Anti-tslp antibodies and uses thereof

Abstract

An anti-TSLP antibody or an antigen binding fragment thereof is provided. Also provided are polynucleotides encoding the antibody or antigen binding protein constructs, as well as expression vectors and host cells comprising the same. Also provided are immunoconjugates, pharmaceutical compositions and medicaments, as well as their use in treating diseases or disorders.

Description

[0001] Priority requirements

[0002] This application claims priority to Chinese application No. 202311318859.2, filed on October 12, 2023. The entire contents of the aforementioned application are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of immunopharmaceuticals, and more particularly to antibodies or antigen-binding fragments thereof capable of specifically binding to TSLP. This disclosure also relates to pharmaceutical compositions comprising them and their uses. Background Technology

[0004] Autoimmune diseases are conditions caused by an abnormal immune response to normally functioning parts of the body. There are at least 80 types of autoimmune diseases. The causes of autoimmune diseases are often unclear. Some autoimmune diseases (such as lupus) are familial, while others may be triggered by infection or other environmental factors. Some common autoimmune diseases include, for example, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0005] Recent clinical and commercial successes of therapeutic antibodies have sparked great interest in using antibodies to treat various immune-related disorders. There is a need to develop antibodies for use in a variety of antibody-based therapies to treat immune disorders. Summary of the Invention

[0006] This disclosure relates to anti-TSLP antibodies or antigen-binding fragments thereof that can specifically bind to TSLP. The antibodies or antigen-binding fragments thereof may be used to treat diseases or disorders (e.g., cancer or immune disorders).

[0007] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TSLP (thymic stromal lymphopoietin), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) 1, 2, and 3, and in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; the light chain variable region comprises CDR1, CDR2, and CDR3, and in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence; in some embodiments, the selected VH The amino acid sequences of CDR1, VH CDR2, and VH CDR3 and the selected amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 are one of the following: (1) The selected amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 are shown in SEQ ID NO:37, 38, and 39, respectively, and the selected amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 are shown in SEQ ID NO:40, 41, and 42, respectively; (2) The selected amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 are shown in SEQ ID NO:86, 38, and 39, respectively, and the selected amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 are shown in SEQ ID NO:40, 41, and 42, respectively; (3) The selected amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 are shown in SEQ ID NO:86, 38, and 39, respectively, and the selected amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 are shown in SEQ ID NO:40, 41, and 42, respectively; NO:117, 118, 119 are shown, and the selected VL CDR1, VL CDR2 and VL CDR3 amino acid sequences are shown as SEQ ID NO:120, 121 and 122, respectively.

[0008] In some embodiments, VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO:37, 38, and 39, respectively, and VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO:40, 41, and 42, respectively. In some embodiments, VH CDR1 is determined according to the AbM definition, and in some embodiments, VH CDR2, VHCDR3 and VL CDR1, VL CDR2, and VL CDR3 are determined according to the Kabat definition. In some embodiments, according to the Kabat definition, VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO:86, 38, and 39, respectively, and VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO:40, 41, and 42, respectively. In some embodiments, according to the definition of Chothia, VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO: 117, 118, and 119, respectively, and VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO: 120, 121, and 122, respectively.

[0009] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TSLP, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to a selected VH sequence, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to a selected VL sequence; in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO:43 and the selected VL sequence is SEQ ID NO:44; and (2) the selected VH sequence is SEQ ID NO:45 and the selected VL sequence is SEQ ID NO:46. In some embodiments, the antibody or antigen-binding fragment specifically binds to human TSLP and / or monkey TSLP. In some embodiments, the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated CDR regions, scFv, and nanobodies.

[0010] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TSLP, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 identical to VH CDR1, VH CDR2, and VHCDR3 of a selected VH sequence, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 identical to VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence; in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO:43 and the selected VL sequence is SEQ ID NO:44; and (2) the selected VH sequence is SEQ ID NO:45 and the selected VL sequence is SEQ ID NO:46.

[0011] In one aspect, this disclosure relates to antibodies or antigen-binding fragments thereof that cross-compete with the antibodies or antigen-binding fragments thereof described herein.

[0012] In some embodiments, the antibody or its antigen-binding fragment is a bispecific antibody or a multispecific antibody or its antigen-binding fragment.

[0013] In one aspect, this disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising: (1) an immunoglobulin heavy chain or a fragment thereof, said heavy chain or fragment thereof comprising VH, said VH comprising CDR1, CDR2 and CDR3, said CDR comprising amino acid sequences shown in SEQ ID NO:37, 38, 39, respectively; amino acid sequences shown in SEQ ID NO:86, 38, 39, respectively; or amino acid sequences shown in SEQ ID NO:117, 118, 119, respectively; and in some embodiments, VH binds to TSLP when it pairs with a light chain variable region (VL) comprising an amino acid sequence shown in SEQ ID NO:44 or 46; or (2) an immunoglobulin light chain or a fragment thereof, said light chain or fragment thereof comprising VL, said VL comprising CDR1, CDR2 and CDR3, said CDR comprising amino acid sequences shown in SEQ ID NO:40, 41, 42, respectively; or amino acid sequences shown in SEQ ID NO:46, 38, 39, respectively; or amino acid sequences shown in SEQ ID NO:47, 38, 39 ... The amino acid sequences shown in ID NO: 120, 121, 122; and in some embodiments, VL binds to TSLP when it pairs with VH containing the amino acid sequence shown in SEQ ID NO: 43 or 45. In some embodiments, VH specifically binds to human TSLP when VH pairs with VL. In some embodiments, the immunoglobulin heavy chain or a fragment thereof is the heavy chain or a fragment thereof of human immunoglobulin, or the heavy chain or a fragment thereof of humanized immunoglobulin. In some embodiments, the nucleic acid encodes a single-stranded variable fragment (scFv), a bispecific or multispecific antibody, or an antigen-binding fragment thereof. In some embodiments, the nucleic acid is cDNA.

[0014] In one aspect, this disclosure relates to a vector comprising one or more nucleic acids described herein, or a nucleic acid encoding an antibody or an antigen-binding fragment thereof described herein.

[0015] In one aspect, this disclosure relates to a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, this disclosure relates to a cell comprising one or more nucleic acids described herein, or nucleic acids encoding antibodies or antigen-binding fragments thereof described herein.

[0016] In one aspect, this disclosure relates to a method for generating an antibody or an antigen-binding fragment thereof, or an antigen-binding protein construct thereof, the method comprising: (a) culturing the cells under conditions sufficient to cause the cells described herein to generate an antibody or an antigen-binding fragment thereof, or an antigen-binding protein construct thereof; and (b) collecting the antibody or antigen-binding fragment thereof, or antigen-binding protein construct generated by the cells.

[0017] In one aspect, this disclosure relates to an antibody-drug conjugate comprising a therapeutic agent covalently bound to an antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

[0018] In one aspect, this disclosure relates to a pharmaceutical composition comprising: a pharmaceutically acceptable carrier, and an antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate, as described herein.

[0019] In one aspect, this disclosure relates to a kit comprising an antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate.

[0020] In one aspect, this disclosure relates to a method of treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, an antibody-drug conjugate, or a pharmaceutical composition.

[0021] In one aspect, this disclosure relates to a method of treating a subject suffering from an immune disorder, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, an antibody-drug conjugate, or a pharmaceutical composition. In some embodiments, the immune disorder is allergy, asthma, or atopic dermatitis. In some embodiments, the immune disorder is type II or mixed allergic disease. In some embodiments, the subject is a human or a non-human animal.

[0022] In one aspect, this disclosure provides an isolated anti-TSLP antibody or an antigen-binding fragment thereof, comprising: HCDR1 shown in SEQ ID NO: 37, HCDR2 shown in SEQ ID NO: 38, HCDR3 shown in SEQ ID NO: 39, LCDR1 shown in SEQ ID NO: 40, LCDR2 shown in SEQ ID NO: 41, and LCDR3 shown in SEQ ID NO: 42.

[0023] In one aspect, this disclosure relates to a polynucleotide encoding the anti-TSLP antibody or its antigen-binding fragment (e.g., any anti-TSLP antibody or its antigen-binding fragment described herein).

[0024] In one aspect, this disclosure relates to expression vectors comprising the said polynucleotide (e.g., any polynucleotide described herein).

[0025] In one aspect, this disclosure relates to a host cell incorporating the said polynucleotide (e.g., any polynucleotide described herein) or its expression vector.

[0026] In one aspect, this disclosure relates to immune conjugates comprising the said anti-TSLP antibody or an antigen-binding fragment thereof (e.g., any anti-TSLP antibody or an antigen-binding fragment thereof described herein).

[0027] In one aspect, this disclosure relates to a pharmaceutical composition comprising: the anti-TSLP antibody or an antigen-binding fragment thereof (e.g., any anti-TSLP antibody or antigen-binding fragment thereof described herein), or an immunoconjugate thereof (e.g., any immunoconjugate described herein), and optionally pharmaceutically acceptable excipients.

[0028] In one aspect, this disclosure relates to a kit comprising the anti-TSLP antibody or an antigen-binding fragment thereof (e.g., any anti-TSLP antibody or antigen-binding fragment thereof described herein), an immunoconjugate thereof (e.g., any immunoconjugate described herein), or a pharmaceutical composition (e.g., any pharmaceutical composition described herein).

[0029] In one aspect, this disclosure relates to the use of the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit in the preparation of a medicament for treating or preventing a disease or disorder (e.g., cancer or autoimmune disease). Alternatively, this disclosure relates to the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit for the preparation of an inhibitor of TSLP. Alternatively, this disclosure relates to a method of inhibiting TSLP, comprising administering the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit to a subject in need.

[0030] In one aspect, this disclosure relates to the use of the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit in the preparation of a medicament for the treatment or prevention of a disease or disorder (e.g., cancer or autoimmune disease). Alternatively, this disclosure relates to a method of treating or preventing a disease or disorder (e.g., cancer or autoimmune disease) comprising administering the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit to a subject in need. Alternatively, this disclosure relates to the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit for the treatment or prevention of a disease or disorder (e.g., cancer or autoimmune disease). In some embodiments, the disease or disorder is a type II or mixed allergic disease, such as asthma and / or atopic dermatitis.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples described are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, the documents of this application (including definitions) shall prevail.

[0032] Other features and advantages of the invention will be apparent from the following detailed description, drawings, and claims. Attached Figure Description

[0033] Figure 1A-Figure 1B The effects of IL-4, IL-13, and / or TSLP on CCL-17 release from PBMCs are shown. Figure 1A The release of CCL-17 from PBMCs was demonstrated by TSLP alone, IL-4 alone, IL-13 alone, and a mixture of the three (3MIX); Figure 1B The inhibitory effects of immunoglobulin IgG1 (control), Dupilumab, Tezepelumab, and the combination of Dupilumab and Tezepelumab on the release of CCL-17 were demonstrated.

[0034] Figures 2A-2D The effect of TSLP-mediated DC maturation on the release of naïve T cell-mediated cytokines IL-5 and IL-13 was demonstrated. Figure 2A A schematic diagram is shown of DC-driven naïve T cells developed into Th2 cells by TSLP stimulation, the cells releasing cytokines such as IL-4, IL-5 and IL-13. Figure 2B The study showed that co-culturing TSLP-stimulated DCs with naïve T cells significantly enhanced the release of Th2 cytokines (e.g., IL-5 / IL-13) compared to TSLP-stimulated DCs or CD4+ T cells alone. Figures 2C-2D The results showed that the inhibition levels of IL-5 release were comparable across the test groups. However, for IL-13 release, administration of either anti-IL-4Rα antibody or anti-TSLP antibody alone was almost ineffective in inhibiting its release. The combined administration of anti-IL-4Rα antibody and anti-TSLP antibody significantly inhibited its release in a dose-dependent manner.

[0035] Figure 3 Three bispecific antibody molecular structures are shown.

[0036] Figures 4A-4BThe blocking function of each bispecific antibody prepared in Example 2 on the IL-4Rα / IL-4 / IL-13 and TSLP / TSLPR pathways is shown. Figure 4A The blocking function of each bispecific antibody prepared in Example 2 on the IL-4Rα / IL-4 / IL-13 pathway is shown. Figure 4B The blocking function of each bispecific antibody prepared in Example 2 on the TSLP / TSLPR pathway is shown.

[0037] Figures 5A-5B The blocking activity of the 11H7E11 chimeric antibody and the scFv antibody against TSLP / TSLPR was demonstrated. Figure 5A The results of detecting the blocking activity of the 11H7E11 chimeric antibody against TSLP / TSLPR using CTLL2-mCD127-hTSLPR-stat5-Luc2 reporter cells are shown. Figure 5B The results show the inhibitory effects of 11H7E11 chimeric antibody and scFv antibody on the secretion of CCL-17 chemokine by TSLP-induced human mDC cells.

[0038] Figures 6A-6B A schematic diagram of an exemplary bispecific antibody structure is shown. Figure 6A ) and the structures of peptide chain #1 and peptide chain #2 therein ( Figure 6B ).

[0039] Figures 7A-7B The CE-SDS results of the candidate molecule in Example 8 are shown. Figure 7A ), and no significant increase in aggregates and fragmentation products was observed in candidate molecule H6-5G-hz11H7-2-scFv ( Figure 7B ).

[0040] Figures 8A-8B The results of in vitro biological function tests show the effect of bispecific antibodies with linkers of different lengths on blocking function. Figure 8A The blocking function of each candidate bispecific antibody in Example 8 on the IL-4Rα / IL-4 / IL-13 pathway is shown. Figure 8B The blocking function of each candidate bispecific antibody in Example 8 on the TSLP / TSLPR pathway is shown.

[0041] Figures 9A-9B This demonstrates the bispecific antibody inhibition of hIL-4 ( Figure 9A ) and hIL-13 ( Figure 9B Results of TF-1 cell proliferation experiments induced by TF-1.

[0042] Figure 10The results of the CTLL2 reporter gene cell activity assay using bispecific antibodies bound to TSLP are shown.

[0043] Figures 11A-11C The bispecific antibodies against hIL-4 and hIL-13 were shown. Figure 11A ), hTSLP ( Figure 11B ), and IL-4 and IL-13 simultaneously with TSLP ( Figure 11C Results of experiments inhibiting the release of CCL-17 activity from PBMCs induced by PBMCs.

[0044] Figures 12A-12D The therapeutic effect of bispecific antibodies on asthmatic mice was demonstrated. Figure 12A The total number of lymphocytes after treatment is shown. Figure 12B The total number of eosinophils after treatment is shown. Figure 12C The total number of monocytes after treatment is shown. Figure 12D The total number of neutrophils after treatment is shown.

[0045] Figure 13 The changes in blood drug concentration of bispecific antibodies at different time points are shown.

[0046] Figure 14 The results show the changes in blood drug concentrations at different time points after FcRn humanized mice were administered bispecific antibodies D5-5G-hz11H7-2-scFv and D5-11H7-YTE at a dose of 10 mg / kg.

[0047] Figures 15A-15C The VH and VL CDR sequences of the antibodies discussed in this disclosure are listed.

[0048] Figure 16 The VH and VL sequences of the antibodies discussed in this disclosure are listed.

[0049] Figures 17A-17C The VH and VL CDR common sequences of the anti-IL-4Rα antibodies discussed in this disclosure are listed.

[0050] Figure 18 Additional sequences discussed in this disclosure are listed. Detailed Implementation

[0051] TSLP (Thymic Stromal Lymphopoietin) is a cytokine primarily derived from epithelial cells. It transmits signals through a heterodimeric receptor complex binding to the TSLP receptor and the IL-7Rα chain, and has been found to promote type 2 helper T (TH2) cell responses, participating in immune responses in various inflammatory diseases. TSLP is involved in the pathogenesis of asthma, and abnormal TSLP signaling is closely associated with other inflammatory allergic diseases, including atopic dermatitis. In 2021, the US FDA approved Tezspire, an anti-TSLP antibody jointly developed by AstraZeneca and Amgen. ® As a medication for treating severe asthma.

[0052] Antibody drugs targeting multiple targets for various allergic diseases, such as atopic dermatitis and asthma, are currently under development.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0054] definition

[0055] The term "antibody" herein refers to a protein or polypeptide capable of specifically recognizing and binding to an antigen, encompassing a wide range of natural and artificial antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, single-domain antibodies, full-length antibodies, and antibody fragments exhibiting the desired biological activity. In some embodiments, antibodies may be classified into five isotypes based on heavy chain class: IgG, IgM, IgD, IgA, and IgE.

[0056] When referring to antibodies, the term "isolated" in this article means that the antibody is essentially free of other cellular components that bind to it in the natural state. For example, an isolated antibody may be an antibody that has been removed from the natural environment.

[0057] As used herein, the term "bispecific" means that an antigen-binding construct (e.g., an antibody) comprises two antigen-binding parts (e.g., antigen-binding fragments) each having a specific binding specificity. For example, a first antigen-binding fragment and a second antigen fragment bind specifically to epitopes on a first antigen and a second antigen, respectively, or bind specifically to different epitopes of the same antigen, respectively.

[0058] The term "antigen-binding fragment" in antibody refers to a portion or segment of a full-length antibody with fewer amino acid residues than a full-length antibody, but capable of binding antigens or competing with full-length antibodies (i.e., full-length antibodies from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv, diabody antibodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting full-length antibodies with papain. Furthermore, digestion of complete antibodies with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. The Fv fragment consists of VL (light chain variable region) and VH (heavy chain variable region) domains on one arm of the antibody. The two domains, VL and VH, can be encoded by independent genes, but they can also be generated as a single protein chain using a recombinant approach, connecting the two domains with a synthetic linker peptide. In this single protein chain, the VL and VH regions pair to form a single-chain Fv (scFv).

[0059] The term "scFv" in this article includes the VH and VL domains of an antibody that are present in a single polypeptide chain.

