展示抗体片段，免疫的文库可提供针对免疫原的高亲和力抗体，不需要构建杂交瘤。或者，可以克隆未免疫全集(例如来自人的)，以在没有任何免疫接种的情况下提供针对大范围非自身和自身抗原的抗体，如Griffiths等，EMBO Journal 12:725-734(1993)描述的。此外，也可以通过自干细胞克隆未重排的V基因区段，并使用含有随机序列的PCR引物扩增编码高度可变的CDR3区，并在体外实现重排来合成生成未免疫文库，如由Hoogenboom and Winter,Journal of Molecular Biology 227:381-388(1992)所描述的。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与本文定义的抗原结合片段相区分。该术语特别指轻链和重链包含恒定区的抗体。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域，VH)和抗体轻链可变结构域(或区域，VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。现有技术已公开众多 适于连接抗体VH和VL的接头，例如由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本披露的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“抗原”是指能够由诸如抗原结合蛋白(包括例如抗体)的选择性结合剂结合，且能够用于动物中以产生能够结合该抗原的抗体的分子或分子部分。抗原可具有一个或多个能够与不同的抗原结合蛋白(例如抗体)相互作用的表位。
术语“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，“结合亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间1:1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些)测量。
术语“特异性地结合”、“特异性结合”或“结合”是指抗体以比针对其他抗原或表位更高的亲和力，结合至某个抗原或其表位。通常，抗体以约1×10 -7M或更小(例如约1×10 -8M或更小、约1×10 -9M或更小、约1×10 -10M或更小、约1×10 -11M或更小，或者约1×10 -12M或更小)的平衡解离常数(KD)结合抗原或其表位。在一些实施方式中，抗体与抗原结合的KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的10％，或1％。可使用标准程序来测量KD，例如通过
Figure PCTCN2021135410-appb-000002
表面等离子体共振测定法测量的。然而，特异性结合至抗原或其表位的抗体可能对其它相关的抗原具有交叉反应性，例如，对来自其它物种(同源)(诸如人或猴，例如食蟹猕猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp))或狨猴(Callithrix jacchus)(commonmarmoset，marmoset)的相应抗原具有交叉反应性。
术语“KD”指解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域公知的方法测定抗体的KD值。用于测定抗体KD的方法包括使用生物传感***例如***测量表面等离子体共振，或通过溶液平衡滴定法(SET)测量溶液中的亲和力。
术语“核酸”在本文中可与术语“多核苷酸”互换使用，并且是指呈单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。所述术语涵盖含有已知核苷酸类似物或修饰的骨架残基或连接的核酸，所述核酸是合成的、天然存在的或非天然存在的，其具有与参考核酸相似的结合特性，并且以类似于参考核苷酸的方式代谢。此类类似物的实例 包括但不限于硫代磷酸酯、氨基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色***置的染色***置处。“编码抗TSLP抗体的分离的核酸”指编码抗体重链和轻链(或其片段)的一个或更多个核酸分子，包括在单一载体或分开的载体中的这样的一个或更多个核酸分子，和存在于宿主细胞中一个或更多个位置的这样的一个或更多个核酸分子。
除非另有说明，否则特定的核酸序列涵盖其保守修饰的变体(例如，简并密码子取代)和互补序列以及明确指明的序列。具体地，如下详述，简并密码子取代可以通过产生如下序列而获得，在这些序列中，一个或多个所选的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代(Batzer等，Nucleic Acid Res.19:5081,1991；Ohtsuka等,J.Biol.Chem.260:2605-2608,1985；和Rossolini等,Mol.Cell.Probes8:91-98,1994)。
