ANTI-HUMAN P40 PROTEIN DOMAIN ANTIBODY AND USE THEREOF
20220298235 · 2022-09-22
Inventors
- Yu XIA (Zhongshan, CN)
- Zhongmin Maxwell WANG (Zhongshan, CN)
- Peng ZHANG (Zhongshan, CN)
- Baiyong LI (Zhongshan, CN)
Cpc classification
A61P1/04
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
A61K47/6845
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
A61P1/00
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
International classification
C07K16/24
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
Abstract
Provided is an antibody for the treatment or prevention of autoimmune diseases, comprising a heavy chain variable region represented by SEQ ID NO: 1 or SEQ ID NO: 24, and a light chain variable region represented by SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 25.
Claims
1. An antibody or an antigen-binding fragment thereof, preferably specifically binding to human IL-12/IL-23 p40, wherein the antibody comprises: (1) an HCDR1, an HCDR2 and an HCDR3 contained in a heavy chain variable region set forth in SEQ ID NO: 1 or SEQ ID NO: 24; and (2) an LCDR1, an LCDR2 and an LCDR3 contained in a light chain variable region set forth in any one of SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID N: 15, SEQ ID NO: 17 and SEQ ID NO: 25; preferably, the HCDR1 comprises or consists of a sequence set forth in SEQ ID NO: 3 or 26, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, the HCDR2 comprises or consists of a sequence set forth in SEQ ID NO: 4, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, and the HCDR3 comprises or consists of a sequence set forth in SEQ ID NO: 5, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence; and the antibody further comprises: an LCDR1, comprising or consisting of a sequence set forth in SEQ ID NO: 8, 19, 20 or 27, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, an LCDR2, comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 9, 21 or 28, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, and an LCDR3, comprising or consisting of a sequence set forth in SEQ ID NO: 10, 22 or 23, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, preferably, the antigen-binding fragment is selected from the group consisting of: Fab, Fab′, F(ab′).sub.2, Fd, Fv, dAb, Fab/c, complementarity determining region (CDR) fragment, single chain antibody (e.g., scFv), bivalent antibody and domain antibody.
2. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody comprises framework regions (FRs) in the heavy chain variable region and FRs in the light chain variable region, wherein (1) the FRs in the heavy chain variable region comprise FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 29, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 29, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 29; the FR-H2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 30, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 30, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 30; the FR-H3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 31, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 31, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 31; the FR-H4 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 32, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 32, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 32; (2) the FRs in the light chain variable region comprise FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 33 or 41, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 33 or 41, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence set forth in SEQ ID NO: 33 or 41; the FR-L2 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 34, 42, 45 or 47, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 34, 42, 45 or 47, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 34, 42, 45 or 47; the FR-L3 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 35, 43, 46 or 48, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 35, 43, 46 or 48, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 35, 43, 46 or 48; the FR-L4 comprises or consists of an amino acid sequence set forth in SEQ ID NO: 36 or 44, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 36 or 44, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 36 or 44.
3. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody comprises: (1) a heavy chain variable region comprising or consisting of: an amino acid sequence set forth in SEQ ID NO: 1 or 24, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 1 or 24, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence set forth in SEQ ID NO: 1 or 24; and a light chain variable region comprising or consisting of: an amino acid sequence set forth in SEQ ID NO: 6, 11, 13, 15, 17 or 25, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 6, 11, 13, 15, 17 or 25, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 6, 11, 13, 15, 17 or 25, preferably, the antibody further comprises a heavy chain constant region and a light chain constant region, and the constant regions are derived from species other than murine, e.g., from a human antibody, preferably from a human IgG, more preferably from IgG1; preferably, the heavy chain constant region is an Ig gamma-1 chain C region (more preferably an Ig gamma-1 chain C region of GenBank ACCESSION No. P01857); the light chain constant region is an Ig kappa chain C region (more preferably an Ig kappa chain C region of GenBank ACCESSION No. P01834), more preferably, the antibody comprises or consists of a heavy chain set forth in SEQ ID NO: 19 and a light chain set forth in SEQ ID NO: 20, preferably, the antibody is a humanized antibody, a chimeric antibody or a multispecific antibody (e.g., a bispecific antibody).
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10. An isolated polypeptide or an isolated polynucleotide encoding a polypeptide, wherein the isolated polypeptide is selected from the group consisting of: (a) an isolated polypeptide comprising sequences set forth in SEQ ID NOs: 3, 4 and 5 or SEQ ID NOs: 26, 4 and 5, wherein the polypeptide specifically binds to human IL-12/IL-23 p40 protein domain as part of an anti-human IL-12/IL-23 p40 protein domain antibody, the antibody further comprising sequences set forth in SEQ ID NO: 8, 19 or 20, SEQ ID NO: 9 or 21, SEQ ID NO: 10, 22 or 23 or SEQ ID NOs: 27, 28 and 22; (b) an isolated polypeptide comprising sequences set forth in SEQ ID NO: 8, 19 or 20, SEQ ID NO: 9 or 21, SEQ ID NO: 10, 22 or 23, or SEQ ID NOs: 27, 28 and 22, wherein the polypeptide specifically binds to human IL-12/IL-23 p40 protein domain as part of an anti-human IL-12/IL-23 p40 protein domain antibody, the antibody further comprising sequences set forth in SEQ ID NOs: 3, 4 and 5; (c) an isolated polypeptide comprising a sequence set forth in SEQ ID NO: 1 or 24, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, wherein the polypeptide specifically binds to human IL-12/IL-23 p40 protein domain as part of an anti-human IL-12/IL-23 p40 protein domain antibody, the antibody further comprising a sequence set forth in SEQ ID NO: 6, 11, 13, 15, 17 or 25, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence; and (d) an isolated polypeptide comprising a sequence set forth in SEQ ID NO: 6, 11, 13, 15, 17 or 25, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, wherein the polypeptide specifically binds to human IL-12/IL-23 p40 protein domain as part of an anti-human IL-12/IL-23 p40 protein domain antibody, the antibody further comprising a sequence set forth in SEQ ID NO: 1 or 24, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence; wherein the isolated polynucleotide encoding a polypeptide is selected from the group consisting of: (a) an isolated polynucleotide encoding a polypeptide comprising sequences set forth in SEQ ID NOs: 3, 4 and 5, wherein the polypeptide specifically binds to human IL-12/IL-23 p40 protein domain as part of an anti-human IL-12/IL-23 p40 protein domain antibody, the antibody further comprising sequences set forth in SEQ ID NO: 8, 19 or 20, SEQ ID NO: 9 or 21, SEQ ID NO: 10, 22 or 23 or SEQ ID NOs: 27, 28 and 22; (b) an isolated polynucleotide encoding a polypeptide comprising sequences set forth in SEQ ID NO: 8, 19 or 20, SEQ ID NO: 9 or 21, SEQ ID NO: 10, 22 or 23, or SEQ ID NOs: 27, 28 and 22, wherein the polypeptide specifically binds to human IL-12/IL-23 p40 protein domain as part of an anti-human IL-12/IL-23 p40 protein domain antibody, the antibody further comprising sequences set forth in SEQ ID NOs: 3, 4 and 5; (c) an isolated polynucleotide encoding a polypeptide comprising a sequence set forth in SEQ ID NO: 1 or 24, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, wherein the polypeptide specifically binds to human IL-12/IL-23 p40 protein domain as part of an anti-human IL-12/IL-23 p40 protein domain antibody, the antibody further comprising a sequence set forth in SEQ ID NO: 6, 11, 13, 15, 17 or 25, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence; and (d) an isolated polynucleotide encoding a polypeptide comprising a sequence set forth in SEQ ID NO: 6, 11, 13, 15, 17 or 25, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, wherein the polypeptide specifically binds to human IL-12/IL-23 p40 protein domain as part of an anti-human IL-12/IL-23 p40 protein domain antibody, the antibody further comprising a sequence set forth in SEQ ID NO: 1 or 24, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence; preferably, the polynucleotide comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 2 or 37, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence; or the polynucleotide comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 7, 12, 14, 16, 18 or 38, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence.
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13. The polynucleotide according to claim 10, wherein the polynucleotide is contained in a vector, a host cell.
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15. The polynucleotide according to claim 10, which is used to prepare a polypeptide by culturing a host cell comprising the polynucleotide according to claim 10 in a suitable condition, and isolating the polypeptide from the cell cultures.
16. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof according to claim 1 is contained in an antibody conjugate, wherein the antibody or the antigen-binding fragment thereof is coupled to a conjugated moiety, wherein the conjugated moiety is a purification tag (e.g., a His tag), a cytotoxic agent or a detectable label; preferably, the conjugated moiety is a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.
17. The antibody or the antigen-binding fragment thereof according to claim 1 wherein the antibody or the antigen-binding fragment thereof is contained in a fusion protein, a multispecific antibody, preferably a bispecific antibody, a kit, wherein preferably, the kit further comprises a second antibody specifically identifying the antibody or the antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol, or a pharmaceutical composition, and optionally, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or an excipient, more preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
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25. An in vivo or in vitro method, comprising administering a cell or a subject comprising the antibody or the antigen-binding fragment thereof according to claim 3, wherein the method is selected from the group consisting of: a method for blocking the binding of human IL-12/IL-23 p40 protein domain to ligand IL-12Rβ1 or IL-23R, a method for down-regulating the activity or the level of human IL-12/IL-23 p40 protein domain, and a method for blocking cellular response mediated by the binding of human IL-12Rβ1 or human IL-23R to p40 protein domain, wherein preferably, a ligand of the IL-12/IL-23 p40 is IL-12Rβ1 or IL-23R.
26. A method for the prevention, treatment, adjuvant treatment and/or diagnosis of autoimmune diseases (e.g., plaque psoriasis or systemic lupus erythematosus) or ulcerative colitis (e.g., refractory or recurrent), comprising administering to a subject in need the antibody or the antigen-binding fragment thereof according to claim 1, wherein preferably, the subject has received conventional treatment or is inadequately responsive, unresponsive or intolerant to biological agents, and thus fails to achieve complete response or partial response.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0273] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. In the cases where the techniques or conditions are not specified, the examples were carried out according to the techniques or conditions described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., Third Edition, Science Press) or according to the product manual. Reagents or instruments used are commercially available conventional products if the manufacturers thereof are not specified.
[0274] In the following examples of the present invention, C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center.
[0275] In the following examples of the present invention, IL-12 (Human IL12 (His Tag)) was purchased from Sino Biological (Cat. No.: CT-050-H08H-20, Lot No.: LC11MC2805).
[0276] In the following examples of the present invention, Ab123FR1, an anti-IL-12/IL-23 p40 antibody, was used as a control antibody, and reference can be made to the Chinese granted patent CN103275222B for its preparation method. It was produced by Akeso Biopharma, Inc, and its sequences were shown as positions 20-468 of SEQ ID NO: 3 and positions 20-233 of SEQ ID NO: 4 in CN103275222B.
[0277] In the following examples of the present invention, ustekinumab (trade name Stelara), a marketed anti-p40 antibody for the same target, was purchased from Johnson & Johnson as a control antibody.
[0278] In the following examples of the present invention, the cell line 293T-IL-12Rβ1&IL-23R used was constructed by Akeso Biopharma, Inc. The cell line 293T-IL-12Rβ1&IL-23R was prepared by viral infection of 293T cells using 3rd generation lentiviral systems (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System, Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D, and Naldini L., J Virol. 1998. 72(11):8463-8471), wherein the lentivirus expression vectors used were pLenti-IL-12Rβ1-BSD (IL-12Rβ1, GenBank accession No. 3549; vector pLenti-BSD, purchased from Invitrogen, Cat. No.: K497000) and pCDH-IL-23R-puro (IL-23R, GenBank accession No. 149233; vector pCDH-CMV-MCS-EF1-Puro, purchased from Youbio, product No. VT1480).