[0060] The term “CDR” (complementarity-determining region), also known as “hypervariant region (HVR)”, as used in this article, refers to each region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. Natural antibodies typically contain three CDRs (i.e., HCDR1 to HCDR3) located in the heavy chain variable region and three CDRs (LCDR1 to LCDR3) located in the light chain variable region. Several well-known definitions in the field can be used to distinguish between heavy and light chain CDRs, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics (IMGT) database (the international ImMunoGeneTics information system, http: / / imgt.cines.fr), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., “A New Clustering of Antibody CDR LoopConformations”, Journal of Molecular Biology, 406, 228-256 (2011)).

[0061] Table 1. CDRs determined using different numbering schemes

[0062]

[0063] *Based on Kabat numbering

[0064] **Based on Chothia / Martin numbering

[0065] When referring to antibodies, the terms "variable region," "V region," or "variable domain" are used interchangeably. They refer to the structural domains of the antibody heavy or light chain that participate in the specific binding of the antibody to the antigen. They typically include an amino acid sequence arranged from the N-terminus to the C-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0066] The term "chimeric antibody" refers to an antibody containing sequences derived from two different antibodies (typically from different species), for example, wherein (a) a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site is linked to a constant region of different or altered class, effector function, and / or species origin, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that provides a new biological function to the chimeric antibody; or (b) a variable region or a portion thereof is altered, replaced, or exchanged with a variable region of different or altered antigen specificity.

[0067] As used herein, the term "humanized antibody" refers to an antibody that retains the antigen-specific reactivity of a non-human antibody (such as an alpaca monoclonal antibody or a murine antibody) while exhibiting low immunogenicity when administered to humans as a therapeutic agent. In some embodiments, it typically includes a CDR derived from a non-human animal, a FR region derived from a human, and optionally a constant region derived from a human.

[0068] The term "epitope," also known as "antigenic determinant," as used in this article refers to an antigenic moiety that can be recognized and specifically bound by antibodies. An antigen may have multiple epitopes, which are typically formed by surface-exposed molecular groups such as amino acids or sugar side chains.

[0069] The terms "flexible linker peptide," "linker peptide," or "connector" used in this article refer to short amino acid sequences used to link peptide segments. Flexible linkers may contain glycine (G), alanine (A), threonine (T) residues, etc.

[0070] As used herein, the terms “binding” or “specific binding” mean that the binding is selective to the target antigen and can be distinguished from unwanted or nonspecific interactions. For example, an antibody that specifically binds to a target antigen means that the antibody has higher affinity, stronger binding activity, easier binding, and / or longer binding duration when binding to the target antigen compared to binding to other non-target molecules.

[0071] "Affinity" or "binding affinity" is an intrinsic binding capacity used to reflect the interaction between members of a binding pair. For example, the affinity of molecule X for its partner Y can usually be expressed by the equilibrium dissociation constant (K0). DThe equilibrium dissociation constant is the dissociation rate constant (K). dis or K off ) and association rate constant (K a or K on The ratio of affinity to kinetic binding affinity. Affinity can be measured using common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay.

[0072] The "percentage of amino acid sequence identity" refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the reference sequence, relative to the total number of amino acid residues in the reference sequence, after aligning a candidate sequence with a reference sequence and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. The percentage of amino acid sequence identity can be determined by aligning two or more sequences using tools known in the art, such as BLASTp, ClustalW2 (see Higgins DG et al., Methods Enzymol 1996, 266:383-402; Larkin MA et al., Bioinformatics 2007, 23:2947-2948), ALIGN, or Megalign (DNASTAR) software.

[0073] For polypeptide sequences, "conservative modification" includes the substitution, deletion, or insertion of amino acids in the polypeptide sequence with other amino acids of the same class (e.g., amino acids with similar chemical properties or functions), without substantially altering the desired functional activity of the polypeptide sequence. For example, conserved substitution often results in a replacement of an amino acid with a similar amino acid. A list of conserved substitutions of functionally similar amino acids is known in the art. The following are eight groups of amino acids containing mutually conserved substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0074] The terms “subject,” “patient,” and “individual” are used interchangeably herein and include mammals or non-mammalian vertebrates (such as chickens, emus, and fish), including but not limited to domesticated animals (e.g., cattle, sheep, cats, dogs, pigs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats, guinea pigs, and hamsters), preferably humans.

[0075] The term "treating" (or "treatment") as used herein refers to reducing or alleviating a disease or symptom, slowing the onset or progression of a disease or symptom, reducing the risk of developing a disease or symptom, delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, achieving complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above. Desired therapeutic effects as described herein include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0076] As used herein, the term “optional” indicates whether the object it modifies is present or not; for example, “a pharmaceutical composition contains optional pharmaceutically acceptable excipients” means that the pharmaceutical composition may or may not contain pharmaceutically acceptable excipients.

[0077] The term "therapeutic effective amount" or "effective dose" refers to a dose or concentration that, at the required dose and for the required duration, is effective in preventing or improving symptoms associated with a disease or condition and / or reducing the severity of the disease or condition. The therapeutically effective amount of the formulations, antibodies, or antigen-binding fragments thereof, bispecific antibodies, or compositions disclosed herein can vary depending on various factors such as disease state, individual age, sex, and weight, and the ability of the antibody or antigen-binding portion to elicit the desired response in an individual. A therapeutically effective amount can also be considered as the therapeutically beneficial effect of the formulation, antibody, or antigen-binding fragment thereof, bispecific antibody, or composition clearly outweighing any toxic or harmful effects it causes. The term "effective amount" refers to the amount of an active ingredient or agent sufficient to provide a clinical benefit to a subject, including but not limited to improvement, relief, or reduction of the disease, condition, or related symptoms, or delay or cessation of disease progression.

[0078] The terms “pharmaceutically acceptable” or “clinically acceptable” in this article mean that the carrier, solvent, diluent, excipient and / or salt are generally chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the subject.

[0079] In this paper, the terms "X" and "Xaa" are equivalent and refer to unspecified amino acids. Their scope is defined in the relevant descriptions. To distinguish multiple "X"s in an amino acid sequence, each consecutive "X" is numbered (e.g., written as X...). n And define the scope of each of them.

[0080] Unless otherwise stated, the terms “comprise”, “comprises”, and “comprising” or their equivalents (contain, contain, containing, include, include, including) used herein are open-ended expressions, meaning that they may cover other unspecified elements, components, and steps in addition to those listed.

[0081] Unless otherwise stated, all figures used in the application documents to represent amounts of components, measurements, or reaction conditions should be understood to be modified by the term "about" in all cases, and the meaning of the term "about" here should be considered as within an acceptable margin of error for the corresponding value. When used with percentages, the term "about" may mean, for example, ±1%, preferably ±0.5%, more preferably ±0.1%.

[0082] Unless the context clearly indicates otherwise, singular terms in this document encompass the plural referents, and vice versa. Similarly, unless the context clearly indicates otherwise, the word "or" in this document is intended to include "and".

[0083] In this document, "pharmaceutical composition" means that the active ingredients contained herein may be administered simultaneously, separately, or at regular or irregular intervals, and that the active ingredients may be mixed together or exist separately (e.g., in their respective pharmaceutical composition forms).

[0084] For purposes of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided only because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0085] Next, the technical solutions of this disclosure will be described in more detail through exemplary embodiments, but the scope of protection of this disclosure is not limited thereto.

[0086] Anti-TSLP antibodies or their antigen-binding fragments

[0087] This disclosure provides an isolated anti-TSLP antibody or its antigen-binding fragment, which specifically binds to TSLP, inhibiting TSLP-induced dendritic cell (DC) maturation and chemokine (CCL-17) production, thereby blocking TSLP activity and reducing inflammation. The anti-TSLP antibody described in this disclosure can be a murine antibody, chimeric antibody, humanized antibody, or fully human antibody; it can be a monoclonal antibody, polyclonal antibody, monospecific antibody, or multispecific antibody (such as a bispecific antibody), as long as the antibody can specifically recognize and bind to TSLP.

[0088] In some embodiments, this disclosure provides an isolated anti-TSLP antibody or its antigen-binding fragment thereof, comprising: HCDR1 shown in SEQ ID NO: 37, HCDR2 shown in SEQ ID NO: 38, HCDR3 shown in SEQ ID NO: 39; and / or, LCDR1 shown in SEQ ID NO: 40, LCDR2 shown in SEQ ID NO: 41, and LCDR3 shown in SEQ ID NO: 42.

[0089] In this disclosure, the heavy chain variable region CDR1 (HCDR1) in the isolated anti-TSLP antibody or its antigen-binding fragment is defined by the AbM numbering system; the heavy chain variable regions CDR2 and CDR3 (HCDR2 and HCDR3) and the light chain variable regions CDRs (LCDRs) are defined by the Kabat numbering system. Variable regions defined using other numbering systems are also within the scope of this disclosure.

[0090] In some preferred embodiments, the isolated anti-TSLP antibody or its antigen-binding fragment described in this disclosure comprises: a VH having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identity with the amino acid sequence shown in SEQ ID NO: 43 or 45; and a VH having at least 80%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identity with the amino acid sequence shown in SEQ ID NO: 43 or 45. The amino acid sequences shown in 44 or 46 have a VL with at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% or more identity.

[0091] In some specific embodiments, amino acids that differ from the amino acid sequence shown in SEQ ID NO: 43 or 45, having at least 80% identity, are predominantly (or all) present in the FR region (backbone region). In some specific embodiments, amino acids that differ from the amino acid sequence shown in SEQ ID NO: 44 or 46, having at least 80% identity, are predominantly (or all) present in the FR region (backbone region).

[0092] In some preferred embodiments, the isolated anti-TSLP antibody or its antigen-binding fragment described in this disclosure comprises: VH as shown in SEQ ID NO: 43 or 45, and VL as shown in SEQ ID NO: 44 or 46. In some preferred embodiments, the isolated anti-TSLP antibody or its antigen-binding fragment described in this disclosure comprises: HCDRs contained in VH as shown in SEQ ID NO: 43 or 45, and LCDRs contained in VL as shown in SEQ ID NO: 44 or 46.

[0093] In some preferred embodiments, the isolated anti-TSLP antibody or its antigen-binding fragment described in this disclosure comprises:

[0094] (1) A VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44; or

[0095] (2) VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 45, and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46;

[0096] In this region, all amino acids that differ from the amino acid sequences shown in any of SEQ ID NOs: 43, 44, 45 and 46 in sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more are present in the FR region.

[0097] In some preferred embodiments, the isolated anti-TSLP antibody or its antigen-binding fragment described in this disclosure comprises:

[0098] (1) VH shown in SEQ ID NO: 43 and VL shown in SEQ ID NO: 44; or

[0099] (2) VH shown in SEQ ID NO: 45 and VL shown in SEQ ID NO: 46.

[0100] In some embodiments, the anti-TSLP antibody or its antigen-binding fragment described in this disclosure is an anti-TSLP scFv. In some specific embodiments, the scFv includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein each variable region or a portion thereof is derived from the same antibody or different antibodies, preferably from the same antibody. The VH / VL of the scFv may or may not contain disulfide bonds. In some embodiments, the anti-TSLP scFv includes a linker. In some embodiments, the scFv molecule may be constructed in a VH-linker-VL sequence or a VL-linker-VH sequence. In some embodiments, the linker has low immunogenicity and preferably uses a flexible peptide (e.g., 5-25, 10-20 amino acids in length, containing, for example, glycine or glycine and serine) as the linker (e.g., G...). m Connectors, GS connectors, etc.

[0101] In some specific embodiments, the linker includes an amino acid sequence (GGGGS). n That is, (G4S) n Where n is an integer from 1 to 5, preferably n is 2, 3 or 4, and most preferably n is 4. In some examples, the amino acid sequence of the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 63).

[0102] In some embodiments, the anti-TSLP scFv of this disclosure comprises: (1) a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43 or 45; (2) a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44 or 46; and (3) a (G4S)n connector for connecting the VH and VL, where n is an integer from 1 to 5, preferably 2, 3 or 4.

[0103] In some preferred embodiments, the anti-TSLP scFv described in this disclosure includes:

[0104] (1) A VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43, a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44, and a (G4S)n connector for connecting the VH and VL, where n is an integer from 1 to 5, preferably 2, 3 or 4; or

[0105] (2) A VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 45, a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46, and a (G4S)n connector for connecting the VH and VL, where n is an integer from 1 to 5, preferably n is 2, 3 or 4;

[0106] In this region, all amino acids that differ from the amino acid sequences shown in any of SEQ ID NOs: 43, 44, 45 and 46 in sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more are present in the FR region.

[0107] In some specific implementations, the anti-TSLP scFv described in this disclosure includes:

[0108] (1) VH shown in SEQ ID NO: 43, VL shown in SEQ ID NO: 44, and (G4S)n connector for connecting said VH and VL, where n is 2, 3 or 4; or

[0109] (2) VH shown in SEQ ID NO: 45, VL shown in SEQ ID NO: 46, and (G4S)n connector for connecting said VH and VL, where n is 2, 3 or 4.

[0110] In some specific implementations, the anti-TSLP scFv described in this disclosure includes:

[0111] (1) VH shown in SEQ ID NO: 43, VL shown in SEQ ID NO: 44, and the connector shown in SEQ ID NO: 63; or

[0112] (2) VH shown in SEQ ID NO: 45, VL shown in SEQ ID NO: 46, and connector shown in SEQ ID NO: 63.

[0113] In some alternative embodiments, the isolated anti-TSLP antibody or its antigen-binding fragment described herein further includes a heavy chain constant region and a light chain constant region. In a further preferred embodiment, the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4 or variants thereof, and the light chain constant region is selected from the constant regions of human κ and λ chains or variants thereof. Exemplary variants include IgG1, IgG2, or IgG4 heavy chain constant region variants with site-specific modifications and amino acid substitutions, such as the AAA mutation, DLE mutation (Shields et al., 2002; Lazar et al., 2006), YTE mutation, and LS mutation (Ghetie et al., 1997; Zalevsky et al., 2010) known in the art.

[0114] In some embodiments, the heavy chain constant region (CH) is the IgG1 LALA subtype. In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identity with it. In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 83.

[0115] In some embodiments, the light chain constant region is the human κ chain constant region. In some embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 48 or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more identity with it.

[0116] In some embodiments, the isolated anti-TSLP antibody comprises a heavy chain and a light chain, wherein,

[0117] The heavy chain includes VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43 or 45, and CH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 47;

[0118] Furthermore, the light chain includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44 or 46, and a CL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 48.

[0119] In some embodiments, the isolated anti-TSLP antibody comprises a heavy chain and a light chain, wherein,

[0120] (1) The heavy chain comprises VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43 and CH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 47; and the light chain comprises VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44 and CL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 48; or

[0121] (2) The heavy chain comprises VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 45 and CH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 47; and the light chain comprises VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46 and CL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 48;

[0122] In this region, all amino acids that differ from the amino acid sequences shown in any of SEQ ID NOs: 43, 44, 45 and 46 in sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more are present in the FR region.

[0123] In some preferred embodiments, the isolated anti-TSLP antibody comprises a heavy chain and a light chain, wherein,

[0124] (1) The heavy chain comprises VH shown in SEQ ID NO: 43 and CH shown in SEQ ID NO: 47; and the light chain comprises VL shown in SEQ ID NO: 44 and CL shown in SEQ ID NO: 48; or

[0125] (2) The heavy chain includes VH shown in SEQ ID NO: 45 and CH shown in SEQ ID NO: 47; and the light chain includes VL shown in SEQ ID NO: 46 and CL shown in SEQ ID NO: 48.

[0126] In some embodiments, the isolated anti-TSLP antibody or its antigen-binding fragment described in this disclosure is of type IgG1, IgG2, IgG3 or IgG4.

[0127] In some embodiments, the antigen-binding fragment of the isolated anti-TSLP antibody described in this disclosure is selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated CDR regions, and nanobodies.

[0128] The isolated anti-TSLP antibodies or various variants of their antigen-binding fragments described herein retain the ability to specifically bind to the antigen IL-4Rα.

[0129] In some embodiments, the isolated anti-TSLP antibody or its antigen-binding fragment described in this disclosure is bound to the K+ of human TSLP. D Value < 1×10 -9 M, preferred <8×10 -10 M, for example, <5×10 -10 M, and exhibited good blocking activity against TSLP / TSLPR.

[0130] This disclosure provides a variety of antibodies that specifically bind to TSLP and their antigen-binding fragments.

[0131] The antibodies and antigen-binding fragments described herein are capable of binding to TSLP. This disclosure provides, for example, anti-TSLP antibody 11H7E11 and antibodies derived therefrom.

[0132] The CDR sequences of 11H7E11 and 11H7E11-derived antibodies (e.g., chimeric or humanized antibodies) include CDRs of the heavy chain variable domain (SEQ ID NO: 37, 38, 39, respectively) and CDRs of the light chain variable domain (SEQ ID NO: 40, 41, 42, respectively). VH CDR1 is defined according to AbM, while VH CDR2, VH CDR3 and VL CDR1, VLCDR2, VL CDR3 are defined according to Kabat. CDRs can also be defined using the Kabat or Chothia system. Under Kabat numbering, the CDR sequences of the heavy chain variable domain are shown as SEQ ID NO: 86, 38, 39, and the CDR sequences of the light chain variable domain are shown as SEQ ID NO: 40, 41, 42, respectively. Under the Chothia designation, the CDR sequences of the heavy chain variable structural domains are shown as SEQ ID NO:117, 118, and 119, respectively, and the CDR sequences of the light chain variable structural domains are shown as SEQ ID NO:120, 121, and 122, respectively.

[0133] Furthermore, in some embodiments, the antibody or its antigen-binding fragment described herein may also include one, two, or three heavy chain variable regions (CDRs) and / or one, two, or three light chain variable regions (CDRs), wherein the heavy chain variable regions (CDRs) are selected from the group consisting of SEQ ID NO:37, 38, 39, SEQ ID NO:86, 38, 39, and SEQ ID NO:117, 118, 119; and the light chain variable regions (CDRs) are selected from the group consisting of SEQ ID NO:40, 41, 42, and SEQ ID NO:120, 121, 122.