氨基酸序列“同一性”指在比对氨基酸序列时，必要时引入间隙以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基相同的氨基酸残基的百分比。为测定氨基酸序列同一性的百分比，可以通过本领域技术的范围内的多种比对序列的方式来实现，例如，使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，例如腺相关病毒载体(AAV或AAV2)，其中另外的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、293细胞 和NS0细胞。
本披露的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人TSLP特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。包含抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物通常需要冷藏，或冷冻，如-70℃，或者冻干。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本披露的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行 改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。
表3.示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本披露靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本披露靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个 位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必然发生，该说明包括该事件或环境发生或不发生的场合。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂，例如糖、多元醇、山梨糖醇和氯化钠。
此外，本披露包括用于治疗与TSLP相关的疾病的药剂，所述药剂包含本披露的抗TSLP抗体作为活性成分。
本披露中与TSLP相关的疾病没有限制，只要它是与TSLP相关的疾病即可，例如利 用本披露的分子诱导的治疗反应可通过结合人类TSLP，然后阻遏TSLP与其受体结合，进而阻断TSLP相关的信号通路，抑制疾病的进展。
上述与TSLP相关的疾病可以通过用本披露的抗体检测或测定表达TSLP的细胞来诊断。
此外，本披露涉及用于免疫检测或测定目标抗原(例如TSLP)的方法、用于免疫检测或测定目标抗原(例如TSLP)的试剂、用于免疫检测或测定表达目标抗原(例如TSLP)的细胞的方法和用于诊断与目标抗原(例如TSLP)阳性细胞相关的疾病的诊断剂，其包含本披露的特异性识别目标抗原(例如人TSLP)并与胞外区的氨基酸序列或其三维结构结合的抗体或抗体片段作为活性成分。
在本披露中，用于检测或测定目标抗原(例如TSLP)的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
在本披露中，对用于检测或测定目标抗原(例如TSLP)的活体样品没有特别限制，只要它具有包含表达目标抗原(例如TSLP)的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本披露的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
实施例
以下结合实施例用于进一步描述本披露，但这些实施例并非限制本披露的范围。
本披露实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，《分子克隆——实验室手册》，冷泉港实验室；《当代分子生物学方法》，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.TSLP和TSLP受体的表达