[0279] The isotype control antibody was human anti-hen egg lysozyme IgG (anti-HEL, i.e., human IgG, abbreviated as hIgG1), and its sequence was from Affinity Maturation Increases the Stability and Plasticity of the Fv Domain of Anti-Protein Antibodies (Acierno et al., J Mol Biol., 2007, 374(1):130-46, wherein the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 21, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 22). The isotype control antibody was prepared in the laboratory of Akeso Biopharma, Inc.
[0280] The hIL-23 recombinant protein (IL-23, GeneBank accession No. 51561) was prepared in the laboratory of Akeso Biopharma, Inc.
[0281] The following examples are further illustration of the present invention and are not intended to limit the present invention.
Example 1. Design, Expression and Purification of Heavy Chain and Light Chain Sequences of Anti-Human p40 Antibody H8L15
[0282] 1. Design of Antibodies
[0283] To prepare anti-human IL-12/IL-23 p40 antibody H8L15, the inventors determined amino acid sequences of the CDR regions where the antibody binds to the antigen by quantum simulation calculation based on the structure of IL-12/IL-23 p40 protein domain and by means of structural biology-based three-dimensional space structure simulation technology for antigen-antibody binding and the interaction between the CDR regions of the antibody and the antigen. Meanwhile, the framework part of the antibody was optimized correspondingly without influencing the three-dimensional structure of the CDR regions, and finally antibodies H5L9, H5L10, H5L11, H5L12, H5L14 and H8L15 specifically biding to human IL-12/IL-23 p40 were obtained.
[0284] The amino acid sequences of the heavy chain variable regions and the light chain variable regions of the antibodies and the encoding DNA sequences thereof are as follows:
[0285] H5L9: the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 2;
[0286] H5L9: the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 6, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 7;
[0287] H5L10: the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 2;
[0288] H5L10: the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 12;
[0289] H5L11: the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 2;
[0290] H5L11: the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 14;
[0291] H5L12: the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 2;
[0292] H5L12: the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 15, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 16;
[0293] H5L14: the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 2;
[0294] H5L14: the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 17, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 18;
[0295] H8L15: the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 24, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 37;
[0296] H8L15: the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 25, and the encoding DNA sequence thereof is set forth in SEQ ID NO: 38
[0297] 2. Expression and Purification of Antibodies
[0298] The encoding nucleotide sequence of the heavy chain variable region (set forth in SEQ ID NO: 37; constant region: Ig gamma-1 chain C region; ACCESSION: P01857) and the encoding nucleotide sequence of the light chain variable region (set forth in SEQ ID NO: 38; constant region: Ig kappa chain C region; ACCESSION: P01834) of the above-described antibodies, such as H8L15, were each cloned into vector pUC57simple (provided by Genscript) to obtain pUC57simple-H8L15H containing the full-length heavy chain nucleotide of H8L15 and pUC57simple-H8L15L containing the full-length light chain nucleotide of H8L15, respectively.
[0299] Plasmids pUC57simple-H8L15H and pUC57simple-H8L15L were digested (HindIII&EcoRI), and heavy and light chain nucleotide sequences isolated by electrophoresis were subcloned into vector pcDNA3.1, and recombinant plasmids were extracted to co-transfect 293F cells. After the transfected 293F cells were cultured for 7 days, the culture medium was centrifuged at high speed, and the obtained supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in one step with the eluent to isolate the target sample. The antibody sample was stored in PBS buffer.
[0300] Other antibodies were prepared in the same way. The antibodies H5L9, H5L10, H5L11, H5L12, H5L14 and H8L15 prepared in this example were used in the following Examples 2-4.
Example 2. Detection of Binding Activity of Antibodies H5L9, H5L10, H5L11, H5L12, H5L14, H8L15, Ab123FR1 and Ustekinumab to Antigen Human IL-12/IL-23 p40 by ELISA
[0301] A microplate was coated with human p40-His (Akeso Biopharma, Inc.; gene of p40: GeneBank NM002187). After incubation at 4° C. for no less than 12 h, the plate was washed with PBST, patted dry and blocked with a solution of 1% BSA in PBS. After blocking was completed, the plate was washed with PBST and patted dry. The antibody diluted with PBST solution in gradient was added into the wells of the plate, and the antibody dilution gradient is detailed in Table 1. The plate containing the test antibody was incubated at 37° C. for 30 min and then washed with PBST and patted dry. HRP-labeled goat anti-human IgG (H+L) (purchased from Jackson ImmunoResearch Inc., Cat. No.: 109-035-088) secondary antibody working solution diluted at a ratio of 1:5000 was added, and the resulting mixture was incubated at 37° C. for 30 min After the incubation, the plate was washed with PBST and patted dry. TMB (Neogen, 308177) was added for color developing for 5 min in the absence of light, and then stop solution was added to terminate the chromogenic reaction. Then the plate was put into a plate reader immediately, and the OD value of each well in the plate was read at 450 nm. The data were analyzed by SoftMax Pro 6.2.1.
[0302] The detection results of the binding of antibodies H5L9 and Ab123FR1 to the antigen human p40-His are shown in
[0303] The detection results of the binding of antibodies H5L10 and Ab123FR1 to the antigen human p40-His are shown in
[0304] The detection results of the binding of antibodies H5L11, H5L12, H5L14 and Ab123FR1 to the antigen human p40-His are shown in
[0305] The detection results of the binding of antibodies H8L15 and Ab123FR1 to the antigen human p40-His are shown in
[0306] The results show that the binding efficiency of H5L9, H5L10, H5L11, H5L12, H5L14 and H8L15 to the antigen human p40-His is dose-dependent.