[0134] In some embodiments, the anti-TSLP antibody may have a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains or is composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or constitutes the amino acid sequence of the selected VH CDR1; CDR2 contains or is composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or constitutes the amino acid sequence of the selected VH CDR2; and CDR3 contains or is composed of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to or constitutes the amino acid sequence of the selected VH CDR3. The light chain variable region includes CDR1, CDR2, and CDR3, wherein CDR1 contains or is composed of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to or constitutes the amino acid sequence of the selected VH CDR3. The CDR1 amino acid sequence is at least 80%, 85%, 90%, or 95% identical to or composed of the same amino acid sequence as the selected VL CDR2 amino acid sequence, and the CDR3 region contains at least 80%, 85%, 90%, or 95% identical to or composed of the same amino acid sequence as the selected VL CDR3 amino acid sequence. Figures 15A-15C The selected amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 and the selected amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 are shown in the figure.

[0135] In some embodiments, the anti-TSLP antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of CDR1, CDR2, and CDR3, wherein CDR1 has zero, one, or two amino acid insertions, deletions, or substitutions; CDR2 has zero, one, or two amino acid insertions, deletions, or substitutions; and CDR3 has zero, one, or two amino acid insertions, deletions, or substitutions, and VH CDR1, VH CDR2, and VH CDR3 are... Figures 15A-15C As shown in the diagram. In some embodiments, the antibody or antigen-binding fragment described herein may include a light chain variable domain comprising one, two, or three of CDR1, CDR2, and CDR3, wherein CDR1 has zero, one, or two amino acid insertions, deletions, or substitutions; CDR2 has zero, one, or two amino acid insertions, deletions, or substitutions; and CDR3 has zero, one, or two amino acid insertions, deletions, or substitutions, and VL CDR1, VL CDR2, and VLCDR3 are... Figures 15A-15C As shown in the image.

[0136] Insertion, deletion, and substitution can occur within the CDR sequence, or at one or both ends of the CDR sequence.

[0137] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to TSLP. The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises or is composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a selected VH sequence, and the VL comprises or is composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO:43 and the selected VL sequence is SEQ ID NO:44. In some embodiments, the selected VH sequence is SEQ ID NO:45 and the selected VL sequence is SEQ ID NO:46.

[0138] In some embodiments, the antibody or its antigen-binding fragment may have three VH CDRs, which are identical to the CDRs of any VH sequence described herein. In some embodiments, the antibody or its antigen-binding fragment may have three VLCDRs, which are identical to the CDRs of any VL sequence described herein.

[0139] This disclosure also provides nucleic acids comprising polynucleotides, said polynucleotides encoding polypeptides comprising immunoglobulin heavy chains or immunoglobulin light chains. The immunoglobulin heavy chains or immunoglobulin light chains comprise, for example... Figures 15A-15C The CDR shown, or having the following characteristics Figure 16 The sequence is shown. When a polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to TSLP (e.g., human TSLP or monkey TSLP).

[0140] Anti-TSLP antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies described herein are polyclonal, monoclonal, multispecific (multimers, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly prepared antibodies (i.e., intracellular antibodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0141] Antibody fragments are suitable for the provided methods, as long as they retain the desired affinity and specificity of a full-length antibody. Therefore, antibody fragments that bind to TSLP will retain the ability to bind to TSLP. Fv fragments are antibody fragments containing complete antigen recognition and binding sites. This region consists of a dimer of a tightly bound heavy-chain variable domain and a light-chain variable domain, which can be covalently related, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In general, six CDRs or a subset thereof confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three CDRs specific to the antigen) can have the ability to recognize and bind antigens, although typically with lower affinity than a complete binding site.

[0142] Antigen-binding protein construct

[0143] This disclosure provides antibodies targeting TSLP and their antigen-binding fragments. The anti-TSLP antibodies and their antigen-binding fragments can be in various forms.

[0144] Generally, wild-type antibodies (also known as immunoglobulins) can consist of two types of polypeptide chains: light chains and heavy chains. The non-restrictive antibody disclosed herein can be a complete four-chain immunoglobulin antibody containing two heavy chains and two light chains. The heavy chain of the antibody can be any isotype, including IgM, IgG, IgE, IgA, or IgD, or a sub-isotype (IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain can be a κ light chain or a λ light chain. The antibody can contain two identical copies of the light chain and / or two identical copies of the heavy chain. Each heavy chain contains a variable domain (or variable region, VH) and multiple constant domains (or constant regions), which are linked together by disulfide bonds within their constant domains to form the antibody's "stem." Each light chain contains a variable domain (or variable region, VL) and a constant domain (or constant region), and each light chain is linked to one heavy chain by a disulfide bond. Align the variable regions of each light chain with the variable regions of the heavy chain it is attached to. The variable regions of both the light and heavy chains contain three highly variable regions sandwiched between the more conservative frame regions (FR).

[0145] These hypervariable regions (called complementarity-determining regions (CDRs)) form loops that constitute the primary antigen-binding surface of the antibody. The four framework regions primarily adopt a β-sheet conformation, and the CDRs form loops connecting the β-sheet structures (and in some cases, portions of the β-sheet). CDRs in each chain are tightly held together by the framework regions and, together with CDRs from other chains, contribute to the formation of the antigen-binding region.

[0146] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly in their hinge and upper CH2 domain. The sequences and differences of IgG subclasses are known in the art and are described, for example, in the following: Vidarsson et al., “IgG subclasses and allotypes: from structure to effector functions”, Frontiers in Immunology, 5 (2014); Irani et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases”, Molecular Immunology, 67.2 (2015): 171-182; Shakib, Farouk, eds., The human IgG subclasses: molecular analysis of structure, function and regulation, Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0147] Antibodies can also be immunoglobulin molecules derived from any species, such as humans, rodents, mice, rats, and camels. The antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific, and chimeric antibodies, which include an immunoglobulin-binding domain fused to another polypeptide. An antigen-binding domain or antigen-binding fragment is a portion of an antibody that retains the specific binding activity of the intact antibody; that is, any portion of the antibody capable of specifically binding to an epitope on the target molecule of the intact antibody. For example, it includes Fab fragments, Fab' fragments, F(ab')2 fragments, and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, bimeric antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide that includes a binding domain (which is an antibody-binding domain or homologous to an antibody-binding domain). Non-limiting examples of antigen-binding domains include, for example, heavy chain CDRs and / or light chain CDRs of an intact antibody, variable regions of the light chain and / or variable regions of the heavy chain of an intact antibody, the full-length heavy chain or light chain of an intact antibody, or a single CDR of the heavy chain or short chain of an intact antibody.

[0148] In some embodiments, the antigen-binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-stranded variable fragment (scFv) as described herein, fused to both the transmembrane domain and the intracellular domain of CD3ζ. In some embodiments, the chimeric antigen receptor further includes an intracellular signaling domain from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, such as CD3ζ-CD28-41BB or CD3ζ-CD28-OX40, to enhance potency. Therefore, in one aspect, this disclosure further provides cells (e.g., T cells) expressing the chimeric antigen receptor described herein.

[0149] In some implementations, antibodies, their antigen-binding fragments, or antigen-binding protein constructs (e.g., bispecific antibodies) can bind to two different antigens or two different epitopes.

[0150] In some embodiments, the antibody, its antigen-binding fragment, or antigen-binding protein construct (e.g., a bispecific antibody) may contain components selected from... Figures 15A-15C One, two, or three heavy chain variable regions (CDRs). In some embodiments, the antibody, its antigen-binding fragment, or antigen-binding protein construct (e.g., a bispecific antibody) may contain one, two, or three heavy chain variable regions (CDRs). Figures 15A-15C One, two, or three light chain variable regions (CDRs).

[0151] In some embodiments, the antibodies, their antigen-binding fragments, or antigen-binding protein constructs (e.g., bispecific antibodies) described herein may be conjugated to a therapeutic agent. Antibody-drug conjugates comprise antibodies or their antigen-binding fragments that can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracetam, maytansine derivatives such as DM-1 and DM-4, ​​diketones, mitoxantrone, sclerosomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogues).

[0152] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be prepared by engineering the interface between paired antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant structural domain. In this approach, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for improving the yield of heterodimers, rather than other unwanted end products, such as dimers. This approach is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0153] Any antibody, its antigen-binding fragment, or antigen-binding protein construct (e.g., a bispecific antibody) described herein may be conjugated to a stable molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or solution). Non-limiting examples of stable molecules include polymers (such as polyethylene glycol) or proteins (such as serum albumin, e.g., human serum albumin). Conjugation to a stable molecule may increase the half-life of the antibody or antigen-binding fragment or prolong its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in the human body).

[0154] Antibodies, their antigen-binding fragments, or antigen-binding protein constructs (e.g., bispecific antibodies) can also take many forms. Many different forms of antigen-binding constructs are known in the art and are described, for example, in Suurs et al., “A review of bispecific antibodies and antibody constructs in oncology and clinical challenges,” Pharmacology & therapeutics (2019), which is incorporated herein by reference in its entirety.

[0155] In some implementations, the antigen-binding protein construct is BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiaboty-HAS, or tandem scFv. In some embodiments, the antigen-binding protein construct is VHH-scAb, VHH-Fab, double scFab, F(ab')2, bibody antibody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knots-in-holes common light chain, knots-in-holes assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, bivalent antibody-CH3, triple body, microbody, microantibody, TriBi microantibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, bivalent antibody Fc, tandem scFv-Fc, intracellular antibody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body or scFv1-PEG-scFv2.

[0156] In some implementations, the antigen-binding protein construct can be TrioMab. In TrioMab, the two heavy chains are from different species, and the different sequences restrict heavy-light chain pairing.

[0157] In some implementations, the antigen-binding protein construct has two distinct heavy chains and one common light chain. Heterodimerization of the heavy chains can be based on knock-in-holes or other heavy chain pairing techniques.

[0158] In some implementations, the CrossMAb technology can be used to generate bispecific antibodies. The CrossMAb technology can be used to induce correct light chain association in bispecific heterodimeric IgG antibodies, allowing for the generation of various bispecific antibody forms, including bivalent (1+1), trivalent (2+1), and tetravalent (2+2) bispecific antibodies, as well as antibodies based on non-Fc tandem antigen-binding fragments (Fab). These forms can be derived from any existing antibody pair using domain crossover without requiring identification of a common light chain, post-translational processing / in vitro chemical assembly, or the introduction of a set of mutations to induce correct light chain association. This method is described in: Klein et al., “The use of CrossMAb technology for the generation of bi-and multi-specific antibodies,” MAbs. Vol.8. No. 6, Taylor & Francis, 2016; which is incorporated herein by reference in its entirety. In some implementations, the CH1 domain in the heavy chain and the CL domain in the light chain are exchanged.

[0159] Antigen-binding protein constructs can be Duobody. The Fab exchange mechanism naturally present in IgG4 antibodies is mimicked in controlled material of IgG1 antibodies; this mechanism is called controlled Fab exchange. This form ensures specific pairing between heavy and light chains.

[0160] In dual variable domain antibodies (DVD-Ig), an additional VH and variable light chain (VL) domains are added to each N-terminus for bispecific targeting. This form is similar to IgG-scFv, but the added binding domains bind individually to their respective N-termini, instead of scFv binding to the N-terminus of each heavy chain.

[0161] In scFv-IgG, two scFvs are attached to the C-terminus (CH3) of the heavy chain. The scFv-IgG form has two distinct bivalent binding sites and is therefore also referred to as tetravalent. There is no heavy chain and light chain pairing issue in scFv-IgG.

[0162] In some implementations, the antigen-binding protein construct may be in IgG-IgG form. Two complete IgG antibodies are conjugated by chemically linking the C-terminus of the heavy chain.

[0163] Antigen-binding protein constructs can also be in the Fab-scFv-Fc form. In the Fab-scFv-Fc form, the light chain, heavy chain, and third chain containing the Fc region and scFv are assembled. This ensures efficient manufacturing and purification.

[0164] In some implementations, the antigen-binding protein construct can be a TF (fiber-dependent protein). Three Fab fragments are linked by disulfide bridges. Two fragments target tumor-associated antigens (TAAs), and one fragment targets a hapten. The TF form does not have an Fc region.

[0165] ADAPTIR has two scFvs that bind to each side of the Fc region. It abandons the complete IgG as the basis of its construct but retains the Fc region to prolong the half-life and facilitate purification.

[0166] The bispecific T-cell binder (“BiTE”) consists of two scFvs (VLA VHA and VHB VLB) on a single polypeptide chain. It has only a binding domain and no Fc region.

[0167] In BiTE-Fc, the Fc region is fused with the BiTE construct. The addition of the Fc region enhances the half-life, resulting in a longer effective concentration and avoiding consecutive IVs.

[0168] Dual affinity reversal (DART) involves two peptide chains linked by opposite segments, thus fusing VLA with VHB and VLB with VHA, along with their C-terminal sulfur bonds. In DART, the sulfur bonds enhance the stability of BiTE.

[0169] In DART-Fc, the Fc region is attached to the DART structure. It can be generated by assembling three chains, two of which are assembled via disulfide bonds, just like DART. One chain contains half of the Fc region, which dimerizes with the third chain to express only the Fc region. The addition of the Fc region increases the half-life, resulting in a longer effective concentration and avoiding consecutive IVs.

[0170] In tetravalent DART, four peptide chains are assembled. Essentially, two DART molecules are generated using half of the Fc region and dimerize. This form binds divalently to both targets, thus making it a tetravalent molecule.

[0171] Tandem bispecific antibodies (TandAbs) consist of two bispecific antibodies. Each bispecific antibody is composed of covalently linked VHA and VLB fragments, and the two bispecific antibodies are linked by a peptide chain. This enhances the stability of bispecific antibodies composed of two scFvs. It has two bivalent binding sites.

[0172] The ScFv-scFv toxin consists of a toxin and two scFvs with stable connectors. It can be used for the targeted delivery of payloads.

[0173] In the modular scFv-scFv-scFv, a scFv targeting TAA is tagged with a short, recognizable peptide and assembled into a bsAb consisting of two scFvs (one targeting CD3 and one targeting the recognizable peptide).

[0174] In ImmTAC, a stable, soluble T-cell receptor fuses with a CD3-recognizing scFv. By using a TCR, ImmTAC is suitable for targeting processed proteins, such as intracellular proteins.

[0175] Trispecific nanobody has two single variable domains (nanobody) with additional modules for extending half-life. These additional modules are added to improve half-life.

[0176] In Trispecific Killer Engager (TriKE), two scFvs are linked by a peptide linker that incorporates human IL-15. The addition of the IL-15 linker increases NK cell survival and proliferation.

[0177] In some embodiments, the antibody, or its antigen-binding fragment (e.g., anti-TSLP antibody), or related antibody-drug conjugate (ADC) has a light chain constant region and a heavy chain constant region, wherein the light chain constant region is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:48, and the heavy chain constant region is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:47.

[0178] In some embodiments, the antibody or its antigen-binding fragment (e.g., anti-TSLP antibody) is an scFv. The scFv may be in the form of VH-linker-VL or VL-linker-VH. In some embodiments, the linker described herein is a flexible linker, such as the GS linker. In some embodiments, the linker described herein is a flexible linker, such as the GS linker. In some embodiments, the GS linker comprises one or more repeats of GS, SG, GGGGS (SEQ ID NO: 127) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats). In some embodiments, the GS linker comprises at least 80%, 85%, 90%, or 95% of the same amino acid sequence as SEQ ID NO: 63. Detailed information on flexible linkers can be found, for example, Chen, X. et al., “Fusionprotein linkers: property, design and functionality,” Advanced Drug Delivery Reviews, 65.10 (2013): 1357-1369, which is incorporated herein by reference in its entirety.

[0179] In some embodiments, the antigen-binding protein constructs described herein are multispecific antibodies (e.g., bispecific antibodies).

[0180] Polynucleotides, vectors and host cells

[0181] This disclosure relates to an expression vector encoding the aforementioned anti-TSLP antibody or its antigen-binding fragment (e.g., any anti-TSLP antibody or its antigen-binding fragment described herein), comprising the polynucleotide, and a host cell incorporating the polynucleotide or its expression vector.

[0182] In some embodiments, the expression vector may be any expression vector capable of expressing the antibody or its antigen-binding moiety described herein, including but not limited to naked plasmids, phage particles, yeast plasmids, adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), retroviruses (such as lentiviruses), poxviruses, papillomaviruses, papillomaviruses (such as SV40), rod-shaped viruses, or baculoviruses. For ease of production and purification, the expression vector may also contain secretory signal peptides, expression tags, etc.

[0183] It is known to those skilled in the art that, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequences encoding the antibodies or fragments thereof disclosed herein can be synthesized using methods known in the art (e.g., de novo solid-phase DNA synthesis, PCR amplification). Given that specific amino acid sequences have been described in this disclosure, those skilled in the art can easily prepare many different nucleic acids by modifying one or more codons on their respective coding sequences without altering the amino acid sequences of the antibodies or their antigen-binding fragments, or bispecific antibodies disclosed herein.

[0184] Those skilled in the art can use conventional methods known in the art to construct nucleic acids encoding the various antibodies or their antigen-binding fragments, or bispecific antibodies disclosed herein, into suitable vectors for introduction into host cells for expression of the target protein. Vector components may include, but are not limited to, signal sequences, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. In the vector, the nucleic acid encoding the target protein is operatively linked to the promoter.

[0185] In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or any other cells suitable for preparing antigen-binding constructs. In some examples, the mammalian cell is, for example, Chinese hamster ovary (CHO) cells, CHO-S cells, 293 cells, or monkey kidney cells.

[0186] The above-described expression vector can be introduced into suitable host cells using any conventional means known in the art, such as protoplast fusion, calcium phosphate precipitation, electroporation, viral transfection, gene gun, liposome transfection, or other conventional techniques, but not limited thereto.

[0187] Under conditions suitable for the expression of the target protein, the host cells described above are cultured under conventional conditions, and then the antibodies described herein are recovered from the host cells or the culture medium of the host cells by conventional protein separation and purification methods (such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (such as Protein A column affinity chromatography), size exclusion chromatography, etc.).