分别将编码带His标签的人TSLP、食蟹猴TSLP、人TSLP受体胞外区序列克隆至phr载体上，构建成表达质粒，然后转染HEK293。具体转染步骤为：前一天将HEK293E细胞以0.8×10 6/mL接种于Freestyle表达培养基(含有1％FBS)中，放置于37℃恒温摇床(120rpm)继续培养24小时。24小时后，将转染质粒和转染试剂PEI用0.22μm的滤器除菌，然后将转染质粒调整为100μg/100mL细胞，PEI(1mg/mL)和质粒的质量比为3:1，以200mL HEK293E细胞的转染为例，取10mL的Opti-MEM和200μg质粒混匀，静置5分钟(min)；另取10mL的Opti-MEM和600μg PEI混匀，静置5min。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入200mL HEK293E的细胞中，放入8％CO 2，120rpm，37℃的摇床中培养。转染第3天，补充10％体积的补料培养基。待转染第6天，取样4500rpm离心10min收集细胞上清，过滤，将重组的TSLP和TSLP受体蛋白上清通过实施例2进行纯化，纯化的蛋白可用于下述各实施例实验中。相关序列如下所示：
1.带his标签的人TSLP抗原(huTSLP-His)氨基酸序列
Figure PCTCN2021135410-appb-000003
注释：下划线为信号肽序列；斜体部分为Flag-His6-tag标记。
SEQ ID NO:1
2.带his标签的食蟹猴TSLP抗原(cynoTSLP-His)氨基酸序列
Figure PCTCN2021135410-appb-000004
注释：下划线为信号肽序列；斜体部分为flag-His6-tag标记。
SEQ ID NO:2
3.带Fc标签的人TSLP受体胞外区(人-TSLPR-Fc-ECD)氨基酸序列：
Figure PCTCN2021135410-appb-000005
Figure PCTCN2021135410-appb-000006
注释：下划线部分为人-TSLPR的胞外区，斜体部分为接头-人Fc-tag。
SEQ ID NO:3
4.人TSLP受体全长序列氨基酸序列：
Figure PCTCN2021135410-appb-000007
注释：下划线部分为信号肽
SEQ ID NO:4
5.人IL7Rα全长序列氨基酸序列(Uniprot编号：P16871)
Figure PCTCN2021135410-appb-000008
注释：下划线部分为信号肽
SEQ ID NO:5
实施例2.TSLP和TSLP受体(TSLPR)重组蛋白的纯化
2.1带His标签的各种属TSLP重组蛋白的纯化
将细胞表达上清高速离心去除杂质，过滤，PBS溶液平衡镍柱，冲洗10倍柱体积。将过滤后的上清上柱。用含有30mM咪唑的PBS溶液冲洗柱子，至A 280读数降至基线。 再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。PBS浓缩换液，LC-MS鉴定为正确后分装备用。得到带His标签的人TSLP和食蟹猴TSLP。
2.2带人Fc标签的各种属TSLP和人TSLP受体胞外区重组蛋白的纯化
将细胞表达上清高速离心去除杂质，重组抗体表达上清用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用100mM乙酸pH3.5洗脱目的蛋白，用1M Tris-HCl，pH8.0中和。浓缩换液，所得到的蛋白经电泳，LC-MS鉴定为正确后分装备用。
实施例3.重组TSLP受体和IL7Rα受体细胞系的构建和鉴定
为筛选可以阻断TSLP结合TSLP受体的抗体，构建了同时表达人TSLP受体和人IL7Rα(TSLPR/IL7Rα)的CHO-K1和BaF3细胞株。采用慢病毒包装目的基因TSLPR/IL7Rα克隆至细胞内形成稳定高表达细胞株。首先分别将人TSLPR和人IL7Rα基因克隆至pCDH-CMV-MCS-EF1-puro和pCDH-CMV-MCS-EF1-Neo(SBI，CD500B-1)质粒内，然后通过慢病毒感染的方法将人TSLPR克隆至CHO-K1和BaF3细胞株内，经10μg/mL嘌呤霉素(puromycin，Gibco，US)筛选压力下选择培养三周。在此基础上进行第二轮感染，再将人IL7Rα基因克隆进去，用1mg/mL G418(Gibco，US)和10μg/mL嘌呤霉素时筛选两至三周。最后通过流式分选的方法，筛选出同时高表达TSLPR和IL7Rα的CHO-K1和BaF3单克隆细胞株。
实施例4.抗人TSLP单克隆抗体的相关序列
本披露中的抗体可以通过杂交瘤筛选、噬菌体展示、亲和力成熟等技术获得。本披露发明人经过大量实验，获得了一系列高亲和力的TSLP抗体。示例性的，抗TSLP抗体的序列如下：
4.1抗TSLP的可变区序列如下：
Figure PCTCN2021135410-appb-000009
Figure PCTCN2021135410-appb-000010
Figure PCTCN2021135410-appb-000011
注释：下划线部分为互补决定区序列。
4.2本披露中抗TSLP抗体的CDR区序列如下表所示：
表4.抗体重链及轻链的CDR区序列
Figure PCTCN2021135410-appb-000012
Figure PCTCN2021135410-appb-000013
4.3本披露中抗TSLP抗体及其对应的可变区序列如下：
