ANTI-TSLP ANTIBODY AND USES THEREOF

Abstract

Related are an anti-TSLP antibody or an antigen-binding fragment thereof, nucleic acid molecules coding the same, and a method for preparing the same. The anti-TSLP antibody or the antigen-binding fragment thereof have high affinity to TSLP, capable of effectively binding with TSLP and blocking the proliferative effect of TSLP with respect to Ba/F3-hTSLPR-hIL7Rα cells, and blocking the capability of TSLP in activating and secreting cytokines with respect to PBMC. At the same time, further related are a medicinal composition comprising the antibody or the antigen-binding fragment thereof, and uses of the composition in preparing a medicament for preventing and/or treating asthma, allergic inflammation, an allergic reaction or autoimmune disease.

Claims

1. An antibody or antigen-binding fragment thereof that binds to thymic stromal lymphopoietin (TSLP), wherein the antibody or antigen-binding fragment thereof comprises complementary determining regions (CDRs) as follows: (a) a CDR-H1 or a sequence variant thereof, a CDR-H2 or a sequence variant thereof, and a CDR-H3 or a sequence variant thereof, contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 1, 17, 30, 40, 53 or 68; and/or (b) a CDR-L1 or a sequence variant thereof, a CDR-L2 or a sequence variant thereof, and a CDR-L3 or a sequence variant thereof, contained in a light chain variable region (VL) as set forth in SEQ ID NO: 2, 18, 31, 41, 54 or 69; preferably, the sequence variant is a CDR having a substitution, deletion or addition of one or more amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to the CDR from which it is derived; preferably, the substitution is a conservative substitution.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (1) a VH and/or a VL, wherein, as defined by the IMGT numbering system: (1a) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 3, a CDR-H2 with the sequence of SEQ ID NO: 4 and a CDR-H3 with the sequence of SEQ ID NO: 5; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 6, a CDR-L2 with the sequence of SEQ ID NO: 7 and a CDR-L3 with the sequence of SEQ ID NO: 8; (1b) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 19, a CDR-H2 with the sequence of SEQ ID NO: 20 and a CDR-H3 with the sequence of SEQ ID NO: 21; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 22, a CDR-L2 with the sequence of SEQ ID NO: 23 and a CDR-L3 with the sequence of SEQ ID NO: 24; (1c) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 32, a CDR-H2 with the sequence of SEQ ID NO: 33 and a CDR-H3 with the sequence of SEQ ID NO: 34; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 35, a CDR-L2 with the sequence of SEQ ID NO: 23 and a CDR-L3 with the sequence of SEQ ID NO: 24; (1d) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 42, a CDR-H2 with the sequence of SEQ ID NO: 43 and a CDR-H3 with the sequence of SEQ ID NO: 44; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 45, a CDR-L2 with the sequence of SEQ ID NO: 46 and a CDR-L3 with the sequence of SEQ ID NO: 47; or (1e) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 55, a CDR-H2 with the sequence of SEQ ID NO: 56 and a CDR-H3 with the sequence of SEQ ID NO: 57; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 58, a CDR-L2 with the sequence of SEQ ID NO: 59 and a CDR-L3 with the sequence of SEQ ID NO: 60; (2) a VH and/or a VL, wherein, as defined by the AbM numbering system: (2a) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 9, a CDR-H2 with the sequence of SEQ ID NO: 10 and a CDR-H3 with the sequence of SEQ ID NO: 11; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 12, a CDR-L2 with the sequence of SEQ ID NO: 13 and a CDR-L3 with the sequence of SEQ ID NO: 8; (2b) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 25, a CDR-H2 with the sequence of SEQ ID NO: 26 and a CDR-H3 with the sequence of SEQ ID NO: 27; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 28, a CDR-L2 with the sequence of SEQ ID NO: 29 and a CDR-L3 with the sequence of SEQ ID NO: 24; (2c) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 36, a CDR-H2 with the sequence of SEQ ID NO: 37 and a CDR-H3 with the sequence of SEQ ID NO: 38; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 39, a CDR-L2 with the sequence of SEQ ID NO: 29 and a CDR-L3 with the sequence of SEQ ID NO: 24; (2d) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 48, a CDR-H2 with the sequence of SEQ ID NO: 49 and a CDR-H3 with the sequence of SEQ ID NO: 50; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 51, a CDR-L2 with the sequence of SEQ ID NO: 52 and a CDR-L3 with the sequence of SEQ ID NO: 47; or (2e) the VH comprises a CDR-H1 with the sequence of SEQ ID NO: 61, a CDR-H2 with the sequence of SEQ ID NO: 62 and a CDR-H3 with the sequence of SEQ ID NO: 63; and/or, the VL comprises a CDR-L1 with the sequence of SEQ ID NO: 64, a CDR-L2 with the sequence of SEQ ID NO: 65 and a CDR-L3 with the sequence of SEQ ID NO: 60; or (3) a VH and/or a VL, wherein, as compared to the VH and/or the VL in any one of (1a), (1b), (1c), (1d), (1e) or (2a), (2b), (2c), (2d), (2e), at least one CDR contains a mutation, wherein the mutation is a substitution, deletion or addition of one or several amino acids or any combination thereof (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids, or any combination thereof); preferably, the substitution is a conservative substitution; preferably, the antibody or antigen-binding fragment thereof binds to human TSLP and/or monkey TSLP.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH as set forth in SEQ ID NO: 1, 17, 30, 40, 53 or 68, and/or a VL as set forth in any one of SEQ ID NO: 2, 18, 31, 41, 54 or 69; (b) a VH having at least 70%, at least 80%, at least 85%, 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% sequence identity to any VH in (a); and/or, a VL having at least 70%, at least 80%, at least 85%, 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% sequence identity to any VL in (a); or (c) a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (for example, a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) as compare to any VH in (a); and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (for example, a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) as compare to any VL in (a); preferably, the substitution is a conservative substitution.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH as set forth in SEQ ID NO: 1 and a VL as set forth in SEQ ID NO: 2; (b) a VH as set forth in SEQ ID NO: 17 and a VL as set forth in SEQ ID NO: 18; (c) a VH as set forth in SEQ ID NO: 30 and a VL as set forth in SEQ ID NO: 31; (d) a VH as set forth in SEQ ID NO: 40 and a VL as set forth in SEQ ID NO: 41; (e) a VH as set forth in SEQ ID NO: 53 and a VL as set forth in SEQ ID NO: 54; (f) a VH as set forth in SEQ ID NO: 68 and a VL as set forth in SEQ ID NO: 69; (g) a VH and a VL, wherein the VH has at least 70%, at least 80%, at least 85%, 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% sequence identity to; and/or, the VL has at least 70%, at least 80%, at least 85%, 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% sequence identity to the VH and the VL in any of (a) to (f); or (h) a VH and a VL, wherein the VH has a substitution, deletion or addition of one or several amino acids or any combination thereof (for example, a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof); and/or the VL has a substitution, deletion or addition of one or several amino acids or any combination thereof (for example, a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof), as compared to the VH and VL in any of (a) to (f); preferably, the substitution is a conservative substitution.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody, humanized antibody, or fully human antibody.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof; and/or (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, 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% sequence identity to the wild-type sequence from which it is derived; alternatively, the variant has a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g. a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or any combination thereof) as compared to the wild-type sequence from which it is derived; preferably, the substitution is a conservative substitution; preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG1; preferably, the light chain constant region is kappa or lambda light chain constant region, and more preferably, the antibody or antigen-binding fragment thereof comprises a human kappa light chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the heavy chain constant region or the variant thereof comprises: (1) a heavy chain constant region of human IgG1 or a variant thereof, wherein the variant is mutated at least one of positions 234, 235, 237, 265, 297, 331, 329 and 434 according to the EU numbering system, preferably, the variant comprises at least one of the following mutations: L234A, L235A, D265A, N297A, L234F, L235E, P331S, P329G, N434A, N434Y, N434F, N434W, N434S, N434G, N434H and N434Q; more preferably, the variant comprises at least one of the following mutations: L234A, L235A, G237A and N434A; more preferably, the IgG1 variant comprises the mutations of L234A, L235A and G237A; and more preferably, the IgG1 variant comprises the mutations of L234A, L235A, G237A and N434A; (2) a CH as set forth in SEQ ID NO: 14 or a variant thereof, wherein the variant comprises conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) as compared to SEQ ID NO: 14, or having at least 70%, at least 80%, at least 85%, 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% sequence identity to SEQ ID NO: 14; the variant comprises N297A and/or N434A according to the EU numbering system, preferably, the variant comprises N434A; (3) a CH as set forth in SEQ ID NO: 15 or a variant thereof, wherein the variant comprises conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) as compared to SEQ ID NO: 15, or having at least 70%, at least 80%, at least 85%, 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% sequence identity to SEQ ID NO: 15; (4) a heavy chain constant region of human IgG4 or a variant thereof, wherein the variant is mutated at least one of positions 228 and 434 according to the EU numbering system; preferably, the variant comprises S228P and/or N434A; preferably, the variant comprises S228P and N434A; or (5) a CH as set forth in SEQ ID NO: 70 or a variant thereof, wherein the variant comprises conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) as compared to SEQ ID NO: 70, or having at least 70%, at least 80%, at least 85%, 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% sequence identity to SEQ ID NO: 70; and/or the light chain constant region or the variant thereof comprises: (6) a kappa light chain constant region; or (7) a light chain constant region (CL) as set forth in SEQ ID NO: 16 or a variant thereof, wherein the variant comprises conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) as compared to SEQ ID NO: 16, or having at least 70%, at least 80%, at least 85%, 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% sequence identity to SEQ ID NO: 16; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 14 and a light chain constant region (CL) as set forth in SEQ ID NO: 16; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 15 and a light chain constant region (CL) as set forth in SEQ ID NO: 16; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 70 and a light chain constant region (CL) as set forth in SEQ ID NO: 16; preferably, the mutation or substitution renders the antibody or antigen-binding fragment thereof to have no or reduced ADCP, ADCC and/or CDC activity, as compared to the corresponding antibody or antigen-binding fragment thereof not comprising said mutation or substitution.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises: (a) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a CH as set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL as set forth in SEQ ID NO: 2 and a CL as set forth in SEQ ID NO: 16; (b) a heavy chain comprising a VH as set forth in SEQ ID NO: 17 and a CH as set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL as set forth in SEQ ID NO: 18 and a CL as set forth in SEQ ID NO: 16; (c) a heavy chain comprising a VH as set forth in SEQ ID NO: 30 and a CH as set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL as set forth in SEQ ID NO: 31 and a CL as set forth in SEQ ID NO: 16; (d) a heavy chain comprising a VH as set forth in SEQ ID NO: 40 and a CH as set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL as set forth in SEQ ID NO: 41 and a CL as set forth in SEQ ID NO: 16; (e) a heavy chain comprising a VH as set forth in SEQ ID NO: 53 and a CH as set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL as set forth in SEQ ID NO: 54 and a CL as set forth in SEQ ID NO: 16; (f) a heavy chain comprising a VH as set forth in SEQ ID NO: 68 and a CH as set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL as set forth in SEQ ID NO: 69 and a CL as set forth in SEQ ID NO: 16; or (g) a heavy chain and a light chain, wherein, the heavy chain has at least 80%, at least 85%, 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% sequence identity to, and/or, the light chain has at least 70%, at least 80%, at least 85%, 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% sequence identity to the heavy chain and the light chain in any of (a)-(f).