[0307] As shown in
[0308] As shown in
[0309] As shown in
[0310] As shown in
TABLE-US-00008 TABLE 1 Binding activity of H5L9 to human p40-His Antibody Antigen coating: Concentration p40-His (0.125 μg/mL) (μg/mL) Ab123FR1 H5L9 1.000 2.884 2.863 2.817 2.860 0.333 2.818 2.838 2.864 2.776 0.111 2.806 2.833 2.768 2.831 0.037 2.543 2.650 2.318 2.463 0.012 1.751 1.792 1.649 1.622 0.004 0.849 0.918 0.801 0.820 0.001 0.357 0.441 0.386 0.497 PBS 0.046 0.046 0.052 0.052 Second HRP-labeled goat anti-human IgG antibody (H+L) (1:5000) EC.sub.50(nM) 0.063 0.079
TABLE-US-00009 TABLE 2 Binding activity of H5L10 to human p40-His Antibody Antigen coating: Concentration p40-His (0.125 μg/mL) (μg/mL) Ab123FR1 H5L10 1.000 3.070 3.071 3.052 3.094 0.333 3.074 3.081 3.066 3.030 0.111 3.085 3.043 2.977 3.022 0.037 2.853 2.788 2.794 2.784 0.012 2.164 1.894 1.981 2.104 0.004 1.229 0.935 1.058 1.030 0.001 0.532 0.438 0.490 0.464 PBS 0.062 0.059 0.059 0.057 Second HRP-labeled goat anti-human IgG antibody (H+L) (1:5000) EC.sub.50(nM) 0.051 0.057
TABLE-US-00010 TABLE 3 Binding activity of H5L11, H5L12 and H5L14 to human p40 Antibody Concentration Antigen coating: p40-His (0.125 μg/mL) (μg/mL) H5L11 H5L12 H5L14 Ab123FR1 1 2.474 2.410 2.518 2.542 2.475 2.429 2.386 2.342 2.353 2.282 0.3 2.472 2.409 2.541 2.473 2.576 2.471 1.815 1.736 1.801 1.862 0.1 2.392 2.382 2.486 2.371 2.488 2.386 0.864 0.946 0.971 1.091 0.03 2.132 2.074 2.096 2.111 1.995 1.964 0.356 0.372 0.408 0.438 0.01 1.279 1.310 1.334 1.309 1.196 0.995 0.149 0.168 0.165 0.180 0.003 0.613 0.593 0.625 0.665 0.603 0.580 0.082 0.083 0.084 0.089 0.001 0.269 0.242 0.286 0.263 0.237 0.256 0.056 0.060 0.056 0.062 0 0.045 0.046 0.045 0.047 0.046 0.049 0.048 0.037 0.041 0.046 Second antibody HRP-labeled goat anti-human IgG (H + L) (1:5000) EC.sub.50(nM) 0.082 0.082 0.107 1.181
TABLE-US-00011 TABLE 4 Binding activity of H8L15 to human p40-His Antibody Concentration Antigen coating: p40-His (0.25 μg/mL) (μg/mL) H8L15 Ab123FR1 Ustekinumab 1.0000 2.584 2.470 2.466 2.425 2.686 2.690 0.3333 2.519 2.551 2.325 2.425 2.521 2.555 0.1111 2.520 2.490 2.241 2.301 2.473 2.334 0.0370 2.376 2.280 2.190 2.055 2.317 2.329 0.0123 1.602 1.537 1.300 1.373 1.377 1.386 0.0041 0.835 0.801 0.663 0.675 0.686 0.656 0.0014 0.370 0.351 0.312 0.340 0.310 0.284 0.0000 0.069 0.068 0.096 0.087 0.090 0.078 Second antibody HRP-labeled goat anti-human IgG (H + L) (1:5000) EC.sub.50(nM) 0.059 0.074 0.077
Example 3. Determination of Affinity Constants of H5L9, H5L10, H5L11, H5L12, H5L14, H8L15, Ab123FR1 and Ustekinumab for Antigen Human IL-12/IL-23 p40 by Fortebio
[0311] The sample dilution buffer for H5L9, H5L10, H5L11, H5L12, H5L14, H8L15, Ab123FR1 and ustekinumab was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). p40-His was immobilized on a HIS1K (manufacturer: Fortebio, Cat. No.: 18-5120) sensor at a concentration of 1 μg/mL for 40 s. The sensor was equilibrated in a buffer for 60 s, and the p40-His immobilized on the sensor bound to the antibody at a concentration of 5-0.31 nM (two-fold dilution) for 120 s, and then the protein dissociated in the buffer for 300 s. The sensor was refreshed with 10 mM glycine solution (pH=1.5). The detection temperature was 37° C., the detection frequency was 0.3 Hz, and the sample plate shaking rate was 500 rpm. The data were analyzed by 1:1 model fitting to obtain affinity constants.
[0312] The determination results of the affinity constants of humanized antibodies H5L9, H5L10, H5L11, H5L12, H5L14, Ab123FR1, H8L15 and ustekinumab (as control antibody) for human p40-His are shown in Table 5, and the detection results are shown in
[0313] The results show that: the affinity constant of H5L9 for human p40-His is 8.49E-10M, the affinity constant of H5L10 for human p40-His is 1.21E-10M, the affinity constant of H5L11 for human p40-His is 1.36E-10M, the affinity constant of H5L12 for human p40-His is 9.05E-11M, the affinity constant of H5L14 for human p40-His is 6.20E-11M, the affinity constant of Ab123FR1 for human p40-His is 7.40E-11M, the affinity constant of H8L15 for human p40-His is 6.09E-11M, and the affinity constant of ustekinumab for human p40-His is 8.64E-11M.
[0314] In terms of affinity, the antibodies were ranked as follows from strong to weak: H8L15, H5L14, Ab123FR1, ustekinumab, H5L12, H5L10, H5L11 and H5L9 H8L15 and H5L14 show stronger affinity for human p40-His than Ab123FR1 and ustekinumab.
TABLE-US-00012 TABLE 5 Affinity constants of H5L9, H5L10, H5L11, H5L12, H5L14, H8L15, Ab123FR1 and ustekinumab for human p40-His Test antibodies K.sub.D (M) kon(l/Ms) S E (kon) kdis(1/s) S E(kdis) Rmax(nm) H5L9 8.49E−10 1.08E+07 8.50E+05 9.20E−03 3.07E−04 0.11-0.13 H5L10 1.21E−10 3.58E+06 3.64E+05 4.35E−04 2.22E−04 0.14-0.18 H5L11 1.36E−10 3.15E+06 2.32E+05 4.28E−04 1.44E−04 0.16-0.19 H5L12 9.05E−11 3.84E+06 2.12E+05 3.48E−04 1.24E−04 0.17-0.21 H5L14 6.20E−11 2.94E+06 2.36E+05 1.82E−04 1.58E−04 0.11-0.15 Ab123FR1 7.40E−11 2.00E+06 2.89E+05 1.48E−04 2.15E−04 0.15-0.18 H8L15 6.09E−11 2.95E+06 2.13E+05 1.80E−04 1.37E−04 0.18-0.21 Ustekinumab 8.64E−11 1.99E+06 2.51E+05 1.72E−04 1.81E−04 0.16-0.17 K.sub.D is the affinity constant; K.sub.D = k.sub.dis/k.sub.on.