[0188] This disclosure also provides a nucleic acid sequence and an amino acid sequence, wherein the nucleic acid sequence is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any nucleotide sequence described herein, and the amino acid sequence is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any amino acid sequence described herein.

[0189] This disclosure also provides a nucleic acid sequence and an amino acid sequence, wherein the nucleic acid sequence has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with any amino acid sequence described herein, and wherein the amino acid sequence has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with any amino acid sequence described herein.

[0190] In some embodiments, this disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is fewer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence has fewer than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0191] In some embodiments, the amino acid sequence (i) includes an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any of the sequences described herein.

[0192] In some embodiments, the nucleic acid sequence (i) includes a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0193] The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. How to determine the percentage of sequence homology is known in the art. In some embodiments, conserved amino acid residues (e.g., leucine and isoleucine) having similar physicochemical properties (homology percentage) can be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include, for example, amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percentage of homology is higher than the percentage of identity.

[0194] This disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding a heavy-chain polypeptide as described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding a light-chain polypeptide as described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding a scFv polypeptide as described herein.

[0195] Immunoconjugates

[0196] This disclosure relates to immunoconjugates comprising the aforementioned anti-TSLP antibody or its antigen-binding fragment (such as any anti-TSLP antibody or its antigen-binding fragment described herein).

[0197] In some embodiments, this disclosure provides antibody-drug conjugates (ADCs) comprising the aforementioned anti-TSLP antibody or its antigen-binding fragment conjugated to an inflammatory therapeutic agent. In some embodiments, the inflammatory therapeutic agent may be an asthma treatment drug. In some embodiments, the anti-TSLP antibody or its antigen-binding fragment is directly conjugated to the therapeutic agent. In some embodiments, the anti-TSLP antibody or its antigen-binding fragment is conjugated to the therapeutic agent via a linker. In the ADC, the linker for antibody-therapeutic agent conjugation may be a cleavable linker, such as a peptide linker, disulfide bond, or hydrazone linker, or a non-cleavable linker.

[0198] Pharmaceutical Compositions and Routes of Administration

[0199] This document also provides pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antigen-binding protein constructs, antibodies (e.g., bispecific antibodies), antigen-binding fragments, or antibody-drug conjugates described herein. Two or more (e.g., two, three, or four) of any of the antigen-binding protein constructs, antibodies, antigen-binding fragments, or antibody-drug conjugates described herein may be present in any combination of the pharmaceutical compositions. The pharmaceutical compositions may be formulated in any manner known in the art.

[0200] This disclosure relates to a pharmaceutical composition comprising: the anti-TSLP antibody or its antigen-binding fragment, or its immunoconjugate, and optionally pharmaceutically acceptable excipients. In this document, the pharmaceutical composition comprises a therapeutically effective amount of each of the above-mentioned components.

[0201] In some embodiments, the pharmaceutical composition further includes an anti-inflammatory agent, such as a conventional asthma treatment, such as a glucocorticoid anti-inflammatory drug.

[0202] The excipients described herein can be any pharmaceutically acceptable excipient, such as, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc. For other pharmaceutically acceptable pharmaceutical excipients, please refer to, for example, *Handbook of Pharmaceutical Excipients* (4th Edition), by RC Luo et al., translated by Zheng Junmin, Chemical Industry Press, 2005.

[0203] In some embodiments, the pharmaceutical composition may be in the form of a sterile aqueous solution, microemulsion, liposome, or powder. In some embodiments, the pharmaceutical composition may be in the form of a unit dose to facilitate administration to the patient at the desired dosage.

[0204] The dosage range of the pharmaceutical compositions described in this disclosure can be determined by clinicians based on experience, taking into account factors such as the method of administration (including administration time, administration interval, and route of administration), the patient's age, weight, sex or pathological condition, diet, excretion rate, and sensitivity to the drug.

[0205] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may include sterile diluents (such as sterile water or saline), fixative oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetates, citrates, or phosphates), and isotonic agents (such as sugars (e.g., dextrose), polyols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent No. 4522811). The composition may be formulated and packaged into ampoules, disposable syringes, or multi-dose vials. Where necessary (e.g., in injectable formulations), adequate flowability can be maintained, for example, by using coatings such as lecithin or surfactants. The absorption of antibodies or their antigen-binding fragments can be prolonged by incorporating agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved via implants and microcapsule delivery systems that may include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0206] Compositions containing one or more of the antigen-binding protein constructs, antibodies, antigen-binding fragments, and antibody-drug conjugates described herein can be formulated for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) in dose-unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and dose uniformity).

[0207] The toxicity and efficacy of the composition can be determined using standard pharmaceutical procedures in cell culture or laboratory animals (such as monkeys). The LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50 to ED50. Agents exhibiting a high therapeutic index are preferred. If an agent exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce unwanted side effects). Toxicity and efficacy can also be determined using other standard pharmaceutical procedures.

[0208] Exemplary doses include milligrams or micrograms per kilogram of the subject’s body weight of any antigen-binding protein construct, antibody, or antigen-binding fragment or antibody-drug conjugate described herein (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg).

[0209] The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use. This disclosure also provides methods for preparing antibodies or antigen-binding fragments thereof or antibody-drug conjugates for the various uses described herein.

[0210] Drug combination products

[0211] In some embodiments, the pharmaceutical compositions described herein contain additional therapeutic agents, such as additional anti-inflammatory agents, such as conventional asthma medications, such as glucocorticoid anti-inflammatory drugs, etc.

[0212] medicine box

[0213] This disclosure relates to a kit comprising the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, or a pharmaceutical composition thereof. In this document, the kit contains therapeutically effective amounts of the aforementioned components.

[0214] In some embodiments, the pillbox may further include instructions for use. In some embodiments, the pillbox may further include reagents for diagnosing the patient. In some embodiments, the pillbox may further include a device for administering medication to the patient, such as a syringe.

[0215] In some embodiments, the kit may further include pharmaceutical excipients, such as sterile water or saline, for assisting in administering the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, or its pharmaceutical composition to the patient.

[0216] Methods for preparing antibodies, antigen-binding fragments, and antigen-binding protein constructs

[0217] Isolated human protein fragments can be used as immunogens to generate antibodies using standard techniques for the preparation of polyclonal and monoclonal antibodies. Polyclonal antibodies can be produced in an animal through multiple injections (e.g., subcutaneous or intraperitoneal injections) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to a reagent that is immunogenic in the species to be immunized. The antigenic peptide or protein can be injected into the animal more than once (e.g., twice, three times, or four times).

[0218] Full-length peptides or proteins can be used as immunogens, or fragments of their antigenic peptides can be used as immunogens. The antigenic peptide of a protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the protein's amino acid sequence and covers the protein's epitope, such that antibodies generated against the peptide form specific immune complexes with the protein.

[0219] Immunogens are commonly used to prepare antibodies by immunizing a suitable subject (e.g., a person or transgenic animal expressing at least one human immunoglobulin locus). Appropriate immunogenic formulations may contain, for example, recombinantly expressed or chemically synthesized peptides. The formulation may also include adjuvants, such as Freund's complete or incomplete adjuvants, or similar immunostimulants.

[0220] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide or its antigenic peptide (e.g., a fraction of a protein) as an immunogen. Antibody titers in the immunized subject can be monitored over time using standard techniques, such as enzyme-linked immunosorbent assays (ELISA) using immobilized polypeptides or peptides. If desired, antibody molecules can be isolated from mammals (e.g., from blood) and further purified using well-known techniques, such as protein G chromatography or protein A chromatography to obtain IgG fractions. At an appropriate time following immunization, for example when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies using standard techniques, such as hybridoma techniques originally described by Kohler et al. (Nature 256:495-497, 1975), human B-cell hybridoma techniques (Kozbor et al., Immunol. Today 4:72, 1983), EBV hybridoma techniques (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or tri-source hybridoma techniques. Techniques for producing hybridomas are well known (see Current Protocols in Immunology, 1994, Colligan et al. (eds.), John Wiley & Sons, Inc., New York, NY). For example, using a standard ELISA assay, hybridoma cells producing monoclonal antibodies are detected by screening for antibodies that bind to peptides or epitopes of interest in the hybridoma culture supernatant.

[0221] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human antibody, humanized antibody, or chimeric antibody or its antigen-binding fragment, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence constituting the antigen-binding domain or antigen-binding site of the antibody. Among this population of variants, some antibody or antigen-binding fragments exhibit increased affinity for the target protein. Any combination of deletions, insertions, and / or combinations can be made to obtain an antibody or its antigen-binding fragment with increased binding affinity to the target. Introducing amino acid changes into the antibody or antigen-binding fragment can also alter post-translational modifications in the antibody or antigen-binding fragment or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars attach via enzymes present in the cell), or introducing new glycosylation sites.

[0222] The antibodies disclosed herein can be derived from any kind of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (such as monkeys and apes), cattle, pigs, horses, sheep, camels (such as camels and llamas), chickens, goats, and rodents (such as rats, mice, hamsters, and rabbits), including genetically engineered rodents to produce human antibodies.

[0223] Phage display (panning) can be used to optimize antibody sequences with desired binding affinity. In this technique, a gene encoding a single-stranded Fv (including VH or VL) is inserted into a phage coat protein gene, causing the phage to "display" the scFv externally while containing the protein gene internally, creating a link between genotype and phenotype. These displaying phages can then be screened against a target antigen to detect the interaction between the displayed antigen-binding site and the target antigen. Therefore, large protein libraries can be screened and amplified in a process called in vitro selection, yielding antibody sequences with desired binding affinity.

[0224] Human antibodies and humanized antibodies include antibodies having variable and constant regions derived from human immunoglobulin sequences (or having the same amino acid sequence as those derived from human immunoglobulin sequences). Human antibodies may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR.

[0225] In some embodiments, antibodies, their antigen-binding fragments, or antigen-binding protein constructs (e.g., bispecific antibodies) can be covalently modified. These covalent modifications can be performed through chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications to antibodies or antibody fragments can be introduced into the molecule by reacting target amino acid residues of the antibody or antibody fragment with an organic derivatizing agent capable of reacting with selected side chains or N- or C-terminal residues.

[0226] In some embodiments, the provided antibody variants have a carbohydrate structure lacking (directly or indirectly) fucose linked to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn297 (relative to the sum of all sugar structures attached to Asn 297 as measured by MALDI-TOF mass spectrometry, such as complexes, hybrids, and high-mannose structures). Asn297 refers to the asparagine residue located approximately at position 297 in the Fc region (Eu number of the Fc region residue; or position 314 in the Kabat number); however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. This fucosylated variant can have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0227] In some implementations, to improve production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further engineered to replace serine at position 228 (EU number) of IgG4 with proline (S228P). For example, a detailed description of the S228 mutation is described below: Silva et al., “The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation,” Journal of Biological Chemistry, 290.9 (2015): 5462-5469, which is incorporated herein by reference in its entirety.

[0228] In some embodiments, the methods described herein are designed for the preparation of bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between paired antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant structural domain. In this approach, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for improving the yield of heterodimers, rather than other unwanted end products, such as dimers. This method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0229] In some implementations, the knife-into-hole (KIH) technique can be used, which involves engineered CH3 domains to create a “knob” or “hole” in each heavy chain, thereby promoting heterodimerization. For example, the KIH technique is described in Xu, Yiren, et al., “Production of bispecific antibodies in 'knobs-into-holes' using a cell-free expression system,” MAbs. Vol. 7. No. 1, Taylor & Francis, 2015, which is incorporated herein by reference in its entirety. In some implementations, one heavy chain has a T366W and / or S354C (knob) substitution (EU number), and another heavy chain has a Y349C, T366S, L368A, and / or Y407V (hole) substitution (EU number). In some implementations, one heavy chain has one or more of the following substitutions: Y349C and T366W (EU number). The other heavy chain may have one or more of the following substitutions: E356C, T366S, L368A, and Y407V (EU number). Additionally, substitutions (-ppcpScp-->-ppcpPcp-) may be introduced into the hinge region of the two substituted IgGs.

[0230] In addition, anion exchange chromatography can be used to purify bispecific antibodies. Anion exchange chromatography is a process of separating substances based on their charges using an ion exchange resin containing positively charged groups (such as diethylaminoethyl (DEAE)). In solution, the resin is coated with positively charged counterions (cations). The anion exchange resin will bind to negatively charged molecules, displacing the counterions. Anion exchange chromatography can be used to purify proteins according to their isoelectric point (pI). The isoelectric point is defined as the pH at which a protein has no net charge. When pH > pI, the protein has a net negative charge, and when pH < pI, the protein has a net positive charge. Thus, in some embodiments, different amino acid substitutions can be introduced into the two heavy chains such that the pI of the homodimer containing two Arm A's and the pI of the homodimer containing two Arm B's are different. The pI of the bispecific antibody having Arm A and Arm B will be between the two pI's of the homologous dimers. Thus, the two homodimers and the bispecific antibody can be released under different pH conditions. The present disclosure shows that some amino acid residue substitutions can be introduced into the heavy chain to regulate the pI.

[0231] Therapeutic method

[0232] The present disclosure relates to the use of the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit in the preparation of an inhibitor of TSLP.

[0233] The present disclosure relates to the use of the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit in the preparation of a medicament for treating or preventing type II or mixed allergic diseases. Alternatively, the present disclosure relates to a method for treating type II or mixed allergic diseases, comprising administering to a subject in need thereof the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit. Alternatively, the present disclosure relates to the anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit for treating or preventing type II or mixed allergic diseases.

[0234] In some embodiments, the type II and mixed allergic diseases include asthma, atopic dermatitis, etc.

[0235] The anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit can effectively inhibit the binding of TSLP to TSLPR / IL7R on the surface of CTLL2 reporter cells, inhibit the TSLP-induced release of CCL-17, and can show a therapeutic effect on inflammation in animals.

[0236] The anti-TSLP antibody or its antigen-binding fragment, its immunoconjugate, pharmaceutical composition, or kit can also be used to diagnose the presence of a relevant antigen (such as TSLP) in a diagnostic sample.

[0237] The anti-TSLP antibodies or their antigen-binding fragments, their immunoconjugates, or pharmaceutical compositions disclosed herein can be prepared into any dosage form known in the art, such as injections, suspensions, solutions, powders, emulsions, sprays, tablets, pills, capsules, granules, ointments, suppositories, gels, etc.

[0238] The anti-TSLP antibodies or antigen-binding fragments thereof, their immunoconjugates, or pharmaceutical compositions disclosed herein are suitable for intravenous, intramuscular, intra-articular, intra-articular, subcapsular, subarachnoid, intraorbital, intracardiac, subcutaneous, extracorporeal, intraperitoneal, intraspinal, intranasal, or epidermal administration (e.g., by injection or infusion). The anti-TSLP antibodies or antigen-binding fragments thereof, their immunoconjugates, or pharmaceutical compositions disclosed herein can be formulated as sterile aqueous solutions, microemulsions, liposomes, or powders.

[0239] The methods described herein include approaches for treating cancer-related disorders. Typically, these methods involve administering a therapeutically effective amount of an engineered antibody, its antigen-binding fragment, an antigen-binding protein construct (e.g., a bispecific antibody), or an antibody-drug conjugate, as described herein, to a subject who requires or has been identified as requiring such treatment.

[0240] As used herein, the term “cancer” refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. This term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignant transformation of cells, tissues, or organs, regardless of their histopathological type or stage of invasion. The term “tumor” as used herein refers to cancerous cells, such as clumps of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignant tumors of various organ systems. In some embodiments, the reagents described herein are designed for treating or diagnosing cancer in a subject. The term “cancer” is generally accepted to refer to a malignant tumor of epithelial or endocrine tissue. This term also includes carcinosarcoma, for example, a malignant tumor comprising both cancerous and sarcomatous tissue. “Adenocarcinoma” refers to cancer originating from or within glandular tissue in which tumor cells form recognizable glandular structures. The term “sarcoma” is generally accepted to refer to a malignant tumor of mesenchymal origin. In some embodiments, the cancer is chemotherapy-resistant cancer.

[0241] In one aspect, this disclosure also provides methods for treating cancer in a subject, methods for reducing the rate of increase in tumor volume over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, the treatment can prevent, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment can cause a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0242] In one aspect, this disclosure is characterized by a method comprising administering a therapeutically effective amount of the antibody described herein, its antigen-binding fragment, antigen-binding protein construct (e.g., a bispecific antibody), or antibody-drug conjugate to a subject in need, such as a subject with cancer or identified or diagnosed with cancer.

[0243] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Cancer patients can be identified using various methods known in the art.

[0244] Effective amounts can be administered in single or multiple doses. For example, an effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate is an amount sufficient to improve, stop, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to improve, stop, stabilize, reverse, slow, and / or delay the in vitro proliferation of cells (e.g., biopsy cells, any cancer cells described herein, or cell lines (e.g., cancer cell lines)). As understood in the art, the effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate can vary depending on other factors, particularly the patient's medical history and the type (and / or dosage) of the composition used.

[0245] The effective amount and schedule for administering the antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and / or compositions disclosed herein can be determined empirically, and such determinations are within the competence of those skilled in the art. Those skilled in the art will understand that the dosage must be administered will vary depending on, for example, the mammal to which the antibodies, antibody-encoding polynucleotides, antibody-drug conjugates, and / or compositions disclosed herein will be received, the route of administration, the specific type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, antibody-drug conjugate, and / or composition disclosed herein used, and any other drugs administered to the mammal.

[0246] Example

[0247] The present disclosure will be further described in detail below through embodiments. However, the scope of protection of the present disclosure is not limited to these embodiments. Those skilled in the art can make any adjustments, combinations or modifications to the various embodiments and implementations of the present disclosure without departing from the spirit or concept of the present disclosure, and the resulting solutions will still fall within the scope of protection of the present disclosure.

[0248] Example 1. Proof of Concept

[0249] To verify that simultaneous blocking of IL-4Rα and TSLP can bring stronger blocking and inhibitory effects against type II immunity, two different functional experiments were used in this embodiment.