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain and a light chain, the heavy chain comprising: (i) a sequence as set forth in SEQ ID NO: 66 or 73; (ii) a sequence having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or any combination thereof) as compared to the sequence in (i); or (iii) a sequence having at least 80%, at least 85%, 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% sequence identity to the sequence in (i); and the light chain comprising: (iv) a sequence as set forth in SEQ ID NO: 67; (v) a sequence having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or any combination thereof) as compared to the sequence in (iv); or (vi) a sequence having at least 80%, at least 85%, 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% sequence identity to the sequence in (iv); preferably, the substitution in (ii) or (v) is a conservative substitution; or (b) a heavy chain and a light chain, the heavy chain comprising: (i) a sequence as set forth in SEQ ID NO: 71; (ii) a sequence having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or any combination thereof) as compared to the sequence in (i); or (iii) a sequence having at least 80%, at least 85%, 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% sequence identity to the sequence in (i); and the light chain comprising: (iv) a sequence as set forth in SEQ ID NO: 72; (v) a sequence having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or any combination thereof) as compared to the sequence in (iv); or (vi) a sequence having at least 80%, at least 85%, 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% sequence identity to the sequence in (iv); preferably, the substitution in (ii) or (v) is a conservative substitution.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, Fab, Fab′, F(ab′).sub.2, Fv fragments, disulfide linked Fv (dsFv) and a diabody.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof carries a marker; preferably, the antibody or antigen-binding fragment thereof carries a detectable marker such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance) or biotin.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof exhibits at least one of the following characteristics: (a) binding to TSLP (e.g., human TSLP) with a K.sub.D of less than about 50 nM, e.g., less than about 20 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or less, wherein the K.sub.D is measured by Fortebio or ELISA; (b) binding to TSLP (e.g., human TSLP) with an EC50 of less than about 50 nM, e.g., less than about 20 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or less, wherein the EC50 is measured by flow cytometry or ELISA; (c) inhibiting the binding of TSLP to IL7Rα/TSLPR with an IC50 of less than about 50 nM, e.g., about 50 nM, 20 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or less, wherein the IC50 is measured by ELISA; (d) inhibiting or blocking TSLP-induced activation and/or proliferation of mast cells, DC, NKT cells; (e) inhibiting or blocking TSLP-induced OX40L expression; (f) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion; (g) inhibiting or blocking TSLP-induced secretion of Th2 Cytokines, such as TARC, CCL22, IL-4, IL-13 or IL-5; (h) having a good affinity for binding to FcRn; or (i) having an isoelectric point (PI) of about 6.5 to about 8.5, such as about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.7, about 7.9, about 8.0, about 8.2 or about 8.5.

13. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to claim 1.

14. A vector comprising the isolated nucleic acid molecule according to claim 13; preferably, the vector is a cloning vector or an expression vector.

15. A host cell comprising the isolated nucleic acid molecule according to claim 13, or a vector comprising the isolated nucleic acid molecule according to claim 13.

16. A method for preparing the antibody or antigen-binding fragment thereof according to claim 1, comprising: culturing a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to claim 1 or a vector comprising the isolated nucleic acid molecule under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

17. A multispecific antibody, comprising the antibody or antigen-binding fragment thereof binding to TSLP according to claim 1, and an additional antibody or fragment thereof or an antibody mimetic; preferably, the multispecific antibody is a bispecific antibody or trispecific antibody or tetraspecific antibody.

18. A conjugate comprising the antibody or antigen-binding fragment thereof according to claim 1 and a coupling moiety, wherein the coupling moiety is a detectable marker, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or enzyme, or the coupling moiety is a therapeutic agent.

19. A pharmaceutical composition comprising: (i) the antibody or antigen-binding fragment thereof according to claim 1, (ii) a nucleic acid encoding (i), (iii) a vector comprising (ii), (iv) a host cell comprising (i) or (ii), (v) a multispecific antibody comprising (i) and an additional antibody or fragment thereof or an antibody mimetic, and/or (vi) a conjugate comprising (i) and a coupling moiety that is a detectable marker or a therapeutic agent, and a pharmaceutically acceptable carrier and/or excipient.

20. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is used in at least one of the following biological activities in a subject: (a) inhibiting or blocking the binding of TSLP to IL7Rα-TSLPR, (b) down-regulating or eliminating the activity of TSLP, (c) down-regulating or blocking OX40L expression, (d) inhibiting or blocking TSLP-induced activation and/or proliferation of mast cells, DC, NKT cells, (e) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion, or (f) inhibiting or blocking TSLP-induced secretion of Th2 Cytokines, such as TARC, CCL22, IL-4, IL-13 or IL-5.