表5.抗体的可变区
抗体 VH VL 抗体 VH VL
Ab-1 VH1 VL1 Ab-14 VH8 VL1
Ab-2 VH2 VL1 Ab-23 VH17 VL1
Ab-6 VH3 VL2 Ab-24 VH18 VL1
Ab-9 VH3 VL3      
Ab-25 VH19 VL1      
Ab-27 VH21 VL1      
将上述抗体的轻、重链可变区与抗体的轻、重链恒定区连接后形成全长抗体。如，Ab-1抗体的重链可变区为VH1，轻链可变区为VL1，其他类推；示例性的，可使用IgG1重链恒定区(如：SEQ ID NO:34)和λ轻链恒定区(如SEQ ID NO:35)作为本披露中抗体的恒定区。
4.4.抗体恒定区
抗体的重链恒定区可选自IgG1、IgG2、IgG4及其变体的恒定区。示例性的，本披露中使用了IgG1恒定区，其序列如SEQ ID NO:34所示。轻链恒定区可选自人源κ、λ链或其变体的轻链恒定区。示例性的，本披露中使用了人源λ链的恒定区，其序列如SEQ ID NO:35所示。
>IgG1恒定区
Figure PCTCN2021135410-appb-000014
>λ轻链恒定区
Figure PCTCN2021135410-appb-000015
将本披露中的重、轻链可变区与上述恒定区重组，得到重、轻链的全长序列。示例性的，抗体的序列如下所示：
>Ab-14抗体重链：
Figure PCTCN2021135410-appb-000016
Figure PCTCN2021135410-appb-000017
>Ab-14抗体轻链：
Figure PCTCN2021135410-appb-000018
>Ab-23抗体重链：
Figure PCTCN2021135410-appb-000019
>Ab-23抗体轻链：
Figure PCTCN2021135410-appb-000020
>Ab-24抗体重链：
Figure PCTCN2021135410-appb-000021
Figure PCTCN2021135410-appb-000022
>Ab-24抗体轻链：
Figure PCTCN2021135410-appb-000023
本披露中使用的对照抗体为Amg157，其抗体序列如下：>Amg157的重链序列
Figure PCTCN2021135410-appb-000024
>Amg157的轻链序列
Figure PCTCN2021135410-appb-000025
本披露的抗体可用常规基因克隆、重组表达的方法进行克隆、表达和纯化。
测试例
测试例1：Biacore测定抗TSLP抗体与人和食蟹猴TSLP的结合
用Biacore T200(GE)仪器测定人源化TSLP抗体与人和食蟹猴TSLP的亲和力。
用Protein A生物传感芯片(Cat.#29127556,GE)亲和捕获待测分子，然后于芯片表面流经抗原(huTSLP-his，cynoTSLP-his，实施例1制备所得)，用Biacore T200仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用甘氨酸-盐酸再生溶液(pH 1.5Cat.#BR-1003-54,GE)将生物传感芯片洗净再生。用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，如下表所示。
表6.抗体与人和食蟹猴TSLP的亲和力
Figure PCTCN2021135410-appb-000026
结果显示，本披露中的抗TSLP抗体与人和食蟹猴TSLP具有很高的亲和力，优于对照Amg157抗体。
测试例2：基于ELSA的抗TSLP抗体阻断TSLP结合TSLP受体实验
TSLP受体有两个亚基，TSLPR和IL7R，其中TSLPR为TSLP特异性受体，IL7R为TSLP与IL7的共用受体。TSLP先与TSLPR结合，再与IL7R结合。本测试例用来鉴定TSLP抗体是否可以阻断TSLP结合到重组表达的TSLPR受体蛋白胞外区。
将人-TSLPR-Fc-ECD(2μg/mL，SEQ ID NO:3)包被ELISA板，4℃孵育过夜，弃去液体后，加入用PBS稀释的5％脱脂牛奶封闭液200μL/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％吐温-20)洗板3次，将生物素标记的huTSLP-Fc抗原配制成3nM，待测抗体从200nM开始梯度稀释，抗原和抗体1：1混匀后37℃放置15min，100μL每孔加入酶标板中，37℃放置1小时(h)，用PBST洗板3次后，加入100μL/孔用样品稀释液以1：4000浓度稀释的 Streptavidin-Peroxidase Polymer，于37℃孵育1小时。用PBST洗板5次后，加入100μL/孔TMB显色底物(KPL，52-00-03)，于室温孵育3-10min，加入100μL/孔1MH 2SO 4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算TSLP抗体阻断TSLP与TSLPR结合的IC50值，结果见下表。