Example 4. Detection of Anti-Human IL-12/IL-23 p40 Antibodies Competitively Blocking the Binding of Human IL-12 and IL-23 to 293T-IL-12Rβ1&IL-23R Cells by Flow Cytometry
[0315] 1.1. Detection of Antibodies H5L9, H5L10, H5L11, H5L12, H5L14 and Ustekinumab Competitively Blocking the Binding of Human IL-12 to 293T-IL-12Rβ1&IL-23R Cells by Flow Cytometry
[0316] 293T-IL-12Rβ1&IL-23R cells were digested in a conventional way and divided into several samples with 300,000 cells for each. 200 μL of 1% PBSA was added into each sample, and the mixture was centrifuged at 700×g for 5 min to discard the supernatant. According to experiment design, correspondingly diluted antibody (highest final concentration of 30 μg/mL, 3-fold dilution, 8 concentrations in total) and human IL-12 (Sino Biological, Cat. No.: CT-050-H08H-20) (final concentration of 20 nM) were mixed at a ratio of 1:1, and a blank control was set. The mixture of antibody and human IL12 was incubated on ice for 30 min and then added to the cell precipitate at 100 μL/sample. The resulting mixture was well mixed and incubated on ice for 60 min 200 μL of 1% PBSA was added, and the mixture was centrifuged at 700×g for 5 min to discard the supernatant, and then washed twice. Alexa Fluor® 488 anti-His tag antibody (Biolegend, Cat. No.: 652509) was diluted at a ratio of 1:400 and added to each tube at 100 μL. The mixture was well mixed and incubated on ice for 40 min in the absence of light. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 700×g for 5 min to discard the supernatant, and then washed twice. 1% PBSA was added at 200 μL/tube, and the cells were resuspended and transferred to a flow cytometry tube for testing. The results of antibodies H5L9, H5L10, H5L11, H5L12, H5L14 and ustekinumab competitively blocking the binding of human IL-12 to 293T-IL-12Rβ1&IL-23R cells detected by FACS are shown in Table 6 and
[0317] According to the results shown in Table 6 and
[0318] In terms of strength for competitively blocking the binding of IL-12 to IL-12Rβ1 on the cell membrane surface of 293T-IL-12Rβ1&IL-23R, the antibodies were ranked as follows from strong to weak: H5L12, H5L10, ustekinumab, H5L11, H5L14 and H5L9.
[0319] The above results show that the competitive binding activity of H5L12 and H5L10 for competitively blocking the binding of IL-12 to IL-12Rβ1 on the cell membrane surface is better than that of ustekinumab.
TABLE-US-00013 TABLE 6 Results of antibodies H5L9, H5L10, H5L11, H5L12, H5L14 and ustekinumab competitively blocking the binding of human IL-12 to 293T-IL-12Rβ1&IL-23R cells detected by FACS Concentration EC.sub.50 (μg/mL)/MFI 30 10 3.33 1.11 0.37 0.12 0.04 0.01 (μg/mL) H5L9 117 134 146 157 159 160 158 169 \ H5L10 31.6 34.1 40.2 44.6 87.5 151 154 163 0.3312 H5L11 33 33.9 42.2 54.3 103 145 151 166 0.414 H5L12 31.8 32.4 31.8 38.5 86.6 133 149 161 0.3172 H5L14 34.9 46.3 51.9 66 113 146 155 149 0.5320 Ustekinumab 28.7 31 29.7 31.9 95.7 137 150 160 0.3770
[0320] 1.2. Detection of Antibodies Ab123FR1, H8L15 and Ustekinumab Competitively Blocking the Binding of Human IL-12 to 293T-IL-12Rβ1&IL-23R Cells by Flow Cytometry
[0321] 293T-IL-12Rβ1 &IL-23R cells were digested in a conventional way and divided into several samples with 300,000 cells for each. 200 μL of 1% PBSA was added into each sample, and the mixture was centrifuged at 1200 rpm for 5 min to discard the supernatant. According to experiment design, correspondingly diluted antibody (highest final concentration of 60 μg/mL, 3-fold dilution, 8 concentrations in total) and IL12-His (Sino Biological, Cat. No.: CT-050-H08H-20) (40 nM) were mixed at a ratio of 1:1, and a blank control was set. The mixture of antibody and IL12 was incubated on ice for 30 min and then added to the cell precipitate at 100 μL/sample. The resulting mixture was well mixed and incubated on ice for 60 min 200 μL of 1% PBSA was added, and the mixture was centrifuged at 1200 rpm for 5 min to discard the supernatant, and then washed twice. THETM His tag antibody (FITC) (Genscript, Cat. No.: A01620) was diluted at a ratio of 1:500 and added to each tube at 100 μL. The mixture was well mixed and incubated on ice for 40 min in the absence of light. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 1200 rpm for 5 min to discard the supernatant, and then washed twice. 1% PBSA was added at 200 μL/tube, and the cells were resuspended and transferred to a flow cytometry tube for testing. The results of antibodies Ab123FR1, H8L15 and ustekinumab competitively blocking the binding of human IL-12 to 293T-IL-12Rβ1&IL-23R cells detected by FACS are shown in Table 7 and
[0322] According to the results shown in Table 7 and
[0323] The above results show that the competitive binding activity of H8L15 for competitively blocking the binding of IL-12 to IL-12Rβ1 on the cell membrane surface is better than that of Ab123FR1 and ustekinumab.