21. A kit comprising: (i) the antibody or antigen-binding fragment thereof according to claim 1, and/or (ii) a nucleic acid encoding (i), and/or (iii) a vector comprising (ii), and/or (iv) a host cell comprising (ii) or (iii), and/or (v) a multispecific antibody comprising (i) and an additional antibody or fragment thereof or an antibody mimetic, and/or (vi) a conjugate comprising (i) and a coupling moiety that is a detectable marker or a therapeutic agent, and/or (vii) a pharmaceutical composition comprising any one or more of (i) to (vi) and a pharmaceutically acceptable carrier and/or excipient, and optionally an instruction of use.

22. (canceled)

23. A method of in vivo/in vitro at least one of: (a) inhibiting or blocking the binding of TSLP to IL7Rα-TSLPR; (b) down-regulating or eliminating the activity of TSLP; (c) down-regulating or blocking OX40L expression; (d) inhibiting or blocking TSLP-induced activation and/or proliferation of mast cells, DC, NKT cells, (e) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion; or (f) inhibiting or blocking TSLP-induced secretion of Th2 cytokines, such as TARC, CCL22, IL-4, IL-13 or IL-5, comprising: administering an effective amount of: (i) the antibody or antigen-binding fragment thereof according to claim 1, (ii) a nucleic acid encoding (i), (iii) a vector comprising (ii), (vi) a host cell comprising (ii) or (iii), (v) a multispecific antibody comprising (i) and an additional antibody or fragment thereof or an antibody mimetic, (vi) a conjugate comprising (i) and a coupling moiety that is a detectable marker or a therapeutic agent, or (vii) a pharmaceutical composition comprising any one or more of (i) to (vi) and a pharmaceutically acceptable carrier and/or excipient, optionally, an additional therapeutic is administered simultaneously with, before or after administration of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the multispecific antibody, the conjugate, or the pharmaceutical composition.

24. A method of preventing and/or treating an allergic inflammation or an autoimmune disease in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to claim 19, wherein the subject is a mammal; preferably, the subject is a human, optionally, an additional therapeutic is administered simultaneously with, before or after administration of the pharmaceutical composition, optionally, the allergic inflammation is selected from the group consisting of: asthma, idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, COPD, systemic sclerosis, keloid, ulcerative colitis, chronic sinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; Churg-Strauss syndrome, eosinophilia, eosinophilic granuloma with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema; optionally, the autoimmune disease is selected from the group consisting of: diabetes, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid diseases, psoriasis and thyroid diseases; optionally, the additional therapeutic immunosuppressants (for example, corticosteroids, non-steroidal glucocorticoid receptor agonists, leukotriene D4 antagonists, leukotriene B4 antagonists, A2A agonists, A2B antagonists, dopamine receptor agonists, pirfenidone, nintedanib, or avB6 antagonists), bronchodilators (for example, beta-2 adrenergic receptor agonists, muscarinic antagonists, short-acting β2 receptor agonists, long-acting β2 receptor agonists, short-acting anticholinergic drugs, methyl xanthine drugs, long-acting anticholinergic drugs), other cytokine or cytokine receptor antagonists or antibodies (for example, IL-13 antagonists, IL-6 antagonists, antagonists of IL-1, IL-33, IL-25 or TNF-alpha, anti-IgE antibodies, anti-IL31 antibodies, anti-IL31R antibodies, anti-IL13 antibodies, anti-endoglin antibodies, anti-IL1b antibodies, another anti-TSLP antibody or anti-hTSLPR antibodies), antibiotics, radiotherapy, leukotriene antagonists (for example, montelukast, zafirlukast or pranlukast), PDE4 inhibitors (for example, roflumilast, xanthene), antihistamines or antitussive drugs.

25. A method of detecting the presence or level of TSLP in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof according to claim 1 under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof and TSLP, and detecting the formation of the complex.

26. A medicament or kit for diagnosing an allergic inflammation or an autoimmune disease, comprising: (i) the antibody or antigen-binding fragment thereof according to claim 1, (ii) a nucleic acid encoding (i), (iii) a vector comprising (ii), (iv) a host cell comprising (ii) or (iii), (v) a multispecific antibody comprising (i) and an additional antibody or fragment thereof or an antibody mimetic, (vi) a conjugate comprising (i) and a coupling moiety that is a detectable marker or a therapeutic agent, or (vii) a pharmaceutical composition comprising any one or more of (i) to (vi) and a pharmaceutically acceptable carrier and/or excipient, optionally, the allergic inflammation is selected from the group consisting of: asthma, idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, COPD, systemic sclerosis, keloid, ulcerative colitis, chronic sinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; Churg-Strauss syndrome, eosinophilia, eosinophilic granuloma with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema; optionally, the autoimmune disease is selected from the group consisting of: diabetes, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid diseases, psoriasis and thyroid diseases.

27. A method of diagnosing asthma, allergic inflammation, allergic reactions, or autoimmune diseases in a subject, comprising: contacting a sample from the subject with the antibody or antigen-binding fragment thereof according to claim 1 under conditions allowing to form a complex between the antibody or antigen-binding fragment thereof and TSLP; and detecting the formation of the complex, wherein, an increased level of TSLP is indicative of asthma, allergic inflammation, allergic reactions, or autoimmune diseases, as compared with a healthy control.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0363] FIG. 1: Detection of Ba/F3 cell line overexpressing both human TSLPR/IL7Rα genes.

[0364] FIG. 2A: Detection of inhibitory activity of chimeric antibody 25A5C5 on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0365] FIG. 2B: Detection of inhibitory activity of chimeric antibodies 27C2B6 and 37C2D10 on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0366] FIG. 2C: Detection of inhibitory activity of chimeric antibodies 43B1A8 and 90H3H11 on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0367] FIG. 3A: Detection of binding affinity of recombinant fully human antibodies 25A5C5-hIgG, 27C2B6-hIgG and 37C2D10-hIgG to human TSLP protein.

[0368] FIG. 3B: Detection of binding affinity of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG to human TSLP protein.

[0369] FIG. 4A: Detection of inhibitory activity of recombinant fully human antibodies 25A5C5-hIgG, 27C2B6-hIgG and 37C2D10-hIgG on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0370] FIG. 4B: Detection of inhibitory activity of recombinant fully human antibody 43B1A8-hIgG on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0371] FIG. 4C: Detection of inhibitory activity of recombinant fully human antibody 90H3H11-hIgG on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0372] FIG. 5: Detection of inhibitory activity of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG on secretion of TARC by PBMC.

[0373] FIG. 6: Detection of thermal stability (Tm) of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG.

[0374] FIG. 7A: Detection of inhibitory activity of recombinant fully human antibody 43B1-H2L2 on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0375] FIG. 7B: Detection of inhibitory activity of recombinant fully human antibody 43B1-H6L1 on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0376] FIG. 8: Detection of inhibitory activity of recombinant fully human antibody 43B1-H2L2 on secretion of MDC cytokines by PBMC cells.

[0377] FIG. 9: Pharmacokinetic analysis of recombinant fully human antibody 43B1-H2L2 molecule in cynomolgus monkeys.

SEQUENCE INFORMATION

[0378] The sequence information related to the invention is described in the following table.