表7.抗体阻断活性结果
抗体 IC50(nM)
Amg157 0.61
Ab-1 0.18
Ab-2 0.18
Ab-3 0.2
Ab-6 0.2
Ab-9 0.26
Ab-14 0.2
Ab-23 0.19
Ab-24 0.21
Ab-25 1.12
Ab-27 5.98
结果显示，本披露抗体Ab-1、Ab-2、Ab-3、Ab-6、Ab-9、Ab-14、Ab-23和Ab-24均可较强地抑制TSLP与其受体TSLPR的结合；其抑制活性优于对照Amg157抗体。
测试例3：抗TSLP抗体抑制TSLP诱导的过表达TSLPR/IL7R的BaF3细胞增殖实验
TSLP可以与BaF3表面的TSLPR/IL7R结合，进而促进BaF3的增殖。本测试例用来鉴定本披露抗体可以阻断TSLP的诱导BaF3增殖的活性。
实验过程如下：
过表达TSLPR/IL7R的BaF3细胞培养于10％FBS以及2ng/mL rhIL3(联科生物，Catalog No.96-AF-300-03-20)的RPMI1640培养基中，于37℃，5％CO 2培养箱中培养，细胞密度不超过1×10 6个/mL。检测抗体时，取对数生长期的细胞用PBS洗三遍800rpm离心5min，用RPMI1640(2％FBS，重组人TSLP-Fc：40ng/mL)调整细胞密度8000个/孔/90μL，并加入10μL梯度稀释的待测抗体到96孔板中。培养2天后，加入30μL cell titer，混匀后进行检测。根据读值计算IC50。结果如表8所示。
表8.抗体抑制BaF3细胞增殖活性
抗体 IC50(nM)
Ab-1 2.24
Ab-2 2.63
Ab-3 2.5
Ab-6 12.34
Ab-9 13
Ab-14 0.43
Ab-23 0.43
Ab-24 0.37
结果显示，本披露的抗体Ab-14、Ab-23和Ab-24具有较强的抑制TSLP介导的BaF3细胞增殖的能力。
测试例4：抗TSLP抗体阻断TSLP诱导的天然CD4 +T细胞往Th2细胞的分化
TSLP可以诱导原代髓样mDC细胞成熟，成熟的mDC细胞高表达OX40配体，OX40配体可以与天然CD4 +T细胞表面的OX40结合，进而使天然CD4 +T分化成Th2细胞，产生IL4/IL5/IL13等免疫应答相关因子，使机体发生Th2炎症反应。本测试例用来检测本披露中的抗体可以阻断TSLP诱导的Th2细胞的分化。
采用磁珠分选的方法，(CD1c(BDCA-1) +树突细胞分离试剂盒Miltenyi Biotec)从人外周血单核细胞(PBMC)中分离纯化初始髓样DC，将得到的mDC种植于96孔细胞培养板内。梯度稀释的抗体和重组表达的人TSLP(huTSLP-his，终浓度50ng/mL)预先孵育(37℃)45分钟左右，再分别加入到含有mDC的各细胞培养孔内，37℃培养24小时。收集刺激成熟的mDC，用PBS洗两次。用磁珠分离法(Myltenyi，Biotec)从PBMC中提取CD4 +CD45RA +天然T细胞。将分离得到的天然T细胞和成熟的mDC以5:1的比例混合种植于96孔细胞培养板内，共同培养6天。收集细胞，种植于anti-CD3(10μg/mL)预包被的96孔板内，并加入anti-CD28(1μg/mL)，再刺激分化的T细胞，培养24小时，最后收集细胞培养上清。ELISA检测上清中细胞分泌的Th2相关的细胞因子。IL-4和IL-5细胞因子用的是R&D的ELISA试剂盒检测，TNF-α和IL-13用欣博盛的ELISA试剂盒检测。结果如表9和图1A-1D所示。
表9.Th2相关的细胞因子浓度(给药量1μg/mL)
细胞因子 Amg157 Ab-23
IL-13(pg/mL) 1789.9 537.9
IL-4(pg/mL) 50.1 38.7
IL-5(pg/mL) 99.1 68.1
TNF-α 263.2 194.2
结果显示，本披露所得抗体Ab-23可以显著抑制Th2细胞因子IL4、IL5、IL13和TNF-α的产生，表明本披露所得抗体可以阻断TSLP诱导的Th2细胞的分化。
虽然以上描述了本披露的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本披露的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本披露的保护范围由所附权利要求书限定。

Claims (15)

  1. 一种抗TSLP抗体，其包含重链可变区和轻链可变区，其中：
    i)重链可变区包含氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；且轻链可变区包含氨基酸序列分别如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的LCDR1、LCDR2和LCDR3；