TABLE-US-00014 TABLE 7 Results of antibodies Ab123FR1, H8L15 and ustekinumab competitively blocking the binding of IL-12 to 293T-IL-12Rβ1&IL-23R cells detected by FACS Concentrationx EC.sub.50 (μg/mL)/MFI 30 10 3.33 1.11 0.37 0.12 0.04 0.01 (μg/mL) H8L15 34.92 46.83 57.84 90.8 118.86 127.45 136.89 117.45 1.46 Ab123FR1 36.29 47.78 54.84 105.2 115.6 108.16 123.57 118.88 1.99 Ustekinumab 40.32 40.9 46.98 99.64 120.43 123.28 114.93 114.66 1.58
[0324] 2.1. Detection of Antibodies H5L9, H5L10, H5L11, H5L12, H5L14 and Ustekinumab Competitively Blocking the Binding of Human IL-23 to 293T-IL-12Rβ1&IL-23R Cells by Flow Cytometry
[0325] 293T-IL-12Rβ1&IL-23R cells were digested in a conventional way and divided into several samples with 300,000 cells for each. 200 μL of 1% PBSA was added into each sample, and the mixture was centrifuged at 700×g for 5 min to discard the supernatant. According to experiment design, correspondingly diluted antibody (highest final concentration of 30 μg/mL, 3-fold dilution, 8 concentrations in total) and human IL-23 (IL-23-His-Biotin, Akesobio, 20161209) (final concentration of 2 μg/mL) were mixed at a ratio of 1:1, and a blank control was set. The mixture of antibody and human IL23 was incubated on ice for 30 min and then added to the cell precipitate at 100 μL/sample. The resulting mixture was well mixed and incubated on ice for 60 min. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 700×g for 5 min to discard the supernatant, and then washed twice. FITC Steptavidin (Biolegend, Cat. No.: 405202) was diluted at a ratio of 1:500 and added to each tube at 100 μL. The mixture was well mixed and incubated on ice for 40 min in the absence of light. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 700×g for 5 min to discard the supernatant, and then washed twice. 1% PBSA was added at 200 μL/tube, and the cells were resuspended and transferred to a flow cytometry tube for testing. The results of antibodies H5L9, H5L10, H5L11, H5L12, H5L14 and ustekinumab competitively blocking the binding of human IL-23 to 293T-IL-12Rβ1&IL-23R cells detected by FACS are shown in Table 8 and
[0326] According to the results shown in Table 8 and
[0327] In terms of strength for competitively blocking the binding of IL-23 to IL-23 receptor complex on the cell membrane surface of 293T-IL-12Rβ1&IL-23R, the antibodies were ranked as follows from strong to weak: H5L10, H5L12, H5L11, H5L14, ustekinumab and H5L9.
[0328] The above results show that the competitive binding activity of H5L10, H5L12, H5L11 and H5L14 for competitively blocking the binding of IL-23 to IL-23 receptor complex on the cell membrane surface of 293T-IL-12Rβ1&IL-23R is better than that of ustekinumab.
TABLE-US-00015 TABLE 8 Results of antibodies H5L9, H5L10, H5L11, H5L12, H5L14 and ustekinumab competitively blocking the binding of human IL-23 to 293T-IL-12Rβ1&IL-23R cells detected by FACS Concentration EC.sub.50 (μg/mL)/MFI 30 10 3.33 1.11 0.37 0.12 0.04 0.01 (μg/mL) H5L9 104 136 153 184 206 217 221 222 4.252 H5L10 11.9 12.7 15.7 81.6 169 221 230 223 0.6995 H5L11 12.9 14.5 20.2 97.6 179 219 224 219 0.8643 H5L12 12.3 12.7 15.8 88.5 177 211 234 221 0.7748 H5L14 14.1 16.7 23.6 95.6 179 203 216 218 0.8806 Ustekinumab 9.64 11.5 13.8 121 186 211 216 219 1.158
[0329] 2.2. Detection of Antibodies Ab123FR1, H8L15 and Ustekinumab Competitively Blocking the Binding of Human IL-23 to 293T-IL-12Rβ1&IL-23R Cells by Flow Cytometry
[0330] 293T-IL-12Rβ1&IL-23R cells were digested in a conventional way and divided into several samples with 300,000 cells for each. 200 μL of 1% PBSA was added into each sample, and the mixture was centrifuged at 1200 rpm for 5 min to discard the supernatant. According to experiment design, correspondingly diluted antibody (highest concentration of 60 μg/mL, 3-fold dilution, 8 concentrations in total) and human IL23-His-Biotin (Akeso Biopharma, Inc., Lot. No.: 20161209) (4 μg/mL) were mixed at a ratio of 1:1, and a blank control was set. The mixture of antibody and IL23-His-Biotin was incubated on ice for 30 min and then added to the cell precipitate at 100 μL/sample. The resulting mixture was well mixed and incubated on ice for 60 min. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 1200 rpm for 5 min to discard the supernatant, and then washed twice. FITC Steptavidin (Biolegend, Cat. No.: 405202) was diluted at a ratio of 1:500 and added to each tube at 100 μL. The mixture was well mixed and incubated on ice for 40 min in the absence of light. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 1200 rpm for 5 min to discard the supernatant, and then washed twice. 1% PBSA was added at 200 μL/tube, and the cells were resuspended and transferred to a flow cytometry tube for testing. The results of antibodies Ab123FR1, H8L15 and ustekinumab competitively blocking the binding of human IL-23 to 293T-IL-12Rβ1&IL-23R cells detected by FACS are shown in Table 9 and
[0331] According to the results shown in Table 9 and
[0332] The above results show that the competitive binding activity of H8L15 for competitively blocking the binding of IL-23 to IL-23R on the cell membrane surface of 293T-IL-12Rβ1&IL-23R is better than that of Ab123FR1 and ustekinumab.