TABLE-US-00001 SEQ ID NO. Description 1 Heavy chain variable region of antibody 25A5C5 2 Light chain variable region of antibody 25A5C5 3 IMGT 25A5C5 CDR-H1 4 IMGT 25A5C5 CDR-H2 5 IMGT 25A5C5 CDR-H3 6 IMGT 25A5C5 CDR-L1 7 IMGT 25A5C5 CDR-L2 8 IMGT/AbM 25A5C5 CDR-L3 9 AbM 25A5C5 CDR-H1 10 AbM 25A5C5 CDR-H2 11 AbM 25A5C5 CDR-H3 12 AbM 25A5C5 CDR-L1 13 AbM 25A5C5 CDR-L2 14 Modified heavy chain constant region of human IgG1 15 Modified heavy chain constant region of human IgG1 16 Human κ light chain constant region 17 Heavy chain variable region of antibody 27C2B6 18 Light chain variable region of antibody 27C2B6 19 IMGT 27C2B6 CDR-H1 20 IMGT 27C2B6 CDR-H2 21 IMGT 27C2B6 CDR-H3 22 IMGT 27C2B6 CDR-L1 23 IMGT 27C2B6 CDR-L2/ 37C2D10 CDR-L2 24 IMGT/AbM 27C2B6 CDR-L3/ IMGT/AbM 37C2D10 CDR-L3 25 AbM 27C2B6 CDR-H1 26 AbM 27C2B6 CDR-H2 27 AbM 27C2B6 CDR-H3 28 AbM 27C2B6 CDR-L1 29 AbM 27C2B6 CDR-L2/ 37C2D10 CDR-L2 30 Heavy chain variable region of antibody 37C2D10 31 Light chain variable region of antibody 37C2D10 32 IMGT 37C2D10 CDR-H1 33 IMGT 37C2D10 CDR-H2 34 IMGT 37C2D10 CDR-H3 35 IMGT 37C2D10 CDR-L1 36 AbM 37C2D10 CDR-H1 37 AbM 37C2D10 CDR-H2 38 AbM 37C2D10 CDR-H3 39 AbM 37C2D10 CDR-L1 40 Heavy chain variable region of antibody 43B1A8/ 43B1A8-hIgG/43B1-H6L1 41 Light chain variable region of antibody 43B1A8/ 43B1A8-hIgG/43B1-H6L1 42 IMGT 43B1A8/43B1A8-hIgG/43B1- H6L1 CDR-H1 43 IMGT 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-H2 44 IMGT 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-H3 45 IMGT 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-L1 46 IMGT 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-L2 47 IMGT/AbM 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-L3 48 AbM 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-H1 49 AbM 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-H2 50 AbM 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-H3 51 AbM 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-L1 52 AbM 43B1A8/43B1A8-hIgG/43B1- H6L1/43B1-H2L2 CDR-L2 53 Heavy chain variable region of antibody 90H3H11 54 Light chain variable region of antibody 90H3H11 55 IMGT 90H3H11 CDR-H1 56 IMGT 90H3H11 CDR-H2 57 IMGT 90H3H11 CDR-H3 58 IMGT 90H3H11 CDR-L1 59 IMGT 90H3H11 CDR-L2 60 IMGT/AbM 90H3H11 CDR-L3 61 AbM 90H3H11 CDR-H1 62 AbM 90H3H11 CDR-H2 63 AbM 90H3H11 CDR-H3 64 AbM 90H3H11 CDR-L1 65 AbM 90H3H11 CDR-L2 66 43B1A8-hIgG heavy chain sequence 67 43B1A8-hIgG/43B1-H6L1 light chain sequence 68 Heavy chain variable region of antibody 43B1-H2L2 69 Light chain variable region of antibody 43B1-H2L2 70 Heavy chain constant region of modified human IgG4 71 Heavy chain amino acid sequence of antibody 43B1-H2L2 72 Light chain amino acid sequence of antibody 43B1-H2L2 73 Heavy chain amino acid sequence of antibody 43B1-H6L1 74 Human IgG1 constant region 75 Human IgG4 heavy chain constant region

DETAILED DESCRIPTION OF THE INVENTION

[0379] The invention is now illustrated with reference to the following examples which are intended to exemplify but not limit the invention.

[0380] Unless indicated otherwise, the processes of molecular biology experiments and immunoassays used in the invention are essentially carried out with reference to the processes described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989; and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, Inc., 1995. Skilled persons in the art will appreciate that the examples are intended to exemplify the invention and are not intended to limit the scope of the invention.

Example 1: Preparation of Antigens

[0381] Human TSLP or monkey TSLP was expressed in Escherichia coli or mammalian cells. The amino acid sequence of human TSLP refers to NP_149024.1 in the protein database of NCBI. The amino acid sequence of cynomolgus monkey (Macaca fascicularis) TSLP refers to XP_005557555.1 in the protein database of NCBI. Antigens used in the present application include TSLP expressed in a modified form, such as a fusion of 6 consecutive histidines as a label fused to the C-terminal of the TSLP sequence (TSLP-His). The above-mentioned human and monkey TSLP sequences were codon optimized by GenScript (Nanjing) Co., Ltd, synthesized in an expression vector (i.e., plasmids containing the complete coding sequence of human TSLP), expressed in Escherichia coli or mammalian cells HEK293F and purified.

[0382] The natural receptor of TSLP is a heterodimer, which is composed of human TSLPR and human IL7R alpha (IL7Rα) subunit. The sequence of human TSLPR refers to Uniprot: Q9HC73.1; the sequence of human IL7Rα subunit refers to GenBank: AAR08908.1. Human hIL7Rα-hTSLPR-hFc fusion protein was composed of the extracellular domain of human IL7Rα (E21-D239), the extracellular domain of human TSLPR receptor (Q23-K231) and part of the coding sequence of human IgG1 Fc region (Hinge-CH2-CH3) in tandem. It was constructed into the expression vector pLVX after codon optimization. The stable expressing cell line of HEK293F was established by using the pLVX vector and finally the fusion protein of hIL7Rα-hTSLPR-hFc was obtained by purification.

Example 2: Construction and Identification of the Cell Line Overexpressing Both Human TSLPR/IL7Rα Genes

[0383] 2.1: Construction of a Cell Line Overexpressing Both Human TSLPR/IL7Rα Genes

[0384] In order to verify the efficacy of human TSLP antibodies in blocking the binding of human TSLP to human TSLPR/IL7Rα receptor, the complete amino acid coding sequence of human TSLPR (Gene ID: UniProtKB/Swiss-Prot: Q9HC73.1, synthesized by GenScript (Nanjing) Co., Ltd) and the complete amino acid coding sequence of human IL7R alpha subunit (Gene ID: GenBank: AAR08908.1, synthesized by GenScript (Nanjing) Co., Ltd) were codon optimized by GenScript (Nanjing) Co., Ltd and cloned into lentiviral vectors pLVX-IRES-puro and pLVX-IRES-zeocin, respectively, and mouse pro-B cell line Ba/F3 cells (purchased from COBIOER BIOSCIENCES CO., LTD) were infected by the virus obtained by the preparation method using a lentivirus packaging system described in Mohammadi Z et al., Mol Biotechnol. 2015 September; 57(9): 793-800, followed by selecting via puromycin+bleomycin and screening single clones, to obtain monoclonal stable cell line Ba/F3-hTSLPR-hIL7Rα.

[0385] 2.2 Detection of Ba/F3 Cell Line Overexpressing Both Human TSLPR/IL7Rα Genes

[0386] The cell line was detected by flow cytometry (flow cytometry: Beckman, CytoFlex; detection antibodies: APC-anti-human TSLPR (Biolegend), APC-anti-human IL7Rα (Biolegend)), to determine if the monoclonal cells correctly expressed human TSLPR and human IL7Rα. As shown in FIG. 1, the flow cytometry results showed that Ba/F3-hTSLPR-hIL7Rα was a monoclonal cell line expressing both genes (nearly 100%) with good homogeneity, which can be used for subsequent experiments.

Example 3: Mouse Immunization and Hybridoma Fusion

[0387] 3.1 Mouse Immunization

[0388] Fully human transgenic mice H2L2 (Harbour BioMed) were immunized for multiple times using human TSLP (NP_149024.1) expressed by Escherichia coli, human TSLP (NP_149024.1) expressed by mammalian cells, monkey TSLP (XP_005557555.1) expressed by mammalian cells and plasmids containing the complete coding sequence of human TSLP, respectively. Booster immunization was carried out every two weeks, for 5-6 times in total. During immunization, the serum titer of anti-human TSLP antibody was detected by ELISA every two weeks (see Example 4.1), and after multiple rounds of immunization, depending on the titer, mice having the best titer were selected for fusion to produce hybridoma.