[0250] IL-4 / IL-13 / TSLP mixed cytokines stimulate human PBMCs to produce the chemokine CCL-17

[0251] Chemokine 17, also known as thymic and activating regulatory chemokine (TARC, CCL17, or CCL-17), is mainly produced by dendritic cells, endothelial cells, and keratinocytes derived from monocytes. It is expressed on keratinocytes, vascular endothelial cells, T cells, and dendritic cells of the epidermis. It recruits Th2 cells to the site of inflammation by interacting with the chemokine receptor CCR4. The level of CCL-17 in the serum of healthy adults gradually decreases with age, with a concentration below 450 pg / mL. However, the level is significantly elevated in the serum of patients with atopic dermatitis. The concentration of CCL-17 in the serum of adult patients is approximately 1000-90000 pg / mL, which is 2-20 times that of healthy individuals, and its serum level is significantly positively correlated with disease recurrence.

[0252] This study used human peripheral blood mononuclear cells (PBMCs) to determine the CCL-17 release activity induced by IL-4 / IL-13 via IL-4Rα and TSLP via TSLPR, and the synergistic effect among the three. Specifically, PBMCs (AllCell, #FPB004F-C) were resuscitated, resuspended in RPMI 1640 cell culture medium (Gibco, #22400-071), and the cells in the culture medium were cultured at a density of 4 × 10⁶ cells per well. 5 Cells were added to the wells of a 96-well plate. Different concentrations of IL-4 (R&D, #204-IL-050), IL-13 (R&D, #213-ILB-100 / CF), and TSLP (Acro Biosystems, #TSP-H52Hb) were added to the cells. The cells were then cultured at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected, and the release of expressed CCL-17 in the supernatant was detected using a CCL17 / TARC ELISA kit (R&D, #SDN00), and a dose-response curve was plotted. Figure 1AAs shown, TSLP stimulation of PBMCs alone induced CCL-17 release at very low concentrations (~0.01 nM), quickly reaching a plateau, but the overall release was low (<100 pg / mL). In contrast, with IL-4 alone, CCL-17 release increased significantly with increasing IL-4 concentration, but did not reach a plateau within the tested range (0.0001–1 nM). Under IL-13 stimulation alone, CCL-17 release reached a plateau at approximately 0.3 nM, with an overall release lower than IL-4, reaching a plateau of approximately 100 pg / mL. When the three cytokines were mixed for cell treatment, they exhibited a significant synergistic effect in increasing CCL-17 release at lower concentrations (0.1–0.3 nM).

[0253] To better simulate CCL-17 release under physiological disease conditions, we evaluated the inhibitory effects of control IgG1 (the amino acid sequences of its heavy and light chains are shown in SEQ ID NOs: 72-73), anti-TSLP antibody, anti-IL-4Rα antibody, and a combination of anti-TSLP antibody and anti-IL-4Rα antibody on CCL-17 release under stimulation at the concentration (0.3 nM) where the synergistic effect of the mixed cytokines was strongest, as determined above.

[0254] like Figure 1B As shown, the inhibitory effect of anti-IL-4Rα antibody (Dupilumab) on CCL-17 release was significantly weaker than that of anti-TSLP antibody (Tezepelumab) and other test groups. The combination of anti-TSLP antibody and anti-IL-4Rα antibody under these experimental conditions exhibited good inhibitory activity, completely blocking CCL-17 release at a low concentration (0.06 nM), which was superior to the single-drug groups.

[0255] Experiments on the release of IL-5 and IL-13 mediated by TSLP-DC differentiation of naïve T cells.

[0256] TSLP is a pleiotropic cytokine expressed by epithelial cells in response to stimulation by foreign antigens. It can act on various immune cells, such as dendritic cells (DCs), T cells, B cells, neutrophils, mast cells, eosinophils, and innate lymphoid cells, and promote their maturation. Figure 2AAs shown, in allergic inflammation, TSLP can strongly activate dendritic cells, subsequently driving naïve T cells to develop into inflammatory effector Th2 cells, releasing large amounts of Th2 cytokines, such as IL-4, IL-5, and IL-13. The IL-4 released by naïve T cells can further promote T cell maturation and differentiation into Th2 cells, ultimately mediating the massive production of Th2 cells and inducing local inflammatory responses. For example, skin epidermal cells stimulated by antigens can produce an inflammatory response, mediating atopic dermatitis; lung epithelial cells stimulated by antigens can produce a large inflammatory response, inducing asthma; and digestive tract epithelial cells stimulated by antigens can mediate an inflammatory response, inducing eosinophilic esophagitis and other disorders. Based on these scientific mechanisms, this study used TSLP-mediated DC maturation to differentiate naïve T cells into Th2 cells, and assessed the TSLP differentiation function by detecting the release of Th2 cytokines.

[0257] The specific experimental procedure is as follows. Initial myeloid DCs (mDCs) were isolated and purified from human peripheral blood mononuclear cells (PBMCs) using the EasySep™ Human Myeloid DC Enrichment Kit (Stemcell, #19061). The obtained mDCs were then distributed in batches of 2 × 10⁶ cells per well. 5 Cells were seeded at a density suitable for optimal growth in 96-well cell culture plates, and 50 ng / mL of human TSLP protein (AcroBiosystems, #TSP-H52Hb) was added. The plates were then incubated at 37°C for 24 hours. Matured mDCs were collected and washed twice with 1×PBS. CD4+ naïve T cells were isolated from PBMCs from another donor using the EasySep™ Human Naïve CD4+ T Cell Isolation Kit (Stemcell, #19555). The isolated naïve T cells and mature mDCs were mixed at a 5:1 cell ratio and seeded in 96-well cell culture plates. The cells were co-cultured at 37°C for 7 days. After co-culture, the cells were collected, stimulated with CD3 / CD28 beads (Thermo, #11132D), and incubated at 37°C for 24 hours. Finally, the cell culture supernatant was collected. The levels of IL-5 and IL-13 secreted by cells in the supernatant were detected using the corresponding ELISA kits (Mabtech, #3490-1H-20 for IL-5 detection and Mabtech, #3471-1H-20 for IL-13 detection, respectively). Figure 2BAs shown, co-culturing DCs with naïve T cells after TSLP stimulation significantly enhances the release of Th2 cytokines (e.g., IL-5 / IL-13). In contrast, TSLP stimulation of DCs alone or CD4+ T cells alone releases only some or no IL-5 or IL-13.

[0258] like Figure 2C As shown, the inhibition levels of IL-5 release were comparable across the test groups. The anti-TSLP antibody (Tezepelumab) showed lower inhibition levels at low doses, suggesting that inhibiting TSLP alone cannot completely suppress DC-mediated naïve T cell differentiation and maturation. However, as... Figure 2D As shown, for IL-13 release, administration of anti-IL-4Rα antibody (Dupilumab) or anti-TSLP antibody alone is almost ineffective in inhibiting its release, while the combination of anti-IL-4Rα antibody and anti-TSLP antibody significantly inhibits its release in a dose-dependent manner. These data demonstrate that simultaneously blocking TSLP and IL-4Rα can significantly inhibit the activity, recruitment, and release of related cytokines and chemokines by monocytes, especially antigen-presenting cells such as dendritic cells, of Th2 cells. Furthermore, it is expected that anti-IL-4Rα / TSLP bispecific antibodies will exert a stronger effect in Th2-mediated inflammatory diseases.

[0259] Example 2. Selection of Bispecific Antibody Molecular Structure

[0260] Bispecific molecular structure design

[0261] Having established that dual blockade of TSLP and IL-4Rα has a stronger therapeutic effect on Th2-mediated inflammation than monoclonal antibodies, this study used benchmark sequences (the amino acid sequences of the heavy and light chains of Dupilumab are shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively; the amino acid sequences of the heavy and light chains of Tezepelumab are shown in SEQ ID NO: 65 and SEQ ID NO: 66, respectively) to test the effects of different molecular forms on biological function. Figure 3As shown, this disclosure constructs three different test molecules, F-1, F-2, and F-3 (anti-IL-4Rα-TSLP trap, 2+2, and 1+1, respectively), for molecular form selection. In F-1, TSLPR and IL7Ra (the amino acid sequence of TSLPR is shown in SEQ ID NO: 70; the amino acid sequence of IL7Ra is shown in SEQ ID NO: 71) are ligated to the C-terminus of the heavy chain of Dupilumab via (G4S)5, and the corresponding coding gene sequences are synthesized. The corresponding heavy chain plasmid is obtained by cloning the homologous recombinase (Exnase® II, # C112-01) from Novizan Pharmaceuticals of Nanjing into the pcDNA3.1 vector. The coding gene sequence of the light chain of Dupilumab is then cloned into the pcDNA3.1 vector using the same homologous recombinase to obtain the corresponding light chain plasmid. In F-2, Teze_scFV (the amino acid sequence of Teze_scFV is shown in SEQ ID NO: 67) was ligated to the C-terminus of the heavy chain of Dupilumab using (G4S)5 to synthesize the corresponding coding gene sequence. This sequence was then cloned into the pcDNA3.1 vector using the homologous recombinase to obtain the corresponding heavy chain plasmid. Similarly, the coding gene sequence of the light chain of Dupilumab was cloned into the pcDNA3.1 vector using the same homologous recombinase to obtain the corresponding light chain plasmid. In F-3, the coding gene sequences of the heavy chains of Dupilumab and Tezepelumab were cloned into the pcDNA3.1 vector using the same homologous recombinase to obtain the corresponding heavy chain plasmids. Likewise, the coding gene sequences of the light chains of Dupilumab and Tezepelumab were cloned into the pcDNA3.1 vector using the same homologous recombinase to obtain the corresponding light chain plasmids.

[0262] Bispecific antibody expression

[0263] The above plasmid was transfected into Expi293F cells, and the target protein was obtained through purification. The specific procedures are as follows:

[0264] Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, # A14351-01). Cell density (viability > 95%) was checked one day before transfection, and the cells were adjusted to 3 × 10⁶ cells / day with fresh Expi293F medium. 6 Continue culturing at 100 cells / mL, and adjust the cell density to 3×100 on the day of transfection. 6 Cells / mL.

[0265] One-tenth (1 / 10) of the final transfection volume of Opti-MEM medium (Gibco, #31985-070) was used as the transfection buffer. Each group of plasmids to be transfected was added at 1 mg / L, with a light chain to heavy chain molar ratio of 1:1. The plasmids were mixed thoroughly, and PEIMax (Polysciences Inc. #24765-1) was added at a DNA:PEI mass ratio of 1:3. The mixture was mixed and incubated at room temperature for 20 min. Then, the mixture was gently poured into the Expi293F cell suspension while shaking. The cells were cultured on a shaker under the following conditions: 8% CO2, 36.5 °C, 120 rpm.

[0266] After culturing for 16-18 hours, 2% (v / L) of a 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a final concentration of 5 g / L glucose solution, and a final concentration of 2.2 mM Valproic acid sodium salt (Merk, # P4543-100G) were added to the cell suspension. After gentle mixing, the mixture was incubated for 7 days at 8% CO2, 36.5 °C, and 120 rpm, followed by centrifugation. The collected samples were then filtered through a disposable vacuum filter with a 0.22 µm pore size. Antibodies were processed using HiTrap. ® Affinity capture was performed using a MabSelect PrismA (GE Healthcare, #17549853) affinity chromatography column. Before purification, the tubing and the affinity chromatography column were cleaned with 10-20 column volumes of 0.1M NaOH, followed by 10-20 column volumes of distilled water, and then equilibrated with 5 column volumes of 1×PBS (Gibco). Subsequently, the filtered cell sample was added to the column, and the column was washed with 10 column volumes of 1×PBS to remove non-specifically bound proteins. The column was then rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluent was collected. The pH was adjusted to 6.0 with 2 M Tris, filtered for sterility, and sent for size exclusion chromatography (SEC) to confirm that the protein purity met the requirements.

[0267] Bispecific antibody activity verification

[0268] This study used CHO cells overexpressing IL-4Rα to test the blocking function of the prepared bispecific antibodies on the IL-4Rα / IL-4 / IL-13 pathway. The specific experimental procedure is as follows: cDNA encoding human IL-4Rα was cloned into the pCHO1.0 vector (Invitrogen), transfected into CHO-S cells (Invitrogen), and CHO-S cells overexpressing human IL-4Rα (CHO-S-human IL-4Rα) were generated. Cells were counted and diluted to 2×10⁶ cells. 6 Cells were added at a rate of 100 µL / well to 96-well U-bottom plates. The plates were centrifuged at 500 g for 5 min to remove the cell culture medium. The test samples (each bispecific antibody) were diluted with FACS buffer to an initial concentration of 20 nM, and then serially diluted three times to prepare a total of 12 concentration gradients. 50 μL of each gradient was added to the cells in the 96-well plates and mixed well. Biotinylated Human IL-4 protein (Acro Biosystems, #IL4-H82E0) was prepared to a concentration of 50 ng / mL, and 50 μL was added to each well of the U-bottom plate. The cells were resuspended and incubated at 4°C for 30 min. The cells were centrifuged at 500 g for 5 min to remove the supernatant, and washed twice with FACS buffer. After centrifugation at 500 g for 5 min, the FACS buffer was removed, and 100 µL of PE-Streptavidin secondary antibody (Biolegend, #554061) (1:200 diluted in FACS buffer) was added to each well. The plates were then incubated at 4°C in the dark for 30 min. After incubation, centrifuge at 500 g for 5 min to remove the supernatant, and wash the cells three times with FACS buffer. Resuspend the cells in 200 µL LFACS buffer and analyze by flow cytometry.

[0269] The blocking function of various bispecific antibodies on the TSLP / TSLPR pathway was evaluated using CTLL2-mCD127-hTSLPR-stat5-Luc2 reporter gene cells. cDNA encoding human TSLPR and IL7R was cloned into the Plenti-IRES-Neo vector (Invitrogen) and transfected into CTLL2-stat5-Luc2 cells (Invitrogen) to generate CTLL2-stat5-Luc2 cells overexpressing human TSLPR / IL7R. CTLL2-mCD127-hTSLPR-stat5-Luc2 reporter cells were cultured in IMDM medium (Gibco, #12440-053) containing 10% FBS, 1% NEAA (Gibco, #11140-050), 400 μg / mL Hygromycin B (Invitrogen, #10687010), 500 μg / mL GENETICIN (Gibco, #10131-027), and 30 ng / mL rhuIL-2 (R&D, #202-IL). The cells were washed twice with IMDM medium containing 10% FBS, 1% NEAA, and 0.5 ng / mL rhuIL-2, at a density of 1 × 10⁶ cells per well. 5 Cells were added to 96-well plates and cultured overnight at 37°C and 5% CO2. Antibody samples and the positive control antibody Tezepelumab (initial concentration 200 nM, serially down-diluted 10 times) were pre-incubated with 1 nM TSLP (Acro Biosystems, #TSP-H52Hb) at room temperature for 1 hour, then added to the cells. The cells were then cultured at 37°C and 5% CO2 for 6 hours. After culture, the luminescence signal produced by the cells was detected using the Bio-Lite Luciferase Assay System (Vazyme, #DD1201-03), and a dose-response curve was plotted.

[0270] The results showed that the bivalent form of anti-IL-4Rα antibody exhibited significantly better blocking activity than the monovalent anti-IL-4Rα antibody, while the monovalent anti-TSLP molecule achieved blocking activity completely equivalent to the bivalent form (e.g., Figure 4A and Figure 4B As shown, F-1, F-2, and F-3 are bispecific antibodies against IL-4Rα-TSLP trap, 2+2, and 1+1 forms, respectively, while F-1 is a TSLP trap that blocks TSLP function, but its activity is not superior to the anti-TSLP antibody Tezepelumab (e.g., ...). Figure 4BBased on the above data, the final molecular form was selected as the 2+2 Morris form (IgG-ScFv) (Coloma, MJ, Morrison, SL, NatBiotechnol 1997; 15: 159–63; Siwei Nie et al., Antib Ther. 2020; 3(1): 18–62.). However, considering that some antibodies will experience a decrease in activity after being converted to scFv form, TSLP antibody screening will be conducted in the scFv form in order to obtain clones with high blocking activity.

[0271] Example 3. Preparation and humanization of anti-IL-4Rα antibodies from hybridomas

[0272] Screening for anti-IL-4Rα antibodies at 10H4.6

[0273] In this study, hybridoma technology was used to immunize Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) with the extracellular fragment of human IL-4 Rα (SinoBiologica, #10402-H08H). After the serum titer met the requirements, the spleens of the mice were isolated to prepare a suspension of B lymphocytes, which were then electrofused with SP2 / 0 myeloma cells (ATCC).

[0274] Hybridoma supernatants were collected, and hybridoma cells specifically expressing anti-IL-4Rα antibodies were screened using fluorescence activated cell sorting (FACS). Supernatants from positive clones were collected for cellular-level IL-4Rα and its ligand IL-4 blocking experiments. Clones with strong blocking function were used in TF-1 assays for further cell proliferation testing, ultimately yielding one clone, 10H4, which showed excellent blocking activity against both IL-4 and IL-13.

[0275] The light and heavy chain gene sequences of the hybridoma candidate clone 10H4 were retrieved, and the human-mouse chimeric antibody Ch10H4 was constructed according to the following steps: 6. Take approximately 5 × 10⁶ freshly cultured cells from each cell line. 6 RNA was extracted from samples (Macherey-Nagel, #740984.250). cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Upstream primers were designed based on the base sequence located in the 5' FR1 region, and downstream primers were designed based on the base sequence located in the antibody constant region or FR4 region to amplify the variable region gene fragments of the antibody light and heavy chains. These fragments were ligated into a T-vector (Mighty TA-cloning Kit, Takara), and single clones were selected for sequencing. The sequencing results were analyzed and compared using MEGA7 software.

[0276] Clones with correctly matched and paired antibody light and heavy chain variable region sequences were ligated into the pcDNA3.1 vector using the homologous recombinase (Exnase® II, # C112-01) from Novizumi (Nanjing) company. Specifically, the heavy chain constant region was the IgG1 LALA subtype (shown as SEQ ID NO: 47), and the light chain constant region was the human κ chain constant region (shown as SEQ ID NO: 48). Expression plasmids for the light and heavy chain antibodies were obtained.