TABLE-US-00016 TABLE 9 Results of antibodies Ab123FR1, H8L15 and ustekinumab competitively blocking the binding of IL-23 to 293T-IL-12Rβ1&IL-23R cells detected by FACS Concentration EC.sub.50 (μg/mL)/MFI 30 10 3.33 1.11 0.37 0.12 0.04 0.01 (μg/mL) H8L15 15.67 17.95 20.65 67.47 140.58 151.42 155.60 155.36 0.8942 Ab123FR1 16.62 17.43 21.52 114.17 148.32 154.10 157.47 159.75 1.41 Ustekinumab 17.65 18.37 22.39 124.07 160.96 164.00 156.17 162.10 1.434
Example 5. H8L15 Effectively Inhibiting IL-17A Secretion by Spleen Cells of Mice with Spontaneous Systemic Lupus Erythematosus
[0333] Spontaneous systemic lupus erythematosus model (Jeltsch-David H. Autoimmun Rev. 2014; 13(9):963-973.) mice (MRL/lpr mice, purchased from Shanghai SLAC Laboratory Animal Co., Ltd.) were anesthetized with chloral hydrate, soaked in 75% ethyl alcohol for disinfection, transferred to a biosafety cabinet, and then dissected to take the spleen. The spleen was rinsed in a dish containing 1640 complete medium to remove fat and fascia tissue. The washed mouse spleen was placed in a 70 μm cell strainer and gently ground with a syringe plunger, and the spleen cell suspension was repeatedly washed with the culture solution. The filtrate was collected and centrifuged at 170×g for 5 minutes to discard the supernatant. 7 mL of erythrocyte lysate was added to resuspend the cell precipitate. The mixture was well mixed gently and left to stand on ice for 8 min, and then an equal volume of complete medium was added to stop lysis. The mixture was centrifuged at 170×g for 5 min to discard the supernatant. The cell precipitate was subjected to centrifugation and washing with 1640 complete medium twice, and then the cell precipitate was resuspended in 1640 complete medium, counted, adjusted for cell density, and seeded in a 96-well plate (1×10.sup.6/100 μL). According to experiment design, 50 μL of antibody was preincubated with 50 μL of IL-23 (final concentration of 20 ng/mL) for 1 h, and then 50 μL of IL-2 (final concentration of 100 U/mL) was added, and the mixture was incubated in an incubator at 37° C./5% CO.sub.2 for 6 days. Six days later, cell supernatant was collected by centrifugation, and IL-17A concentration in the supernatant was detected by ELISA.
[0334] As shown in
Example 6. H8L5 Effectively Ameliorating Skin Injury of Psoriasis Model Mice
[0335] After shaving, C57BL/6 mice (purchased from Guangdong Medical Laboratory Animal Center) were randomly divided into a normal group, a model group, a positive control group and H8L15 groups at 10 mice per group. One day prior to the first injection of recombinant human IL-23, the isotype control antibody (i.e., human anti-hen egg lysosome) was injected subcutaneously in the model group, the H8L15 dose groups were injected with H8L15 at corresponding concentrations, and the normal group was injected subcutaneously with an equal volume of normal saline. On day 1 after administration, 3.5% chloral hydrate was intraperitoneally injected at a dose of 7.5 ml/kg to anesthetize C57BL/6 mice, mice in normal group were intradermally injected with normal saline at 25 μL/mouse, and the remaining mice were intradermally injected with recombinant human IL-23 at 10 μg/25 μL/mouse. The injection was performed once daily for 6 consecutive days. On day 2 after the final intradermal injection of recombinant human IL-23, mice in each group were subjected to cervical dislocation, and small pieces of neck skin (about 0.5 cm×0.5 cm) were cut and fixed in formalin tissue fixative. Pathological sections of mouse skin were made 24 h later for 6 mice per group. After the pathological sections were made, 1 representative field of view was selected under a 100× microscope, and 6 sites in an original picture are randomly selected to measure the thickness of the skin epidermis of the mouse. Data were expressed as mean±standard error, and results were evaluated by one-way analysis of variance after the inter-group comparison processed by GraphPad software. P<0.05 suggests significant difference, and P<0.01 suggests very significant difference.
[0336] The results are shown in
Example 7. Treatment of Colitis with Anti-IL-12/IL-23 p40 Antibodies
[0337] Anti-IL-12/IL-23 p40 antibodies such as H8L15 were found to be effective in alleviating pathological changes and clinical symptoms in ulcerative colitis model mice.
[0338] A colitis model was established by inducing C57BL/6 mice with DSS (dextran sulfate sodium). The experimental mice were divided into groups with 3 mice in normal group and 6 mice in each of other groups. A positive control group (DSS group), an isotype control antibody group (anti-HEL), a high-dose H8L15 group (120 mg/kg) and a low-dose H8L15 group (40 mg/kg) were set. The drugs were administered by subcutaneous injection on D0, D3 and D6. In the normal group, the animal models were established by feeding sterile water through drinking bottles; in the DSS (MP Bio, Cat. No.: Q1723) group, the animal models were established by feeding 1% DSS solution (prepared by adding 2.5 g of DSS to 250 mL of sterile water) through drinking bottles for 9 consecutive days; in the experimental group with antibody, the animal models were established by feeding 1% DSS solution (prepared by adding 2.5 g of DSS to 250 mL of sterile water) through drinking bottles and intraperitoneally injecting hIL-23 recombinant protein (200 μL/100 μg/mouse) daily (D1-D5, D7-D9).
[0339] The use and welfare of the laboratory animals were carried out in compliance with the provisions of Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). The health and death of the animals are monitored daily, and routine examinations include observation of the effects of the test substance or drug on the daily performance of the animals, such as behavioral activities, weight changes and appearance.
[0340] The experimental index was to study the influence of the drug on colitis, and the specific index was based on the table of pathological scores of mouse colitis, which is shown in Table 10.