[0389] 3.2 Fusion Method

[0390] After single-cell suspensions of spleen and lymph node were mixed, SP2/0 myeloma cells in an equivalent amount were added and mixed evenly. The mixed solution of cells was washed and resuspended with electrofusion buffer, and after electrofusion using a BTX-ECM2001 electrofusion device, the cell suspension was immediately transferred from the fusion chamber to a complete fusion culture medium and incubated at 37° C. for 1 hour. The cells were plated to a 96-well plate at a density of 2×10.sup.4 cells per well. After culturing for 5 days, the medium was exchanged with the complete fusion medium, and in 7-10 days, the supernatant was collected for screening hybridoma.

Example 4: Hybridoma Screening

[0391] 4.1 ELISA Screening of Human TSLP-Binding

[0392] The soluble human TSLP-His protein was diluted to 1 μg/mL in 1×CBS coating buffer, then added to 96-well plates, and incubated overnight at 4° C. The 96-well plates were washed with PBST and blocked with a blocking solution (PBS+2% BSA) at 37° C. for 2 hours. The hybridomas supernatant, or the same volume of blocking solution were added to plates, and incubated in an incubator at 37° C. for 2 hours. The 96-well plates were added with goat anti-rat IgG-HRP, and incubated in an incubator at 37° C. for 1 hour, then washed and read OD at 450 nm.

[0393] 4.2 ELISA Screening of Monkey TSLP-Binding

[0394] The monkey TSLP-His protein was diluted to 1 μg/mL in CBS coating buffer, then added to 96-well plates, and incubated overnight at 4° C. The 96-well plates were washed with PBST and blocked with a blocking solution (PBS+2% BSA) at 37° C. for 2 hours. The hybridomas supernatant, or the same volume of blocking solution were added to plates, and incubated in an incubator at 37° C. for 2 hours. The 96-well plates were added with goat anti-rat IgG-HRP, and incubated in an incubator at 37° C. for 1 hour, then washed and read OD at 450 nm.

[0395] 4.3 ELISA Screening of Blocking the Binding of Human TSLP-his to Chimeric Receptor IL7Rα-TSLPR-hFc

[0396] According to the following scheme, the hybridoma supernatant or purified antibody was assayed for blocking the binding of human TSLP-His to chimeric receptor IL7Rα-TSLPR-hFc.

[0397] The soluble hIL7Rα-hTSLPR-hFc protein was diluted in 1×CBS coating buffer, then added to 96-well plates, and incubated overnight at CC. The 96-well plate was washed with PBST and blocked with a blocking solution (PBS+2% BSA) at 37° C. for 2 hours. The hTSLP-His protein (+/−) recombinantly expressed by mammalian cells was added to the plates, together with the hybridoma supernatant or the same volume of blocking solution, and incubated in an incubator at 37° C. for 2 hours. The 96-well plates were added with mouse anti-His-HRP, and incubated in an incubator at 37° C. for 1 hour, then washed and read OD at 450 nm. Hybridomas with strong inhibition rate were selected as candidate clones.

[0398] According to the activities assayed by affinity ELISA and competitive ELISA above, positive clones were selected and subcloned by the limited dilution method to obtain subclones. Finally, the hybridoma subclones which can bind to both human TSLP-His and monkey TSLP-His, as well as block the binding of human TSLP-His to chimeric receptor hIL7Rα-hTSLPR-hFc were obtained. As shown in Table 1, five single clones had strong binding ability for both human TSLP (hTSLP) and monkey TSLP (cTSLP), and also blocked the binding ability of human TSLP to its receptor hIL7Rα-hTSLPR-hFc, with an inhibition rate exceeding 60%.

TABLE-US-00002 TABLE 1 Screening of anti-TSLP hybridoma subclone hTSLP/hIL7Rα- hTSLP binding cTSLP binding hTSLPR-hFc Clone (OD450) (OD450) inhibition rate (%) 25A5C5 2.4 0.12 95.9 27C2B6 1.4 1.11 69.8 37C2D10 1.1 1.12 81.0 43B1A8 1.92 1.11 80.7 90H3H11 0.7 1.02 87.0

Example 5: Preparation of Anti-TSLP Chimeric Antibody

[0399] Control antibody expression: the sequence of control antibody refers to chEMBL database (ID: CHEMBL3707229). The base sequences of heavy and light chains of control antibody were synthesized in pTT5 expression vector, and transiently transfected and expressed by CHO-S Cells (Purchased from Thermo), followed by affinity purification using protein A (MabSelect SuRe, GE), to obtain the control antibody.

[0400] Single hybridoma clones were cultured in serum-free medium to obtain 50 mL of supernatant, followed by purification using Protein A (MabSelect SuRe, GE), to obtain chimeric antibodies of hybridomas. Since H2L2 mouse antibody constant region was genetically modified into a rat constant region, the obtained purified antibody was a chimeric antibody with fully human variable regions carrying rat Fc. The purified antibodies were quantified by spectrophotometry to obtain chimeric antibodies 25A5C5, 27C2B6, 37C2D10, 43B1A8 and 90H3H11.

Example 6: ELISA Detection of the Affinity of Anti-TSLP Chimeric Antibodies for TSLP

[0401] The affinity of chimeric antibodies 25A5C5, 27C2B6, 37C2D10, 43B1A8 and 90H3H11 for human or monkey TSLP was assayed by ELISA. The specific method was briefed as follows: coating human or monkey TSLP-His antigen into 96-well plates, after overnight at 4° C., adding antibodies diluted with different concentration gradients, respectively, after incubating for 2 hours, adding goat anti-rat Fc-HRP secondary antibody, and after incubating for 1 hour, reading the absorption value at 450 nm wavelength by microplate reader.

[0402] The results were shown in Table 2, in which chimeric antibodies 25A5C5 and 27C2B6 each had an affinity for human TSLP substantially comparable to the control antibody, while antibodies 43B1A8 and 90H3H11 each had higher affinity for human TSLP than that of the control antibody. Antibodies 25A5C5 and 90H3H11 had no binding to monkey TSLP; while 37C2D10, 27C2B6 and 43B1A8 each strongly bind to monkey TSLP.

TABLE-US-00003 TABLE 2 Affinity of anti-TSLP chimeric antibody to TSLP hTSLP binding cTSLP binding Antibody EC50 (nM) EC50 (nM) 25A5C5 0.208 No binding 27C2B6 0.221 0.216 37C2D10 0.300 0.495 43B1A8 0.115 0.568 90H3H11 0.139 >100 Control antibody 0.233 0.336

Example 7: Detection of Activity of Anti-TSLP Chimeric Antibody of Blocking TSLP/hIL7Rα-hTSLPR-hFc Binding by Competitive ELISA

[0403] Human TSLP binds to human hIL7Rα-hTSLPR-hFc heterodimer receptor to activate downstream signaling pathways. Competitive ELISA was applied to assay the activity of chimeric antibodies of blocking the binding of chimeric receptor hIL7Rα-hTSLPR-hFc to the antigen. The specific steps refers to Example 4.3, the gradient diluted chimeric antibodies were added to a plate. The results were shown in the Table 3, in which all 5 chimeric antibodies effectively blocked the binding of human TSLP to human hIL7Rα-hTSLPR-hFc heterodimer receptor.

TABLE-US-00004 TABLE 3 Activity of anti-TSLP chimeric antibody by competitive ELISA Control Antibody 25A5C5 27C2B6 37C2D10 43B1A8 90H3H11 antibody IC50 (nM) 0.89 0.77 0.51 1.33 1.65 0.93

Example 8: Inhibition of Anti-TSLP Chimeric Antibody on the Proliferation of Ba/F3-hTSLPR-hIL7Rα Cells

[0404] The activity of anti-TSLP chimeric antibody was detected by inhibiting Ba/F3-hTSLPR-hIL7Rα cells proliferation. Receptor proteins hTSLPR and hIL7Rα were expressed on the cell surface of Ba/F3-hTSLPR-hIL7Rα, wherein, the dimer of the extracellular domains of these two receptor proteins can bind to human TSLP, and the intracellular domains thereof can further transduce signal to activate the intracellular STATS phosphorylation, and promote the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

[0405] The specific steps were as follows: taking an appropriate amount of Ba/F3-hTSLPR-hIL7Rα stable cell line for centrifuging and washing with 1640+10% FBS culture medium for 2 times, to remove the recombinant mouse IL3 in the culture medium; incubating hTSLP-His (+/−) together with purified anti-TSLP chimeric antibody/or control anti-TSLP antibody (Control antibody) in each well at room temperature for 30 minutes; adding 1.5×10.sup.4Ba/F3-hTSLPR-hIL7Rα cells per well and incubating for 3 days; adding CCK8 (RHINO BIO, QDY-003-D) to each well, and reading OD at 450 nm, exporting the data and analyzing the inhibition of chimeric antibody on cell proliferation by using Prism Graphpad software.