    或
    ii)重链可变区包含氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示的HCDR1、HCDR2和HCDR3；且轻链可变区包含氨基酸序列分别如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的LCDR1、LCDR2和LCDR3；
    或
    iii)重链可变区包含氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:23和SEQ ID NO:25所示的HCDR1、HCDR2和HCDR3，且轻链可变区包含氨基酸序列分别如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的抗TSLP抗体，其中所述抗TSLP抗体是鼠源抗体、嵌合抗体或人源化抗体。
  3. 根据权利要求1或2所述的抗TSLP抗体，其中：
    所述重链可变区的氨基酸序列如SEQ ID NO:13、SEQ ID NO:12或SEQ ID NO:14所示，或与SEQ ID NO:13、SEQ ID NO:12或SEQ ID NO:14所示的序列分别具有至少90％的序列同一性；且
    所述轻链可变区的氨基酸序列如SEQ ID NO:6所示，或与如SEQ ID NO:6所示的序列具有至少90％的序列同一性。
  4. 根据权利要求3所述的抗TSLP抗体，其包含氨基酸序列如SEQ ID NO:13、SEQ ID NO:12或SEQ ID NO:14所示的重链可变区；和氨基酸序列如SEQ ID NO:6所示的轻链可变区。
  5. 根据权利要求1至4中任一项所述的抗TSLP抗体，其中所述的抗TSLP抗体包含抗体重链恒定区和轻链恒定区。
  6. 根据权利要求5所述的抗TSLP抗体，其中所述的抗TSLP抗体包含序列如SEQ ID NO:34所示的重链恒定区；和/或序列如SEQ ID NO:35所示的轻链恒定区。
  7. 根据权利要求1至6中任一项所述的抗TSLP抗体，其中所述的抗TSLP抗体包含重链和轻链，其中：
    重链氨基酸序列如SEQ ID NO:38、SEQ ID NO:36或SEQ ID NO:39所示，或分别与SEQ ID NO:38、SEQ ID NO:36或SEQ ID NO:39所示的序列具有至少85％的序列同一性；且
    轻链氨基酸序列如SEQ ID NO:37所示，或与SEQ ID NO:37所示的序列具有至少85％的序列同一性。
  8. 根据权利要求7所述的抗TSLP抗体，其中所述的抗TSLP抗体包含：
    如SEQ ID NO:38所示的重链和如SEQ ID NO:37所示的轻链；或
    如SEQ ID NO:36所示的重链和如SEQ ID NO:37所示的轻链；或
    如SEQ ID NO:39所示的重链和如SEQ ID NO:37所示的轻链。
  9. 根据权利要求1至8中任一项所述的抗TSLP抗体，其中所述的抗TSLP抗体具有以下特性中的一种或多种：
    a)以小于9pM的KD值与人TSLP结合；
    b)以小于0.6nM的IC50值阻断TSLP与TSLPR的结合；或
    c)以小于9pM的KD值与食蟹猴TSLP结合。
  10. 核酸分子，其编码权利要求1至9中任一项所述的抗TSLP抗体。
  11. 一种宿主细胞，其包含权利要求10所述的核酸分子。
  12. 一种药物组合物，其含有治疗有效量的根据权利要求1至9中任一项所述的抗TSLP抗体，或根据权利要求10所述的核酸分子，或根据权利要求11所述的宿主细胞，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  13. 一种用于体外或离体免疫检测或测定TSLP的方法，所述方法包括使用权利要求1至9中任一项所述的抗TSLP抗体的步骤。
  14. 一种试剂盒，其包含权利要求1至9中任一项所述的抗TSLP抗体。
  15. 权利要求1至9中任一项所述的抗TSLP抗体，或权利要求10所述的核酸分子，或权利要求11所述的宿主细胞，或权利要求12所述的药物组合物在制备用于治疗炎症疾病或纤维化疾病的药物中的用途；优选地，
    其中所述的炎症疾病选自：过敏症、皮炎、哮喘、过敏性结膜炎、变应性鼻炎、嗜酸性粒细胞性胃肠炎、过敏性支气管肺曲霉病、过敏性真菌鼻窦炎、慢性嗜酸性粒细胞性肺炎、嗜酸性粒细胞性支气管炎、炎性肠病和过敏性鼻窦炎；
    其中所述的纤维化疾病选自：硬皮症、间质性肺病、特发性肺纤维化、B型或C型慢性肝炎引发的纤维化、辐射诱发的纤维化和伤口愈合引发的纤维化；
    更优选地，其中所述的炎症疾病或纤维化疾病与TSLP相关。
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