[0277] Then, the light chain plasmid and heavy chain plasmid of the same antibody were mixed at a 1:1 molar ratio and transfected into 293F cells using polyethyleneimine (PEI) (Polysciences, #23966). After culturing for 5-7 days, when the cell viability was below 60%, the cell culture supernatant was collected and purified using a Protein A affinity column to obtain the monoclonal antibody.

[0278] The activities of the final candidate molecules are shown in Table 2, among which the blocking activity of the chimeric antibody Ch10H4.6 is comparable to that of the control antibody Dupilumab.

[0279] Table 2. Inhibitory effect of IL-4Rα chimeric antibody on IL-4 / IL-13-induced TF-1 proliferation.

[0280]

[0281] Humanization of anti-IL-4Rα antibody 10H4.6

[0282] Using Discovery Studio and PyMOL software, the chimeric antibody Ch10H4.6 obtained above was humanized through the following steps: determining the CDR loop structure; finding the closest homologous sequences for each V / J region of the heavy and light chains in the human germline sequence database; screening for the human germline that best matches the heavy and light chains and the minimum amount of reversion mutations; constructing the CDR region of the chimeric antibody onto the human backbone region; using sequence and structural features to determine the amino acid positions in the backbone region that maintain the CDR function; performing reversion mutations (returning to the input amino acid type) at the identified important sequence positions; and gene synthesis and protein preparation.

[0283] After obtaining the proteins, the equilibrium dissociation constants (K0) of the chimeric antibody Ch10H4.6 and the humanized antibody Hz10H4.6 binding to IL-4Rα were determined using Octet Red96 and ForteBio thin-layer interferometry. DDupilumab (DUP) was used as a control. The BLI experiment was performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013, 5(2): 270-8).

[0284] In short, the AHQ (Pall, #1502051) sensor was equilibrated in analysis buffer for 30 minutes, followed by a 60-second baseline measurement. The purified antibody obtained as described above was immobilized on the AHQ sensor. The antibody-loaded sensor was then exposed to human IL-4Rα or cynomolgus monkey IL-4Rα, after which the sensor was transferred to analysis buffer for dissociation rate measurement. K was analyzed using Octet analysis software. D Values. The results of the antibody affinity test are shown in Table 3.

[0285] Table 3. Dissociation constants (K2) for antigen-antibody binding detection by ForteBio D (Unit: M)

[0286]

[0287] NB: Not combined

[0288] Example 4. Maturation of the anti-IL-4Rα parent Hz10H4.6 affinity

[0289] This study utilized yeast display technology to engineer Hz10H4.6 to achieve affinity maturation. The main procedures included library construction, library screening, yeast clone identification, protein expression, monoclonal antibody property analysis, and in vitro functional identification. First, six CDRs in the light and heavy chains were mutated to construct six affinity-matured libraries, which were then sorted using magnetic beads and flow cytometry. In the magnetic bead-based sorting, 0.3 nM Biotin-IL-4Rα (Acro Biosystems, #ILR-H82E9) was used to screen the six libraries. In the flow cytometry-based sorting, a balanced screening method was used, maintaining the antigen concentration at 0.3 nM. After multiple rounds of sorting, approximately 145 molecules without post-translational modifications (PTM) and exhibiting high affinity at the yeast level were obtained. To further improve affinity, molecules from the above libraries were reconstructed by PCR to obtain CDR combinatorial libraries containing different mutation sites. The screening methods for these combinatorial libraries also employed magnetic bead-based enrichment and flow cytometry-based sorting. In the magnetic bead-based enrichment method, the concentration of the antigen Biotin-IL-4Rα was 0.3 nM. In the flow cytometry-based sorting method, a kinetic competitive method was used to add parental antibody Hz10H4.6 at a concentration ratio of 1:3 to 1:10, i.e., 1 nM Biotin-IL-4Rα : 3 nM-10 nM Hz10H4.6 IgG. After multiple rounds of sorting and yeast monoclonal identification, molecules with relatively high MFI were selected as candidate molecules. Approximately 200 candidate IgG molecules were constructed and expressed for identification.

[0290] IgG protein preparation

[0291] After single-clone analysis, clones with high MFI (Mean Free Filtration Index) were selected as candidate molecules, and yeast plasmids were extracted. The yeast plasmids were extracted using a kit (Tiangen, #DP112-02). Primers were designed upstream of VH and VL, and downstream of CH1 and CL. Using the yeast-extracted plasmids as templates, they were ligated into the pcDNA3.1 vector using Exnase® II (#C112-01) from Novizumi (Nanjing). Specifically, the heavy chain constant region was the IgG1 LALA subtype; and the light chain constant region was the human κ chain constant region. Expression plasmids for light and heavy chain antibodies were obtained.

[0292] Then, the light chain plasmid and heavy chain plasmid of the same antibody were mixed at a 1:1 molar ratio and transfected into 293F cells using polyethyleneimine (PEI) (Polysciences, #23966). After culturing for 5-7 days, when the cell viability was below 60%, the cell culture supernatant was collected and purified using a Protein A affinity column to obtain the monoclonal antibody.

[0293] Affinity testing

[0294] The affinity of the matured antibody molecule for human IL-4Rα (SinoBiological, #10402-H08H) was determined by surface plasmon resonance (SPR).

[0295] Specifically, the immobilization of anti-human Fc IgG was performed using channels 1 and 2 of the CM5 chip. 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were freshly mixed and activated at a flow rate of 10 μL / min for 420 s. Then, the anti-human Fc IgG was diluted in 10 mM sodium acetate (pH 5.0) to a concentration of 20 μg / mL and injected into channels 3 and 4 of the chip, covalently coupling the protein to the chip channel surface at a coupling height of approximately 8000-10000 RU. Next, 1 M ethanolamine was injected at a flow rate of 10 μL / min for 420 seconds to block the remaining activation sites. The chip with the coupled protein was then equilibrated at a flow rate of 10 μL / min.

[0296] Each cycle of the affinity assay for human IL-4Rα between the candidate antibody and the maturation candidate molecule included antibody capture, antigen binding, and chip regeneration. First, the candidate antibody was diluted to 2 μg / mL and captured in channel 4 of the chip at a flow rate of 10 μL / min for 30 seconds. Then, human IL-4Rα antigen was serially diluted in 2-fold concentrations (0, 0.5, 1, 2, 4, 8, and 16 nM) and injected sequentially into channels 3 and 4 of the chip from low to high concentration. The binding time for human IL-4Rα was 180 seconds, and the dissociation time was 1200 seconds. Finally, the chip was regenerated by injection of 10 mM Glycine at pH 1.5 for 30 seconds. The antibody affinity assay results are shown in the table below.

[0297] Through comprehensive analysis, four candidate molecules were identified: H6, 1222D5 (hereinafter referred to as D5), 1222E2 (hereinafter referred to as E2), and 1180C3 (hereinafter referred to as C3). The affinity parameters of the candidate molecules are shown in Table 4, which indicates that the affinity of the candidate molecules is comparable to that of Dupilumab.

[0298] Table 4 Affinity test data for IL-4Rα candidate antibodies

[0299]

[0300] In vitro functional analysis

[0301] The inhibitory activity of the four maturing antibodies against IL-4 or IL-13-induced cell proliferation via IL-4Rα was detected using the TF-1 human erythroid leukemia cell line. Specifically, TF-1 cells were cultured in RPMI 1640 (Gibco, #22400-071) medium containing 10% FBS and 5 ng / mL GM-CSF (Acro Biosystems, #GMF-H4214). The cells were washed twice with RPMI 1640 medium containing 2% FBS, and then cultured at 1.8 × 10⁶ cells per well. 4 Cells were added to the wells of a 96-well plate. The four affinity-matured antibody samples, the negative control IgG1, and the positive control antibody Dupilumab were added to the cells, with an initial antibody concentration of 100 nM. These antibodies were serially diluted five-fold (10 times) and then added. 2 ng / mL of IL-4 (R&D, #204-IL-050) or 50 ng / mL of IL-13 (Acro Biosystems, #IL3-H52H4) were added. The cells were then incubated at 37°C and 5% CO2 for 72 hours. After incubation, the cells were analyzed using CellTiter-Globe assay. ® (Promega, #G7572) Cell proliferation was assessed and the IC50 of the antibody's inhibitory effect on cell proliferation was calculated. The results are shown in Table 5, and the antagonistic activity of the antibody was further analyzed. The results showed that both the candidate molecule and Dupilumab effectively inhibited IL-4 and IL-13-induced TF-1 cell proliferation, and their activities were comparable.

[0302] Table 5. Inhibitory effects of IL-4Rα candidate antibodies on IL-4 / 13-induced TF-1 cell proliferation.

[0303]

[0304] Example 5. Preparation and humanization of anti-TSLP antibodies from hybridomas

[0305] Antibody screening for anti-TSLP antibody 11H7E11

[0306] In this study, hybridoma technology was used to alternately immunize Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) with human TSLP protein (SinoBiological, #16135-H08H) and monkey TSLP protein (ACRO, #3385a-9CHF1-QH). Once the serum titer met the requirements, the spleens of the mice were isolated to prepare a suspension of B lymphocytes, which were then electrofused with SP2 / 0 myeloma cells (ATCC). The fused cells were then diluted to 1–2 × 10⁻⁶ cells / mL using a selective medium (1640 medium containing 20% ​​FBS and 1 × HAT). 4Cells / mL were seeded into 96-well plates, with 100 µL of cell suspension added to each well. Seven days after confluence, the culture medium was replaced with selection medium (1640 medium containing 10% FBS and 1 × HT), and cultured for 10 days (or longer, depending on cell growth status). The supernatant was then collected for analysis.

[0307] Hybridoma cells specifically expressing anti-TSLP antibodies were screened using ELISA. The experimental procedure was as follows: Human TSLP protein was diluted to a concentration of 1 μg / mL with citrate-buffered saline, and 100 μL was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the coated ELISA plates were washed three times with PBST buffer (1×PBS, 0.05% Tween® 20) and blotted dry. Blocking buffer (1×PBS, 0.05% Tween® 20, 1% BSA) was added to the 96-well ELISA plates and incubated at 37°C for 1 hour. After blocking, the plates were washed three times with PBST buffer and blotted dry. 100 μL of hybridoma supernatant was added to each well of the 96-well ELISA plate and incubated at room temperature for 2 hours. Subsequently, the plates were washed three times with PBST buffer and blotted dry. HRP Goat anti-mouse IgG (Biologend, #405306, dilution 1:3000) was diluted with blocking buffer and added to 100 μL per well of a 96-well microplate. The plate was incubated at room temperature in the dark for 1 hour. The plate was then washed three times with PBST buffer and blotted dry. 100 μL of TMB chromogenic buffer was added to each well for development, and the development was stopped using stop solution. The OD450 absorbance was measured using a microplate reader. The supernatant from positive clones was collected for TSLPR and its ligand TSLP blocking assays. Clones with strong blocking function were subcloned using limiting dilution, and single-clone cells were picked (e.g., from a single clone).

[0308] The limiting dilution subcloning procedure is as follows: Prepare a 96-well plate and add 200 µL of culture medium to each well. This culture medium is the same as the screening medium described above, except that HAT is replaced with HT (Gibco, # 11067-030). Prepare a cell suspension of the positive clones selected from the fusion screening. Add 100 µL of this suspension to each well of the first row of the plate and mix well. Then add 100 µL of the cell suspension from the first row to the second row, mix thoroughly, and add 100 µL to the next row; repeat the above steps. Let the 96-well plate stand for 30 min, and then count the cells under a microscope. Add 20 mL of culture medium to the volume corresponding to every 100 cells, mix well, and plate the plate (200 µL per well). After one week, observe under a microscope and identify and mark the wells containing monoclonal cells.

[0309] Once the cell confluence in each well reached over 50%, the target positive clones were selected using the high-throughput screening method described above. Further testing of the TSLP and TSLPR blocking functions of the target positive clones yielded candidate clone 11H7E11, which exhibited strong blocking activity.

[0310] The antibody light and heavy chain gene sequences of the hybridoma candidate clone 11H7E11 were retrieved and used to construct the mouse chimeric antibody 11H7E11 (the method is the same as described in Example 3; the heavy chain constant region is shown in SEQ ID NO: 47, and the light chain constant region is shown in SEQ ID NO: 48). Simultaneously, an adapter sequence (shown in SEQ ID NO: 63) was introduced at the C-terminus of the VH (shown in SEQ ID NO: 43) of the chimeric antibody and linked to the N-terminus of the VL (shown in SEQ ID NO: 44) of the chimeric antibody to construct an scFv antibody. A His tag was added to the C-terminus of the VL to facilitate subsequent purification. Following the method described in Example 2, the encoding gene of the scFv antibody was cloned into the pcDNA3.1 vector and transfected into Expi293F cells. Cell culture and protein purification were then performed to obtain the scFv antibody.

[0311] Affinity testing

[0312] Biofilm thin-layer interferometry (BLI) was used to determine the affinity of humanized molecules for human and monkey TSLP. Half an hour before the experiment, an appropriate number of AHC sensors (Sartorius, #18-5060) were immersed in SD buffer (1×PBS, 0.1% BSA, 0.05% Tween). ® 20). Dilute both the antibody and antigen to 100 nM. Add SD buffer, antibody, and antigen separately to 96-well black polystyrene microplates (Greiner, #655209). Use FortebioOctet... ® The Red96e system was used for detection. The plate was arranged according to the sample location, and the sensor position was selected. The instrument's operating parameters were set as follows: baseline equilibration 120 seconds, antibody loading and solidification 100 seconds, baseline equilibration 120 seconds, antigen binding 100 seconds, and dissociation 120 seconds; rotation speed was 1000 rpm, and temperature was 30℃. After the experiment, a ForteBio Octet was used. ® The analysis software analyzed the affinity and kinetic parameters. The test results of antibody affinity detection are shown in Table 6 below:

[0313] Table 6. Affinity assay results of anti-TSLP antibody candidate clones

[0314]

[0315] In vitro functional analysis

[0316] The blocking activity of 11H7E11 chimeric antibody and scFv antibody against TSLP / TSLPR was detected using CTLL2-mCD127-hTSLPR-stat5-Luc2 reporter gene cells. Specific experimental methods are as described in Example 2, and the results are shown below. Figure 5A The results showed that the blocking activity of the 11H7E11 chimeric antibody was superior to that of Tezepelumab.

[0317] In addition, in vitro functional analysis was performed using TSLP antibody to inhibit TSLP-induced secretion of CCL-17 chemokine by human mDCs. In short, initial myeloid DCs were isolated and purified from human peripheral blood mononuclear cells (PBMCs) using the EasySep™ Human Myeloid DC Enrichment Kit (Stemcell, #19061), and the obtained mDCs were distributed in batches of 5 × 10⁶ cells per well. 5 Cells were seeded at a density suitable for 96-well cell culture plates. Serially diluted antibody samples (initial concentration 300 nM) and a final concentration of 50 ng / mL human TSLP protein (Acro Biosystems, #TSP-H52Hb) were pre-incubated at 37°C for 45 minutes. These were then added to wells containing mDCs (molecularly derived cells). mDCs were stimulated in vitro, and the cells were cultured at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected, and the CCL-17 content in the supernatant was detected using a CCL-17 / TARC ELISA kit (R&D, #SDN00). Results are shown below. Figure 5B The results showed that both the 11H7E11 chimeric antibody and the scFv antibody had stronger blocking activity than Tezepelumab.

[0318] Antibody humanization

[0319] Based on Discovery Studio and PyMOL software, the chimeric antibody Ch11H7 (containing the heavy chain constant region of the IgG1 LALA subtype (shown as SEQ ID NO: 47) and the light chain constant region of the human κ chain constant region (shown as SEQ ID NO: 48)) obtained from hybridoma screening was humanized through the following steps: determining the CDR loop structure; finding the closest homologous sequences for each V / J region of the heavy and light chains in the human germline sequence database; screening for the human germline that best matches the heavy and light chains and the minimum amount of reversion mutations; constructing the CDR region of the chimeric antibody onto the human backbone region; using sequence and structural features to determine the amino acid positions in the backbone region that maintain the CDR function; performing reversion mutations (returning to the input amino acid type) at the determined important sequence positions; gene synthesis and protein preparation.

[0320] After obtaining the antibody, the affinity between the humanized molecule and human TSLP was determined using biofilm thin-layer interferometry (BLI), and the specific method is as described in Example 3.

[0321] As shown in the affinity data in Table 7, the humanized antibody Hz11H7 molecule maintained an affinity comparable to that of the chimeric antibody.

[0322] Table 7. Dissociation constants (K2) for ForteBio detection of antigen-antibody binding D (Unit: M)

[0323]

[0324] Example 6. Construction, expression, and purification of bispecific antibodies

[0325] Bispecific antibody construction

[0326] A bispecific antibody targeting both IL-4Rα and TSLP was prepared, according to Example 2, which has... Figure 3 The molecular structure of "2+2" in the text.

[0327] A schematic diagram of the structure of an exemplary bispecific antibody is shown below. Figure 6A As shown, it includes peptide chain #1 and peptide chain #2. The linear structures of peptide chain #1 and peptide chain #2 are as follows. Figure 6B As shown.

[0328] Peptide chain #1, from N-terminus to C-terminus, comprises the heavy chain variable region of the anti-IL-4Rα antibody, the CH1 and Fc domains of human IgG1, and a single-chain variable fragment (scFv) linked by synthetic peptides, where VH and VL are derived from the anti-TSLP antibody. Peptide chain #2, from N-terminus to C-terminus, comprises the light chain variable domain of the anti-IL-4Rα antibody and the immunoglobulin CL domain (human κ chain constant region). Specifically, the scFv formed by linking the VH and VL of the anti-TSLP antibody via linker 2 (4×G4S) is linked to the C-terminus of the Fc domain of the IL-4Rα monoclonal antibody molecule via linker 1 (6×G4S).

[0329] Bispecific antibody expression

[0330] The constructed light chain plasmid and heavy chain plasmid were transfected into Expi293F cells, and the target protein was obtained by purification, as described in Example 2.