[0406] As shown in FIG. 2A-2C and Table 4, all 5 chimeric antibodies obviously inhibited the proliferation of Ba/F3-hTSLPR-hIL7Rα cells.

TABLE-US-00005 TABLE 4 Assay of activity of chimeric antibody of blocking human TSLP-induced proliferation of Ba/F3-hTSLPR-hlL7Ra cells Control Antibody 25A5C5 27C2B6 37C2D10 43B1A8 90H3H11 antibody EC50 (nM) 0.11 1.22 0.28 0.10 0.25 0.05

Example 9: Amplification of Variable Regions of Anti-TSLP Chimeric Antibody

[0407] Hybridoma cells were cultured to about 2×10.sup.6, lysed by using TRIzol reagent (Thermo Fisher Sci. Cat #15596026) to extract RNA, then a cDNA reverse transcription kit (Thermo Fisher Sci. Cat #18080-200) was applied for the synthesis of the first strand cDNA. With reference to the methods of IMGT and AbM, as well as the sequence analysis of all murine antibodies, multiple pairs of upstream primers for variable regions were designed by selecting the regions with high homology, downstream primers were designed by using CH1 homologous sequence, and the light and heavy chain variable regions of the antibodies were obtained by PCR amplification with primer pool, and the PCR product was purified using a DNA purification kit (Qiagen, Cat #28104) and cloned into pTT-5 vector, about 10 clones were selected for sequencing in each ligation reaction to obtain the sequences of variable regions, which were further analyzed by IMGT and AbM databases.

TABLE-US-00006 TABLE 5 Amino acid sequences and NOs of variable regions and CDRs of anti-human TSLP chimeric antibodies Clone 25A5C5 27C2B6 37C2D10 43B1A8 90H3H11 Heavy Chain 1 17 30 40 53 variable region Light Chain 2 18 31 41 54 variable region Numbering IM Ab IM Ab IM Ab IM Ab IM Ab system GT M GT M GT M GT M GT M Heavy chain 3 9 19 25 32 36 42 48 55 61 CDR1 Heavy chain 4 10 20 26 33 37 43 49 56 62 CDR2 Heavy chain 5 11 21 27 34 38 44 50 57 63 CDR3 Light Chain 6 12 22 28 35 39 45 51 58 64 CDR1 Light Chain 7 13 23 29 23 29 46 52 59 65 CDR2 Light Chain 8 8 24 24 24 24 47 47 60 60 CDR3

Example 10: Expression, Purification and Binding Affinity Detection of Recombinant Antibody

[0408] The amino acid sequences of the light chain variable regions of 25A5C5, 27C2B6, 37C2D10, 43B1A8 and 90H3H11 were fused to the amino acid sequence of the light chain K constant region (SEQ ID NO: 16), respectively, and the amino acid sequences of the heavy chain variable regions thereof were fused to the amino acid sequence of the heavy chain constant region of IgG1 (SEQ ID NO: 14), respectively, and the nucleotide sequences corresponding to these sequences were constructed into pTT5 vectors. The pTT5 vectors corresponding to the heavy and light chains of each recombinant fully human antibody were simultaneously transfected into CHO-S (purchased from Thermo), the supernatant was purified using Protein A (MabSelect SuRe, GE), and the purified recombinant fully human antibodies were named as 25A5C5-hIgG, 27C2B6-hIgG, 37C2D10-hIgG, 43B1A8-hIgG and 90H3H11-hIgG, respectively, each being quantified for protein concentration by spectrophotometry.

[0409] Detection of the affinity of anti-TSLP recombinant fully human antibodies for mammal-expressed recombinant human or monkey TSLP-His. The specific experimental steps were briefed as follows: coating human TSLP-His or monkey TSLP-His antigen into a 96-well plate, after overnight at 4° C., adding serially diluted antibodies, respectively, after incubating for 2 hours, adding HRP-labeled goat anti-human Fc secondary antibody, and after incubating for 1 hour, reading at wavelength of 450 nm.

[0410] The results were shown in FIG. 3A-3B, in which all 5 candidate antibodies can bind to human TSLP. Also, as shown in Table 6, 37C2D10-hIgG, 43B1A8-hIgG and 90H3H11-hIgG each had higher affinity for hTSLP than the control antibody. 25A5C5-hIgG and 27C2B6-hIgG each had comparable affinity for hTSLP with the control antibody. 27C2B6-hIgG, 37C2D10-hIgG and 43B1A8-hIgG each had comparable affinity for monkey TSLP with the control antibody.

TABLE-US-00007 TABLE 6 The binding of recombinant fully human antibodies for human/monkey TSLP 27C2B6- 37C2D10- 43B1A8- 90H3H11- Control Antibody 25A5C5-hIgG hIgG hIgG hIgG hIgG antibody hTSLP binding 1.45 1.62 0.90 0.80 1.36 1.40 EC50 (nM) cTSLP binding No binding 0.18 0.17 0.23 >100 0.19 EC50 (nM)

Example 11: Detection of the Dynamic Affinity of Anti-TSLP Fully Human Antibody for TSLP

[0411] The dynamic affinity of anti-TSLP fully human chimeric antibody for TSLP was detected by Fortebio, a commonly used dynamic affinity detection device. The method was described briefly as follows: carrying out series dilution of human or monkey TSLP with PBST to obtain 100 nM, 50 nM, 25 nM, 12.5 μM, 6.25 nM, 3.125 nM, 1.5625 nM and 0 nM; pre-wetting a ProA biosensor (Pall Life Sciences) with PBST buffer before use; diluting the recombinant fully human antibody to 5 μg/mL with PBST and immobilizing on the ProA sensor; then placing the sensor with immobilized antibody in PBST buffer for equilibrating 60 s to obtain the baseline and transferring to the antigen diluent for binding for 60 s, and then dissociating in PBST for 180 s; regenerating the sensor with 10 mM Gly (pH 1.5) after an analysis cycle. Date analysis 11.0 version (Pall) with 1:1 model was used to determine the association rate constant (Ka) and the dissociation rate constant (Kd), which were then used to calculate the dissociation equilibrium constant (KD).

[0412] As shown in Table 7, the KD values of 25A5C5-hIgG, 43B1A8-hIgG and 90H3H11-hIgG binding to human TSLP were less than that of the control antibody, showing stronger affinity, which were consistent with the affinity ELISA results. For binding to monkey TSLP, 25A5C5-hIgG had no affinity; while 37C2D10-hIgG, 90H3H11-hIgG and 43B1A8-hIgGlt each showed good binding with an affinity in the order of 10.sup.−8 M to 10.sup.−9 M.

TABLE-US-00008 TABLE 7 Dynamic affinity analysis of fully human antibodies Affinity for hTSLP Affinity for cTSLP Antibody KD (M) KD (M) 25A5C5-hIgG 5.58E−10 No binding 37C2D10-hIgG 1.76E−09 2.01E−08 43BlA8-hIgG 5.81E−10 1.02E−09 90H3H11-hIgG 6.04E−10 1.08E−08 Control antibody 8.00E−10 1.15E−09

Example 12: Inhibition of Recombinant Fully Human Antibodies on the Proliferation of Ba/F3-hTSLPR-hIL7a Cells

[0413] The activity of recombinant fully human antibody was assayed by the proliferation inhibition method of Ba/F3-hTSLPR-hIL7a cells. The method refers to the preceding Example 8.

[0414] The results were shown in Table 8, in which 27C2B6-hIgG and 37C2D10-hIgG can inhibit human TSLP-induced proliferation of Ba/F3-hTSLPR-hIL7Rα cells; 25A5C5-hIgG and 90H3H11-hIgG were substantially comparable to the control antibody in terms of inhibiting human TSLP-induced proliferation of Ba/F3-hTSLPR-hIL7Rα cells; while 43B1A8-hIgG had a significantly stronger inhibitory activity than (about 0.25 times) that of the control antibody, as shown in the FIG. 4A-C.

TABLE-US-00009 TABLE 8 Inhibition activity of recombinant fully human antibodies on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells hTSLP Antibody EC50(nM) 25A5C5-hIgG 0.044 27C2B6-hIgG 0.064 37C2D10-hIgG 0.060 43BlA8-hIgG 0.031 90H3H11-hIgG 0.050 Control antibody 0.041

Example 13: Detection of Inhibition Activity of Recombinant Fully Human Antibody on Human TSLP-Induced PBMC Secretion of Thymus and Activation-Regulated Chemokine (TARC)

[0415] The functional activity of fully human anti-TSLP antibody in primary cells was assessed by inhibiting human TSLP-induced secretion of TARC (a Th2-like cytokine) by human PBMC cells. CD11.sup.+ DC cells were contained in the PBMCs, and studies have shown that TSLP can bind to receptor TSLPR/IL7Rα and activate CD11.sup.+ DC cells, up-regulate OX40L and promote CD11.sup.+ DC cells to secrete Th2-like cytokines (e.g., TARC and CCL22). Anti-TSLP antibodies can block the binding of TSLP to TSLPR/IL7Rα on the surface of DC cells, thereby blocking the activation of DC cells and secretion of Th2-like cytokines by DC cell.

[0416] The method was briefly described as follows: isolating human peripheral blood PBMC using Ficoll separation solution (GE), incubating hTSLP-His (+/−) together with recombinant fully human antibodies or the control antibody for 30 minutes at room temperature, and adding 2×10.sup.5 PBMC cells per cell, incubating for 48 hours. The supernatant was collected and analyzed by ELISA for the human TARC, and the inhibition of the hybridoma supernatant or purified antibody on TARC secretion was assayed. TARC ELISA kit (Sino Biological) was used to detect the TARC level in the supernatant.

[0417] The results were shown in Table 9 and FIG. 5, in which the EC50 values of fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG were lower than that of the control antibody, indicating that the activities thereof of inhibiting TSLP-induced secretion of TARC by PBMC were higher than that of control antibody.

TABLE-US-00010 TABLE 9 Inhibition of recombinant fully human antibody on human TSLP-induced PBMC secretion of TARC Antibody 43B1A8-hIgG 90H3H11-hIgG Control antibody EC50(nM) 0.44 0.42 0.51

Example 14: Assay of the Tm Value of Recombinant Fully Human Antibody

[0418] The Tm value of anti-TSLP antibody was measured by DSF (differential fluorescence scanning technology). The specific experimental steps were as follows: mixing 12.5 μL of 40×SYPRO Orange dye (Life Technologies Co., Ltd., Cat No. 56651), 54 of 1 mg/mL fully human anti-TSLP antibody (diluted in PBS) and 7.54 of sterile water in an EP tube, adding the sample mixture to a Q-PCR system (AB Applied Biosystems ABI, 7500) for reaction, Q-PCR parameter settings: Target (ROX), program (25° C., 3 min; a rate of 1%, 95° C.; 95° C., 2 min). The results were inputted into Graph Prism software to calculate the V50 value. As shown in FIG. 6 and Table 10, the Tm values of 43B1A8-hIgG (74.72° C.) and 90H3H11-hIgG (72.58° C.) were higher than that of the control antibody (66.47° C.), clearly indicating that the fully human antibodies prepared by the invention had better thermal stability.

TABLE-US-00011 TABLE 10 Tm value of recombinant fully human anti-TSLP antibody Antibody Control antibody 43B1A8-hIgG 90H3H11-hIgG Tm(° C.) 66.47 74.72 72.58

Example 15: Assay of Hydrophobicity of Recombinant Fully Human Antibody

[0419] The hydrophobicity comparison was analyzed by Agilent 1260 HPLC combined with TOSOH Tskgel Buty-NPR (2.5) chromatographic column. In order to compare the hydrophobicity difference of three antibodies, these three antibodies were directly loaded for analysis. Mobile phase A: 1.5M (NH.sub.4).sub.2SO.sub.4; mobile phase B: 25 mM Na.sub.2HPO.sub.4 (pH7.0)+25% IPA. The retention times of the three antibodies were shown in Table 11, wherein, the longer the retention time, the stronger the hydrophobicity of the antibody, 90H3H11-hIgG had a hydrophilicity comparable to that of the control antibody; 43B1A8-hIgG had a better hydrophilicity than that of the control antibody, indicating that the process development and antibody aggregation will be superior to the control antibody.

TABLE-US-00012 TABLE 11 Detection of hydrophobicity of recombinant fully human antibody Antibody Control antibody 43B1A8-hIgG 90H3H11-hIgG Retention time 13.93 12.2 13.61 (minutes)

Example 16: Preparation of Recombinant Modified Fully Human Antibodies 43B1-H6L1 and 43B1-H2L2

[0420] The in vivo half-life of an antibody was closely related to the isoelectric point, affinity for FcRn, glycosylation modification and immunogenicity thereof. In order to prolong the half-life of 43B1A8-hIgG antibody, the affinity for FcRn was enhanced by modifying the amino acid sequences of the heavy chain and light chain of the antibody.

[0421] Modification scheme 1: hIgG1 Fc (SEQ ID NO: 14) was replaced with IgG4 (SEQ ID NO: 70) containing 2 amino acid mutations, one being from N to A at amino acid position 434 (EU numbering system), and the other from S to P at amino acid position 228 (EU numbering system) of IgG4.

[0422] Modification scheme 2: N was replaced with A at amino acid position 434 (EU numbering system) of hIgG1 Fc (SEQ ID NO: 14) to obtain SEQ ID NO: 15; and R was mutated into G at position 16 (Chothia numbering system) of FR1 of the heavy chain of 43B1A8-hIgG; and R was mutated into Q at position 79 (Chothia numbering system) of FR3 of the light chain of 43B1A8-hIgG.

[0423] The antibodies obtained by the above modification schemes 1 and 2 were named as 43B1-H6L1 and 43B1-H2L2, respectively.

[0424] The heavy chain variable region sequence of 43B1-H2L2 is SEQ ID NO: 68; the heavy chain constant region sequence thereof is SEQ ID NO: 15; the light chain variable region sequence thereof is SEQ ID NO: 69; and the light chain constant region sequence thereof is SEQ ID NO: 16.

[0425] The heavy chain variable region sequence of 43B1-H6L1 is SEQ ID NO: 40; the heavy chain constant region sequence thereof is SEQ ID NO: 70; the light chain variable region sequence thereof is SEQ ID NO: 41; and the light chain constant region sequence thereof is SEQ ID NO: 16.

TABLE-US-00013 TABLE 12 Amino acid sequences of antibodies 43B1-H6L1 and 43B1-H2L2 and NOs (SEQ ID NO) Heavy Heavy Light Light chain chain chain chain Heavy variable constant Light variable constant Antibody chain region region chain region region 43B1A8-hIgG 66 40 14 67 41 16 43B1-H2L2 71 68 15 72 69 16 43B1-H6L1 73 40 70 67 41 16

[0426] The heavy and light chains of the above two modified molecules 43B1-H2L2 and 43B1-H6L1 were constructed into pTT5 expression vectors, respectively, and after extracting plasmids, they were transfected into CHO-S cells (purchased from Thermo), after culturing for about 10 days, the cell supernatant was purified using Protein A (MabSelect SuRe, GE), and the purified recombinant fully human antibodies were quantified for protein content by spectrophotometry.