[0331] In short, the density of Expi293F cells was adjusted to 3×10⁻⁶. 6For each cell / mL cell, mix all plasmids to be transfected (light chain plasmid to heavy chain plasmid molar ratio of 1:1) in transfection buffer (containing polyethyleneimine (PEI)), then mix DNA with PEI at a mass ratio of 1:3. After incubation for 20 min, add the mixture to Expi293F cell suspension and culture on a shaker at 8% CO2, 36.5 °C, and 120 rpm.

[0332] After culturing for 16-18 hours, 2% (v / L) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), 5 g / L glucose solution, and 2.2 mM Valproic acid sodium salt (Merk, # P4543-100G) were added to the cell suspension. The cells were cultured for another 7 days and then collected by centrifugation. After centrifugation, the samples were filtered through a disposable vacuum filter with a 0.22 µm pore size. Antibodies were processed using HiTrap. ® Affinity capture was performed using a MabSelect PrismA (GE Healthcare, #17549853) affinity chromatography column. Before purification, the tubing and column were cleaned with 10-20 column volumes of 0.1 M NaOH, followed by 10-20 column volumes of distilled water. The column was then equilibrated with 5 column volumes of 1×PBS (Gibco). Subsequently, the filtered cell sample was added to the column, and the column was washed with 10 column volumes of 1×PBS to remove non-specifically bound proteins. The column was then rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluent was collected. The pH was adjusted to 6.0 with 2 M Tris, filtered for sterilization, and sent to the SEC for analysis to confirm that the protein purity met the requirements.

[0333] Example 7. Binding kinetics of bispecific antibodies and antigens

[0334] The affinity of the bispecific antibody candidate molecule for human IL-4Rα (SinoBiological, #10402-H08H) was determined by surface plasmon resonance (SPR).

[0335] Specifically, anti-human Fc IgG was conjugated to channels 1 and 2 of the CM5 chip. 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were freshly mixed and activated at a flow rate of 10 μL / min for 420 seconds. Then, the anti-human Fc IgG was diluted in 10 mM sodium acetate (pH 5.0) to a concentration of 20 μg / mL and injected into channels 3 and 4 of the chip, covalently conjugating the protein to the chip channel surface at a conjugation height of approximately 8000-10000 RU. Next, 1 M ethanolamine was injected at a flow rate of 10 μL / min for 420 seconds to block the remaining activation sites. The chip with conjugated protein was then equilibrated at a flow rate of 10 μL / min.

[0336] Each cycle of the affinity assay for the maturation candidate molecule with human IL-4Rα included antibody capture, antigen binding, and chip regeneration. First, the candidate molecule antibody was diluted to 2 μg / mL and captured in channel 4 of the chip at a flow rate of 10 μL / min for 30 s. Then, the antigen human IL-4Rα was serially diluted in 2-fold concentrations (0, 0.5, 1, 2, 4, 8, and 16 nM) and injected sequentially into channels 3 and 4 of the chip from low to high concentration. The human IL-4Rα binding time was 180 seconds, and the dissociation time was 1200 seconds. Finally, the chip was regenerated by injecting 10 mM Glycine pH 1.5 for 30 seconds. The antibody affinity assay results are shown in the table below.

[0337] Table 8 Results of Bispecific Antibody Affinity Test

[0338]

[0339] The affinity of bispecific antibody molecules for human and monkey TSLP was determined using biofilm thin-layer interferometry (BLI). The experimental method is as described in Example 5. The antibody affinity at the anti-TSLP terminus of the bispecific antibody was detected similarly to that of the monoclonal antibody, using the dissociation constants (K0) of the four candidate molecules for TSLP. D The values ​​are both approximately 2.50E-10M, comparable to Tezepelumab.

[0340] Example 8. Linker optimization for bispecific antibodies

[0341] As shown in Example 6 above, this disclosure constructs a bispecific antibody by linking the heavy chain variable region and the light chain variable region of the TSLP antibody to the C-terminus of the Fc domain of the IL-4Rα monoclonal antibody molecule through Linker 2 (4×G4S) to form an scFv.

[0342] To test the stability of candidate molecules, they were subjected to accelerated thermal stability testing at 40°C for two weeks, followed by capillary electrophoresis (CE) and mass spectrometry to detect aggregates, fragmentation, and post-translational modifications. The CE-SDS results showed... Figure 7A The proportion of aggregates increased by 5.1%, the proportion of fragmentation products increased by 9.2%, and approximately 10% of the S residues in linker 1 (6×G4S) underwent O-glycosylation. Based on these results, it is speculated that the excessive length of linker 1 likely caused aggregation and fragmentation during the accelerated thermal stability test at 40°C for 2 weeks. Furthermore, it is expected that replacing the G4S repeat sequence in linker 1 with a GGGGG (SEQ ID NO: 64) repeat sequence could avoid the heterogeneity caused by O-glycosylation of S residues.

[0343] Therefore, the linker length was optimized by replacing 6×G4S with GGGGG(G5), 3×(G5), and 4×(G5), respectively, to generate H6-5G-hz11H7-2-scFv, H6-15G-hz11H7-2-scFv, and H6-20G-hz11H7-2-scFv. In vitro biological function tests were performed according to the method described in Example 2, and the results showed that different linker lengths had no effect on the blocking function. Figure 8A and Figure 8B Subsequently, the H6-11H7 molecule with the shortest linker (H6-5G-hz11H7-2-scFv) was selected for accelerated thermal stability testing at 40 degrees Celsius for two weeks. The results showed no significant increase in aggregates and fracture products. Figure 7B ).

[0344] Example 9. Experiment on the inhibition of hIL-4 and hIL-13-induced TF-1 cell proliferation by bispecific antibodies.

[0345] The inhibitory activity of bispecific antibodies against IL-4 or IL-13-induced cell proliferation via IL-4Rα was detected using the TF-1 human erythroid leukemia cell line. Specifically, TF-1 cells were cultured in RPMI 1640 (Gibco, #22400-071) medium containing 10% FBS and 5 ng / mL GM-CSF (AcroBiosystems, #GMF-H4214). Cells were washed twice with RPMI 1640 medium containing 2% FBS, and cultured at 1.8 × 10⁶ cells per well. 4Cells were added to the wells of a 96-well plate. Bispecific antibody samples (H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv), negative control IgG1 (its heavy and light chain amino acid sequences are shown in SEQ ID NOs: 72 and 73, respectively), and positive control antibody Dupilumab were added to the cells. The initial antibody concentration was 100 nM, and it was diluted 5-fold in 10 gradients. 2 ng / mL of IL-4 (R&D, #204-IL-050) or 50 ng / mL of IL-13 (AcroBiosystems, #IL3-H52H4) was added, followed by incubation at 37°C and 5% CO2 for 72 hours. After incubation, the cells were analyzed using CellTiter-Globe assay. ® (Promega, #G7572) Cell proliferation was detected and dose-response curves were plotted. Figure 9A and Figure 9B The antagonistic activity of the antibodies was then analyzed. The results showed that both the bispecific antibody and Dupilumab effectively inhibited IL-4 and IL-13-induced TF-1 cell proliferation. Specifically, Figure 9A The results showed that the bispecific antibody and Dupilumab inhibited the IL-4-induced proliferation of TF-1 cells. The IC50 values ​​of H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv were approximately 0.32 nM, 0.16 nM, 0.09 nM, and 0.17 nM, respectively, while Dupilumab showed a lower IC50. 50 It is approximately 0.27 nM; Figure 9B The results showed that the bispecific antibody and Dupilumab inhibited the proliferation of IL-13-induced TF-1 cells. The IC50 values ​​of H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv inhibited the proliferation of IL-13-induced TF-1 cells by approximately 0.31 nM, 0.27 nM, 0.15 nM, and 0.31 nM, respectively, while the IC50 of Dupilumab was approximately 0.37 nM.

[0346] Example 10. Assay on the activity of CTLL2 reporter gene in cells by bispecific antibody binding to TSLP.

[0347] The bispecific antibody blocking TSLP binding to the TSLPR / IL7R receptor on the surface of CTLL2 cells was detected using CTLL2-mCD127-hTSLPR-stat5-Luc2 reporter gene cells. The experimental method was as described in Example 2. Bispecific antibody samples (H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv) and the positive control antibody Tezepelumab (initial antibody concentration 300 nM, three-fold dilution, 10 gradients) were pre-incubated with 1 nM TSLP (Acro Biosystems, #TSP-H52Hb) at room temperature for 1 hour, and then added to the cells. The cells were cultured at 37°C and 5% CO2 for 6 hours. After culture, the luminescence signal produced by the cells was detected using the Bio-Lite Luciferase Assay System (Vazyme, #DD1201-03), and a dose-response curve was plotted. Figure 10 The study then analyzed the antagonistic activity of the antibodies. Results showed that both the tested bispecific antibody and Tezepelumab effectively inhibited the binding of TSLP and CTLL2 reporter genes to TSLPR / IL7R on the cell surface. Figure 10 The results showed that the IC50 values ​​of H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv inhibited CTLL2 reporter gene activity in cells at approximately 0.65 nM, 0.49 nM, 0.61 nM, and 0.51 nM, respectively, which were significantly lower than those of the control antibody Tezepelumab (whose IC50 was approximately 2.69 nM). These results indicate that, compared to the control antibody Tezepelumab, the tested bispecific antibodies exhibited superior inhibitory activity against CTLL2 reporter gene activity by binding to TSLP.

[0348] Example 11. Inhibition of CCL-17 release activity induced by bispecific antibodies in hIL-4, hIL-13 and hTSLP-induced PBMCs.

[0349] The activity of bispecific antibodies (H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, D5-5G-hz11H7-2-scFv) antagonizing IL-4 / IL-13-induced CCL-17 release via IL-4Rα was measured using PBMCs. Specifically, PBMCs (AllCell, #FPB004F-C) were resuscitated, resuspended in RPMI 1640 cell culture medium (Gibco, #22400-071), and the cells in the culture medium were cultured at 4 × 10⁶ cells per well. 5 Cells were added to the wells of a 96-well plate. Antibody was added to the cells (antibody dilution method: initial concentration 100 nM, 8-fold dilution in cell culture medium, for a total of 8 dilutions), along with 0.03 nM IL-4 (R&D, #204-IL-050) and 0.3 nM IL-13 (R&D, #213-ILB-100 / CF). The cells were then cultured at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected, and the release of CCL-17 expressed in the supernatant was detected using a CCL17 / TARC ELISA kit (R&D, #SDN00), and a dose-response curve was plotted to analyze the antibody antagonistic activity. Figure 11A As shown, the results indicated that the bispecific antibody inhibited IL-4 / IL-13-induced CCL-17 release from PBMCs. The IC50 of the control antibody Dupilumab in inhibiting IL-4 and IL-13-induced CCL-17 release from PBMCs was approximately 0.09 nM, and the IC50 of the combination of Dupilumab and Tezepelumab was approximately 0.07 nM. In contrast, the IC50 values ​​of the bispecific antibodies H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv in inhibiting IL-4 and IL-13-induced CCL-17 release from PBMCs were approximately 0.18 nM, 0.10 nM, 0.08 nM, and 0.14 nM, respectively.

[0350] The bispecific antibody also significantly inhibited the release of CCL-17 from TSLP-induced PBMCs. The concentration of TSLP (Acro Biosystems, #TSP-H52Hb) added during the assay was 0.3 nM; the remaining steps are described above. Figure 11BAs shown, the bispecific antibodies H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv effectively inhibited TSLP-induced release of CCL-17 from peripheral blood PBMCs. Their inhibitory activity (IC50 values ​​of approximately 0.12 nM, 0.07 nM, 0.08 nM, and 0.18 nM, respectively) was significantly superior to that of the control antibody Tezepelumab and the combination of Dupilumab and Tezepelumab (IC50 values ​​of approximately 0.21 nM and 0.49 nM, respectively).

[0351] Similarly, the bispecific antibody also showed a significant inhibitory effect on the release of CCL-17 from PBMCs induced simultaneously by IL-4, IL-13, and TSLP. The concentrations of IL-4, IL-13, and TSLP added during the assay were consistent with the concentrations used in the previous tests. Figure 11C As shown, the bispecific antibodies H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv effectively inhibited the release of CCL-17 from PBMCs induced by IL-4, IL-13, and TSLP. Their inhibitory activities (with IC50 values ​​of approximately 0.57 nM, 0.22 nM, 0.25 nM, and 0.25 nM, respectively) were significantly superior to those of the control antibody Dupilumab and the combination of Dupilumab and Tezepelumab (with IC50 values ​​of approximately 3.07 nM and 0.58 nM, respectively).

[0352] The experimental results of the above embodiments show that, compared with existing IL-4Rα monoclonal antibodies and TSLP monoclonal antibodies, the bispecific antibody disclosed herein has comparable or superior effects to the currently approved drugs Dupilumab and Tezepelumab.

[0353] Example 12. Therapeutic effect of bispecific antibodies on asthmatic mice

[0354] A mouse asthma model was established as follows. IL-4 / IL-4Rα humanized transgenic mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were used. On days 0, 7, and 14, IL-4 / IL-4Rα humanized mice were sensitized by intraperitoneal injection of 50 μg of ovalbumin (Sigma, #A5503) dissolved in 0.2 mL of solvent (a saline solution containing 2% Al2O3 as an adjuvant). From days 21 to 24, mice were anesthetized and challenged with nasal drops of 200 μg of ovalbumin (OVA) dissolved in 1×PBS daily.

[0355] This experiment included a total of 7 groups, including a blank control group, a model control IgG group, a Dupilumab group, and a bispecific antibody group (H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv and D5-5G-hz11H7-2-scFv, respectively), with 5 mice in each group (3 mice in the blank control group). The drugs were administered intraperitoneally on days 20 and 23 (the blank control group and the model control IgG group were injected with the same amount of IgG antibody (purchased from Equitech-Bio, #SLH56)). On day 25, bronchoalveolar lavage fluid (BALF) of the mice was collected for flow cytometry analysis.

[0356] BALF was centrifuged at 500 g for 5 min, the supernatant was discarded, and the sample was washed twice with FACS buffer. Then, the prepared antibodies (PE-Cy7 anti-CD45 (BioLegend, #103114), AF700 anti-Siglec F (BioLegend, #565183), APC anti-Ly6G (BioLegend, #127610), BUV395 anti-CD11b (BioLegend, #563553), AF488 anti-F4 / 80 (BioLegend, #123120), BV421 anti-Ly6C (BioLegend, #128032), BB700 anti-CD11C (BioLegend, #745852), PE Dazzel anti-MHC-Ⅱ (BioLegend, #107648), BV605) were added. The cells were incubated with anti-CD3 (BioLegend, #100237) and PE anti-B220 (BioLegend, #103208) at 4°C for 30 min. The cells were then washed twice with FACS buffer, and the number of different immune cells was detected by flow cytometry.

[0357] like Figure 12A-12DAs shown, after OVA sensitization and challenge, the number of lymphocytes (CD45+%) in the model control IgG group was higher than that in the blank control group. Figure 12A Compared with the model control IgG group, the number of CD45-positive cells (CD45+%) in the peripheral blood of mice in the Dupilumab group and the bispecific antibody group was significantly reduced, and the number of eosinophils ( Figure 12B ), monocytes ( Figure 12C ) and neutrophils ( Figure 12D The number of all of them has decreased significantly.

[0358] Example 13. Pharmacokinetic Study of Bispecific Antibody in Mice

[0359] The pharmacokinetics of bispecific antibodies were assessed using intravenous injection (IV) in mice. Each group consisted of nine BALB / c mice weighing approximately 20 g. Each mouse was intravenously injected with 10 mg / kg of the bispecific antibody. Blood samples were collected from the orbital cavity at 5 minutes, 0.5 hours, 2 hours, 6 hours, and on days 2, 4, 7, 14, and 21 after the single administration. After the blood clotted naturally, the serum was collected by centrifugation. The method for determining the antibody drug concentration in serum is as follows: Dilute the antigen human IL-4Rα-his protein (Acro Biosystems, #ILR-H5221) to 1 µg / mL with coating buffer (prepared by dissolving one packet of carbonate powder (Thermo, #23282) in 400 mL of ultrapure water, mixing well, and bringing the volume to 500 mL). Add 100 µL to each well of a 96-well ELISA plate (Thermo, #442404) and incubate overnight at 4°C. Discard the coating buffer and wash the plate three times with 1×PBST. Add 200 µL of blocking buffer (PBST solution containing 2% BSA) to each well and block at room temperature for 1 h. Discard the blocking buffer, wash the plate three times with 1×PBST, and then add diluted mouse serum; incubate the plate at room temperature for 2 h. Discard the solution in the ELISA plate and wash five times with 1×PBST. Add diluted Goat anti-human IgG-Fc-HRP (BETHYL, # A80-104P) at 100 µL per well and incubate at room temperature for 1 hour. Discard the solution in the ELISA plate and wash 5 times with 1× PBST. Add 100 µL of TMB chromogenic buffer (Solepro, #PR1200) to each well and incubate for 5–10 min. Stop incubation with 50 µL of stop buffer (Solepro, #C1058) to each well. Measure the OD450 nm and OD620 nm values ​​using an ELISA reader. The changes in plasma drug concentrations at different time points in four groups of mice administered bispecific antibodies (H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv) at a dose of 10 mg / kg are shown below. Figure 13 As shown. Pharmacokinetic parameters were calculated using an Excel PKSolver non-compartmental model, including the main drug exposure parameter (T). 1 / 2 C max AUC 0-t AUC 0-∞ (CL and Vss) are shown in Table 9.

[0360] Table 9. Pharmacokinetic parameters of candidate molecules in mice

[0361]

[0362] Example 14. YTE molecular construction and pharmacokinetic studies

[0363] Three sites in the Fc region of the bispecific antibodies H6-5G-hz11H7-2-scFv, C3-5G-hz11H7-2-scFv, E2-5G-hz11H7-2-scFv, and D5-5G-hz11H7-2-scFv described in the above examples were subjected to site-directed mutation (M252Y / S254T / T256E (EU Numbering), i.e., YTE mutation) to further enhance their binding ability to human FcRn, thereby extending their half-life in vivo. Similar to the method described in Example 6, a CHO-S cell line expressing the above-mentioned mutated antibodies was generated using the Freedom® CHO-S® kit (Invitrogen). The transfected cells underwent two rounds of pressure selection to obtain a cell pool with high antibody expression. The cell pool was then expanded through culture to express the antibody in large quantities, and the cell supernatant was collected and purified using a Protein A column to achieve an antibody purity >95%. The obtained antibodies were named H6-11H7-YTE, D5-11H7-YTE, C3-11H7-YTE, and E2-11H7-YTE (e.g., Figure 6B As shown, the amino acid sequences of its peptide chain #1 are shown in SEQ ID NOs: 74-77.

[0364] The affinity of the above-mentioned antibody for human FcRn was detected using ForteBio, and as expected, the YTE mutation increased the affinity by more than 10 times under specific conditions.

[0365] The pharmacokinetics of bispecific antibodies were detected by intravenous injection (IV) in FcRn humanized transgenic mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.). Specifically, each group consisted of 6 FcRn humanized transgenic mice, weighing approximately 20 g. Each mouse was intravenously injected with 10 mg / kg of the bispecific antibody D5-5G-hz11H7-2-scFv or D5-11H7-YTE. Blood samples were collected from the orbital cavity at 5 minutes, 2 hours, 6 hours, 24 hours, 48 ​​hours, day 4, day 7, day 14, day 21, and day 28 after a single administration. After the blood clotted naturally, the serum was collected by centrifugation.

[0366] The method for determining the antibody drug concentration in serum is as follows: Dilute the antigenic human IL-4Rα-His protein (Acro Biosystems, # ILR-H5221) to 1 µg / mL with coating buffer (prepared by dissolving one packet of carbonate powder (Thermo, # 23282) in 400 mL of ultrapure water, mixing well, and bringing the volume to 500 mL). Add 100 µL to each well of a 96-well ELISA plate (Thermo, Cat # 442404) and incubate overnight at 4°C. Discard the coating buffer and wash the plate three times with 1× PBST. Add 200 µL of blocking buffer (PBST solution containing 2% BSA) to each well and block at room temperature for 1 hour. Remove the blocking buffer, wash the plate three times with 1×PBST, then add 100 μL of diluted standard (D5-5G-hz11H7-2-scFv), quality control samples (standards at different dilutions for inter-laboratory quality control), and test sample (D5-11H7-YTE) to each well. Incubate at room temperature in the dark for 2 hours. Discard the solution in the ELISA plate, and wash five times with 1×PBST. Add 100 μL of diluted Goat anti-human IgG-Fc-HRP (1:80000 dilution, BETHYL, Cat# A80-104P) to each well, and incubate at room temperature for 1 hour (in the dark). Discard the solution in the ELISA plate, and wash five times with 1×PBST. Add 100 μL of TMB chromogenic solution (Solepro, Cat# PR1200) to each well and incubate at room temperature in the dark for 5–10 minutes. Then add stop solution (Solepro, Cat# C1058), gently shake for 10 seconds, and measure OD450 nm and OD620 nm values ​​within 30 minutes. The changes in plasma drug concentration at different time points in mice administered the test samples (drugated at 10 mg / kg) are shown below. Figure 14 As shown. Pharmacokinetic parameters were calculated using an Excel PKSolver non-compartmental model, including the main drug exposure parameter (T). 1 / 2 C max AUC 0-t AUC 0-∞ The values ​​of CL and Vss are shown in Table 10.

[0367] Table 10. Pharmacokinetic parameters of D5-5G-hz11H7-2-scFv and D5-11H7-YTE molecules in FcRn humanized mice

[0368]

[0369] Other YTE molecules constructed in this embodiment (including H6-11H7-YTE, C3-11H7-YTE and E2-11H7-YTE) exhibited pharmacokinetic properties comparable to D5-11H7-YTE.

[0370] Other implementation methods

[0371] Implementation Method 1. An isolated anti-TSLP antibody or its antigen-binding fragment, comprising:

[0372] HCDR1 shown in SEQ ID NO: 37, HCDR2 shown in SEQ ID NO: 38, HCDR3 shown in SEQ ID NO: 39; and / or

[0373] LCDR1 shown in SEQ ID NO: 40, LCDR2 shown in SEQ ID NO: 41, and LCDR3 shown in SEQ ID NO: 42.

[0374] Implementation Method 2. The anti-TSLP antibody or its antigen-binding fragment as described in Implementation Method 1, comprising:

[0375] VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43 or 45, and

[0376] VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44 or 46.

[0377] Implementation Method 3. An anti-TSLP antibody or its antigen-binding fragment as described in Implementation Method 1 or 2, wherein,

[0378] Amino acids differing from those in the amino acid sequence shown in SEQ ID NO: 43 or 45, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity, are predominantly or entirely present in the FR region; or

[0379] Amino acids that differ from the amino acid sequence shown in SEQ ID NO: 44 or 46, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity, are predominantly or entirely present in the FR region.

[0380] Implementation Method 4. The anti-TSLP antibody or its antigen-binding fragment as described in any one of Implementation Methods 1-3, comprising:

[0381] (1) A VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44; or

[0382] (2) VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 45, and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46;

[0383] In this region, all amino acids that differ from the amino acid sequences shown in any of SEQ ID NOs: 43, 44, 45 and 46 in sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more are present in the FR region.

[0384] Implementation Method 5. The anti-TSLP antibody or its antigen-binding fragment as described in any one of Implementation Methods 1-4, comprising:

[0385] (1) VH shown in SEQ ID NO: 43 and VL shown in SEQ ID NO: 44; or

[0386] (2) VH shown in SEQ ID NO: 45 and VL shown in SEQ ID NO: 46.

[0387] Implementation Method 6. An anti-TSLP antibody or antigen-binding fragment thereof as described in any one of Implementation Methods 1-5, wherein the anti-TSLP antibody or antigen-binding fragment thereof is an anti-TSLP scFv.

[0388] Implementation Method 7. The anti-TSLP antibody or its antigen-binding fragment as described in Implementation Method 6, wherein the anti-TSLP scFv includes a linker comprising an amino acid sequence (GGGGS). n n is an integer from 1 to 5, preferably 2, 3 or 4.

[0389] Implementation Method 8. An anti-TSLP antibody or its antigen-binding fragment as described in Implementation Method 6 or 7, wherein the anti-TSLP scFv comprises:

[0390] (1) VH shown in SEQ ID NO: 43, VL shown in SEQ ID NO: 44, and the connector shown in SEQ ID NO: 63; or

[0391] (2) VH shown in SEQ ID NO: 45, VL shown in SEQ ID NO: 46, and connector shown in SEQ ID NO: 63.

[0392] Implementation 9. An anti-TSLP antibody or antigen-binding fragment thereof as described in any one of Implementations 1-5, wherein the anti-TSLP antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 or variants thereof, and the light chain constant region is selected from the constant regions of human κ chain and λ chain or variants thereof.

[0393] Embodiment 10. An anti-TSLP antibody or antigen-binding fragment thereof as described in any one of Embodiments 1-5 and 9, wherein the anti-TSLP antibody comprises a heavy chain and a light chain, wherein,

[0394] The heavy chain includes VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43 or 45, and CH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 47;

[0395] Furthermore, the light chain includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44 or 46, and a CL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 48.

[0396] Embodiment 11. An anti-TSLP antibody or antigen-binding fragment thereof as described in any one of Embodiments 1-5 and 9-10, wherein the anti-TSLP antibody comprises a heavy chain and a light chain, wherein,

[0397] (1) The heavy chain comprises VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 43 and CH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 47; and the light chain comprises VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 44 and CL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 48; or

[0398] (2) The heavy chain comprises VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 45 and CH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 47; and the light chain comprises VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 46 and CL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 48;

[0399] In this region, all amino acids that differ from the amino acid sequences shown in any of SEQ ID NOs: 43, 44, 45 and 46 in sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more are present in the FR region.

[0400] Embodiment 12. An anti-TSLP antibody or antigen-binding fragment thereof as described in any one of Embodiments 1-5 and 9-11, wherein the anti-TSLP antibody comprises a heavy chain and a light chain, wherein,

[0401] (1) The heavy chain comprises VH shown in SEQ ID NO: 43 and CH shown in SEQ ID NO: 47; and

[0402] The light chain includes VL shown in SEQ ID NO: 44 and CL shown in SEQ ID NO: 48; or

[0403] (2) The heavy chain comprises VH shown in SEQ ID NO: 45 and CH shown in SEQ ID NO: 47; and

[0404] The light chain includes VL shown in SEQ ID NO: 46 and CL shown in SEQ ID NO: 48.

[0405] Implementation Method 13. An anti-TSLP antibody or an antigen-binding fragment thereof as described in any one of Implementation Methods 1-5 and 9-12, wherein the antigen-binding fragment is selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated CDR regions, and nanobodies.

[0406] Embodiment 14. A polynucleotide encoding an anti-TSLP antibody or an antigen-binding fragment thereof as described in any one of Embodiments 1-13.

[0407] Embodiment 15. An expression vector comprising the polynucleotide described in Embodiment 14.

[0408] Embodiment 16. A host cell, said cell having integrated the polynucleotide of Embodiment 14 or the expression vector of Embodiment 15.

[0409] Embodiment 17. An immunoconjugate comprising any one of embodiments 1-13 of the present invention, wherein the immunoconjugate comprises an anti-TSLP antibody or an antigen-binding fragment thereof.

[0410] Embodiment 18. A pharmaceutical composition comprising: an anti-TSLP antibody or an antigen-binding fragment thereof as described in any one of Embodiments 1-13, or an immunoconjugate as described in Embodiment 17, and optionally pharmaceutically acceptable excipients.

[0411] Embodiment 19. A kit comprising the anti-TSLP antibody or its antigen-binding fragment as described in any one of Embodiments 1-13, the immunoconjugate as described in Embodiment 17, or the pharmaceutical composition as described in Embodiment 18.

[0412] Example 20. Use of the anti-TSLP antibody or antigen-binding fragment thereof as described in any one of Examples 1-13, the immunoconjugate of Example 17, the pharmaceutical composition of Example 18, or the kit of Example 19 in the preparation of the antibody or antigen-binding fragment thereof.

[0413] Example 21. Use of the anti-TSLP antibody or antigen-binding fragment thereof as described in any one of Examples 1-13, the immunoconjugate described in Example 17, the pharmaceutical composition described in Example 18, or the cassette described in Example 19 in the preparation of a medicament for the treatment or prevention of a disease or disorder (e.g., cancer or autoimmune disease).

[0414] Implementation 22. Use as described in Implementation 21, wherein the disease or disorder is a type II or mixed allergic disease, such as asthma and / or atopic dermatitis.

[0415] It will be understood that although the invention has been described in conjunction with a detailed description of the invention, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to TSLP (thymic stromal lymphopoietin), comprising: The heavy chain variable region (VH) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein... The VH CDR1 region contains at least 80% of the amino acid sequence identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region contains at least 80% of the amino acid sequence identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region contains at least 80% of the amino acid sequence identical to the selected VH CDR3 amino acid sequence; and The light chain variable region (VL) comprises CDR1, CDR2, and CDR3, wherein the VL CDR1 region contains at least 80% of the same amino acid sequence as the selected VL CDR1 amino acid sequence, the VL CDR2 region contains at least 80% of the same amino acid sequence as the selected VL CDR2 amino acid sequence, and the VL CDR3 region contains at least 80% of the same amino acid sequence as the selected VL CDR3 amino acid sequence; The selected VH CDR1, VH CDR2, and VH CDR3 amino acid sequences and the selected VL CDR1, VL CDR2, and VLCDR3 amino acid sequences are one of the following: (1) The selected VH CDR1, VH CDR2 and VH CDR3 amino acid sequences are shown in SEQ ID NO:37, 38 and 39, respectively, and the selected VL CDR1, VL CDR2 and VL CDR3 amino acid sequences are shown in SEQ ID NO:40, 41 and 42, respectively; (2) The selected VH CDR1, VH CDR2 and VH CDR3 amino acid sequences are shown in SEQ ID NO:86, 38 and 39, respectively, and the selected VL CDR1, VL CDR2 and VL CDR3 amino acid sequences are shown in SEQ ID NO:40, 41 and 42, respectively; (3) The selected VH CDR1, VH CDR2 and VH CDR3 amino acid sequences are shown in SEQ ID NO:117, 118 and 119, respectively, and the selected VL CDR1, VL CDR2 and VL CDR3 amino acid sequences are shown in SEQ ID NO:120, 121 and 122, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The VH comprises CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NO:37, 38, and 39, respectively, and the VL comprises CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NO:40, 41, and 42, respectively. VH CDR1 is determined according to the definition of AbM. Among them, VH CDR2, VH CDR3 and VL CDR1, VL CDR2, VL CDR3 are determined according to the Kabat definition.

3. The antibody or its antigen-binding fragment according to claim 1, wherein, According to Kabat's definition, the VH comprises CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NO:86, 38, and 39, respectively, and the VL comprises CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NO:40, 41, and 42, respectively.

4. The antibody or its antigen-binding fragment according to claim 1, wherein, According to Chothia's definition, the VH comprises CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO: 117, 118, and 119, respectively, and the VL comprises CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NO: 120, 121, and 122, respectively.

5. An antibody or antigen-binding fragment thereof that binds to TSLP, comprising: The heavy chain variable region (VH) and the light chain variable region (VL) contain at least 90% of the same amino acid sequence as the selected VH sequence, and the light chain variable region contains at least 90% of the same amino acid sequence as the selected VL sequence. in, The selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO:43, and the selected VL sequence is SEQ ID NO:44; and (2) The selected VH sequence is SEQ ID NO:45 and the selected VL sequence is SEQ ID NO:

46.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein, The antibody or antigen-binding fragment specifically binds to human TSLP and / or monkey TSLP.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein, The antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein, The antigen-binding fragment is selected from Fab fragment, Fab' fragment, F(ab')2 fragment, Fd fragment, Fv fragment, dAb fragment, isolated CDR region, scFv and nanobodies.

9. An antibody or antigen-binding fragment thereof that binds to TSLP, comprising: The heavy chain variable region (VH) and the light chain variable region (VL) contain VH CDR1, VH CDR2, and VH CDR3 that are identical to those of the selected VH sequence, and the light chain variable region contains VL CDR1, VL CDR2, and VL CDR3 that are identical to those of the selected VL sequence. in, The selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO:43, and the selected VL sequence is SEQ ID NO:44; and (2) The selected VH sequence is SEQ ID NO:45 and the selected VL sequence is SEQ ID NO:

46.

10. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 1-9.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein, The antibody or its antigen-binding fragment is a bispecific antibody or a multispecific antibody or its antigen-binding fragment.

12. A nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising: (1) An immunoglobulin heavy chain or a fragment thereof, wherein the heavy chain or fragment thereof comprises VH, wherein the VH comprises CDR1, CDR2 and CDR3, wherein the CDR comprises amino acid sequences shown in SEQ ID NO:37, 38, 39, SEQ ID NO:86, 38, 39, or SEQ ID NO:117, 118, 119, respectively; and wherein, When the VH pairs with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:44 or 46, the VH binds to TSLP; (2) An immunoglobulin light chain or a fragment thereof, wherein the light chain or the fragment thereof comprises a VL, wherein the VL comprises CDR1, CDR2 and CDR3, wherein the CDR comprises the amino acid sequences shown in SEQ ID NO:40, 41 and 42 respectively; or the amino acid sequences shown in SEQ ID NO:120, 121 and 122 respectively; and wherein the VL binds to TSLP when it is paired with a VH comprising the amino acid sequence shown in SEQ ID NO:43 or 45.

13. The nucleic acid according to claim 12, wherein, When the VH pairs with the VL, the VH specifically binds to human TSLP.

14. The nucleic acid according to claim 12 or 13, wherein, The immunoglobulin heavy chain or fragment thereof is a heavy chain or fragment thereof of human immunoglobulin, or a heavy chain or fragment thereof of humanized immunoglobulin.

15. The nucleic acid according to any one of claims 12-14, wherein, The nucleic acid encodes a single-stranded variable fragment (scFv), a bispecific or multispecific antibody, or an antigen-binding fragment thereof.

16. The nucleic acid according to any one of claims 12-15, wherein, The nucleic acid in question is cDNA.

17. A vector comprising one or more nucleic acids according to any one of claims 12-16, or nucleic acids encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-11.

18. A cell comprising the carrier of claim 17.

19. The cell according to claim 18, wherein, The cells in question are CHO cells.

20. A cell comprising one or more nucleic acids according to any one of claims 12-16, or nucleic acids encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-11.

21. A method for generating an antibody or an antigen-binding fragment thereof, or an antigen-binding protein construct thereof, the method comprising: (a) The cells are cultured under conditions sufficient to cause the cells of any one of claims 18-20 to produce the antibody or its antigen-binding fragment, or the antigen-binding protein construct thereof; as well as (b) Collect the antibody or its antigen-binding fragment or the antigen-binding protein construct produced by the cells.

22. An antibody-drug conjugate comprising a therapeutic agent covalently bound to an antibody or an antigen-binding fragment thereof as described in any one of claims 1-11.

23. The antibody-drug conjugate according to claim 22, wherein, The therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

24. A pharmaceutical composition comprising: a pharmaceutically acceptable carrier, and an antibody or antigen-binding fragment thereof as described in any one of claims 1-11, or an antibody-drug conjugate as described in claim 22 or 23.

25. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11, or the antibody-drug conjugate of claim 22 or 23.

26. A method of treating a subject suffering from an immune disorder or cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-11, an antibody-drug conjugate as described in claim 22 or 23, or a pharmaceutical composition as described in claim 24.

27. The method according to claim 26, wherein, The immune disorder mentioned is an allergy, asthma, or atopic dermatitis.

28. The method according to claim 26, wherein, The immune disorder is a type II or mixed allergic disease.

29. The method according to any one of claims 26-28, wherein, The subjects are either humans or non-human animals.