Example 17: Assay of Dynamic Affinity of Recombinant Fully Human Antibodies 43B1-H6L1 and 43B1-H2L2 for TSLP

[0427] The dynamic affinity of anti-TSLP fully human antibody for TSLP was detected by Fortebio. The method refers to the preceding Example 11. The results were shown in Table 13, in which 43B1-H6L1 and 43B1-H2L2 human antibody molecules each had a dynamic affinity for hTSLP not weaker than that of the control antibody.

TABLE-US-00014 TABLE 13 Analysis of dynamic affinity of modified fully human antibody molecules hTSLP affinity Antibody KD (M) 43B1-H2L2 2.33E−10 43B1-H6L1 4.19E−10 Control antibody 3.73E−10

Example 18: Assay of Isoelectric Points (P1) of Recombinant Fully Human Antibodies 43B1-H6L1 and 43B1-H2L2

[0428] The isoelectric points of 43B1-H6L1, 43B1-H2L2 and 43B1A8-hIgG antibodies were detected by the isoelectric focusing method, which was briefly described as follows: Maurice isoelectric focusing system (ProteinSimple) combined with its capillary cartridge was used for analysis. In order to compare the isoelectric point differences of the three antibodies, these three antibodies were diluted with water, and a pH gradient was formed by using ampholyte 3-10 (GE) at a final concentration of 4% in the detection system. The results were shown in Table 14, in which the isoelectric points of 43B1-H6L1 and 43B1-H2L2 were lower than that of 43B1A8-hIgG by 1.3 and 0.4, respectively, indicating that the modification schemes were successful.

TABLE-US-00015 TABLE 14 Comparison of isoelectric points (PI) of recombinant fully human antibodies Antibody 43B1-H2L2 43B1-H6L1 43B1A8-hIgG Isoelectric 8.2 7.3 8.6 point

Example 19: Assay of Dynamic Affinity of Recombinant Fully Human Antibodies 43B1-H6L1 and 43B1-H2L2 for FcRn

[0429] The dynamic affinity of the modified anti-TSLP fully human antibodies for FcRn was detected by Fortebio. The method was described briefly as follows: carrying out series dilution of anti-TSLP antibody with PBST (pH 6.0) to obtain 200 nM, 100 nM, 50 nM, 25 nM, 12.5 μM, 6.25 nM, 3.125 nM and 1.5625 nM and 0 nM; pre-wetting a SA biosensor (Pall Life Sciences) with PBST (pH 6.0) buffer before use; diluting biotin-labeled FcRn to 2.3 μg/mL and immobilizing on the SA sensor; then placing the sensor with immobilized FcRn in PBST (pH 6.0) buffer for equilibrating for 60 s to obtain the baseline and transferring to the antibody diluent for binding for 60 s, and then dissociating in PBST (pH 6.0) for 60 s; regenerating the sensor with PBST (pH 7.4) after an analysis cycle. Date analysis 11.0 version (Pall) using 1:1 model was used to determine the association rate constant (Ka) and the dissociation rate constant (Kd), which were then used to calculate the dissociation equilibrium constant (KD).

[0430] As shown in Table 15, 43B1-H6L1 and 43B1-H2L2 each had a dynamic affinity (KD) for FcRn less than that of the control antibody, showing an affinity for FcRn of about 2 times of that of the control antibody.

TABLE-US-00016 TABLE 15 Analysis of affinity of modified fully human antibodies for FcRn Affinity for FcRn Antibody KD (M) 43B1-H2L2 5.25E−09 43B1-H6L1 4.23E−09 Control antibody 1.12E−08

Example 20: Assay of Inhibitory Activities of Recombinant Fully Human Antibodies 43B1-H6L1 and 43B1-H2L2 on the Proliferation of Ba/F3-hTSLPR-hIL7Rα Cells

[0431] The activities of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 were detected by the proliferation inhibition method of Ba/F3 cells. The method refers to the preceding Example 8, and the activity EC50 of antibody was analyzed based on the data. The results were shown in Table 16 and FIGS. 7A and 7B, in which 43B1-H2L2 and 43B1-H6L1 can inhibit human TSLP-induced proliferation of Ba/F3-hTSLPR-hIL7Rα cells, and the EC50 values thereof were comparable to that of the control antibody, indicating that fully human antibodies 43B1-H2L2 and 43B1-H6L1 each had a comparable ability of inhibiting the cell activity of TSLP.

TABLE-US-00017 TABLE 16 Inhibitory activity of recombinant fully human antibody on the proliferation of Ba/F3-hTSLPR-hIL7Rα cells 43B1-H2L2 43B1-H6L1 Control antibody hTSLP EC50 (nM) 0.64 0.46 0.57

Example 21: Detection of Inhibitory Activity of Recombinant Fully Human Antibody on Human TSLP-Induced PBMC Secretion of Macrophage-Derived Chemokine (MDC)

[0432] The functional activity of the anti-TSLP fully human antibodies in primary cells was assessed by inhibiting human TSLP-induced secretion of MDC by human PBMC cells. PBMC comprises DC cells. Studies have shown that TSLP can bind to receptor TSLPR/IL7Rα on the cell surface and activate DC cells, up-regulate OX40L and promote DC cells to secrete Th2-like cytokines (e.g., TARC and MDC). Anti-TSLP antibodies can block the binding of TSLP to TSLPR/IL7Rα on the surface of DC cells, thereby blocking the activation of DC cells and the secretion of Th2-like cytokines by DC cells.

[0433] The method was briefly described as follows: isolating human peripheral blood PBMC using Ficoll separation solution (GE), incubating hTSLP-His (+/−) together with recombinant fully human antibodies or the control antibody for 30 minutes at room temperature, and adding 2×10.sup.5 PBMC cells per cell, and incubating for 120 hours. The supernatant was collected and analyzed by ELISA for the production of human MDC, and the inhibition of the antibody on the secretion of MDC was measured. MDC ELISA kit (Raybiotech) was used to detect the TARC level in the supernatant.

[0434] The results were shown in Table 17 and FIG. 8, in which 43B1-H2L2 and the control antibody can significantly inhibit TSLP-induced production of MDC, with EC50 values of 7.84 pM and 5.63 pM, respectively.

TABLE-US-00018 TABLE 17 Inhibition of recombinant fully human antibody on human TSLP-induced PBMC secretion of MDC Antibody 43B1-H2L2 Control antibody EC50 (pM) 7.84 5.63

Example 22: In Vivo Pharmacokinetic Analysis of Recombinant Fully Human Antibody in Cynomolgus Monkey

[0435] The sequence of candidate molecule 43B1-H2L2 of the present application contains N434A mutation in order to affect its affinity for FcRn, thereby prolonging its half-life of drug metabolism in vivo. Therefore, in this example, the pharmacokinetics of 43B1-H2L2 molecule in cynomolgus monkey in vivo was studied by subcutaneous injection, and was compared to that of the control antibody. The methods and results were as follows: four cynomolgus monkeys (Macaca fascicularis, available from Hainan Jingang Biotech Co., Ltd.) were selected and divided into two groups, one male and one female per group. The dose of subcutaneous injection was 5 mg/kg. Blood samples were collected at zero, 5 minutes, 30 minutes, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration, and left at room temperature for 1 hour until coagulation, and centrifuged to obtain the serum samples which were then frozen at −80° C. to be tested. The antibody concentration in serum was determined by ELISA, and the results were analyzed as follows: the pharmacokinetic parameters and curves of single subcutaneous administration were shown in Table 18 and FIG. 9. These results showed that 43B1-H2L2 antibody had a longer half-life as well as a higher AUC of blood concentration, about twice of that of the control antibody.

TABLE-US-00019 TABLE 18 Pharmacokinetic study of recombinant fully human antibody in cynomolgus monkey by subcutaneous administration PK parameters t.sub.1/2(h) T.sub.max (h) C.sub.max (ng/ml) AUC.sub.(0−t) (hr*ng/ml) 43B1-H2L2 549.3731 48 67373.4 47271741 Control 267.3537 72 75870.56 25600470 antibody
Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and changes may be made to the details in accordance with all the published teachings, which are also included in the scope of the invention. The protection scope of the invention is defined by the appended claims and any equivalents thereof.