TABLE-US-00017 TABLE 10 Pathological scores of mouse colitis Pathological tissue condition of colon lesion Score Inflammatory infiltration: 0-none 1-relatively mild 2-mild 3-moderate 4-relatively severe 5-severe Crypt damage: 0-none 1-mild 2-moderate 3-severe Ulcer: 0-none 1-mild 2-moderate 3-severe Edema: 0-no l-yes
TABLE-US-00018 TABLE 11 Administration dosage and scheme Group n Animal model Administration Normal group 3 Feeding sterile water through drinking bottle DSS group 6 1% DSS solution was prepared by adding 2.5 g of DSS to 250 mL of sterile water and fed to the mice through drinking bottles for 9 consecutive days (D1-D9), and the experiment was terminated on day 10 DSS+hIL-23+ 6 1% DSS solution was hIgG1, 120 mg/kg, hIgG1 120 mg/kg prepared by adding 2.5 g of SC, D0, D3, D6 DSS+hIL-23+ 6 DSS to 250 mL of sterile H8L15, 120mg/kg, H8L15 120 mg/kg water and fed to the mice SC, D0, D3, D6 DSS+hIL-23+ 6 through drinking bottles, H8L15, 40mg/kg, H8L15 hIL-23 recombinant protein SC, D0, D3, D6 40 mg/kg (200 μL/100 μg/mouse) were intraperitoneally injected daily (D1-D6, D8-D9), and the experiment was terminated on day 10 *Randomly grouped; the time of first administration was D.sub.0; SC: subcutaneous injection.
[0341] For the experimental results, Table 10 is the pathological scoring standard of mouse colitis, Table 11 is the establishment method of mouse models and the administration scheme of antibody in each experimental group, and the pathological scoring results of colitis are shown in
[0342] Conclusion: according to the body weight of mice in the experimental period shown in
[0343] The information about sequences is as follows:
TABLE-US-00019 The amino acid sequence of heavy chain variable region of H5L9, H5L10, H5L11, H5L12 and H5L14 is set forth in SEQ ID NO: 1 (SEQ ID NO: 1) EVQLVQSGAEVKKPGESLKISCQSSGYSFTTYWIGWVRQMPGQGLEWIGI MSPVDSDIRYNPMFRGQVTMSVDKSSSTAYLQWSSLKASDTAMYYCARRR PGQGYFDFWGQGTMVTVSS The nucleotide sequence of heavy chain variable region of H5L9, H5L10, H5L11, H5L12 and H5L14 is set forth in SEQ ID NO: 2 (SEQ ID NO: 2) GAGGTGCAGCTGGTGCAGTCTGGGGCCGAAGTGAAGAAACCCGGGGAGAG TCTGAAGATCTCATGCCAGAGCTCCGGCTACTCCTTCACCACATATTGGA TCGGGTGGGTGAGACAGATGCCTGGCCAGGGGCTGGAATGGATCGGAATT ATGAGCCCAGTGGACTCCGATATTCGCTACAACCCCATGTTTCGAGGCCA GGTGACAATGAGCGTGGACAAGTCTAGTTCAACTGCTTATCTGCAGTGGA GCTCCCTGAAAGCCAGCGATACCGCTATGTACTATTGTGCCCGGAGAAGG CCTGGACAGGGCTACTTCGACTTTTGGGGGCAGGGAACTATGGTGACCGT CTCTAGT
[0344] For H5L9, H5L10, H5L11, H5L12 and H5L14, HCDR1 is set forth in SEQ ID NO: 3, HCDR2 is set forth in SEQ ID NO: 4, and HCDR3 is set forth in SEQ ID NO: 5
TABLE-US-00020 (SEQ ID NO: 3) HCDR1: GYSFTTYW (SEQ ID NO: 4) HCDR2: MSPVDSDI (SEQ ID NO: 5) HCDR3: ARRRPGQGYFDF The amino acid sequence of light chain variable region of H5L9 is set forth in SEQ ID NO: 6 DIQMTQSPSSLSASVGDRVTITCKASQNVGSWLAWYQQKPGKAPKSLIYS ASSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDIYPFTFGQ GTKLEIK The nucleotide sequence of light chain variable region of H5L9 is set forth in SEQ ID NO: 7 GATATTCAGATGACCCAGAGCCCTTCAAGCCTGTCCGCAAGCGTCGGGGA TAGAGTGACCATTACCTGTAAAGCAAGCCAGAACGTGGGAAGCTGGCTGG CCTGGTACCAGCAGAAGCCAGGCAAAGCACCCAAGTCTCTGATCTATAGT GCAAGCTCCCGGCAGTCAGGAGTGCCAAGCAGATTCAGTGGCTCAGGGAG CGGAACAGACTTTACCCTGACAATCTCTAGTCTGCAGCCTGAGGACTTCG CAACTTACTATTGCCAGCAGTACGATATCTACCCATTCACATTTGGCCAG GGGACTAAACTGGAGATCAAG
[0345] For H5L9, LCDR1 is set forth in SEQ ID NO: 8, LCDR2 is set forth in SEQ ID NO: 9, and LCDR3 is set forth in SEQ ID NO: 10
TABLE-US-00021 (SEQ ID NO: 8) LCDR1: QNVGSW (SEQ ID NO: 9) LCDR2: ASS (SEQ ID NO: 10) LCDR3: QQYDIYPFT The amino acid sequence of light chain variable region of H5L10 is set forth in SEQ ID NO: 11 EIVLTQSPATLSASPGERATISCRASQSVGSWLAWYQQKPGQAPRSLIYA ASNLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPYTFGQ GTRLEIK The nucleotide sequence of light chain variable region of H5L10 is set forth in SEQ ID NO: 12 GAGATCGTCCTGACACAGAGTCCTGCTACCCTGAGCGCTTCCCCAGGAGA GAGGGCAACCATCTCCTGCCGCGCCTCTCAGaGCgTTGGCTCCTGGCTGG CTTGGTACCAGCAGAAGCCAGGCCAGGCACCCCGAAGCCTGATCTATGCC GCTTCTAaTCTGCAGAGCGGGATTCCCGCTAGATTCTCTGGCAGTGGGTC AGGAACAGACTTTACCCTGACAATCTCAAGCCTGGAGCCTGAAGATTTCG CCGTGTACTATTGCCAGCAGTACAACATCTACCCATATACATTTGGCCAG GGGACTCGGCTGGAGATCAAG The amino acid sequence of light chain variable region of H5L11 is set forth in SEQ ID NO: 13 EIVLTQSPATLSASPGERATISCRASQSVSSWLAWYQQKPGQAPRSLIYS ASNLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPYTFGQ GTRLEIK The nucleotide sequence of light chain variable region of H5L11 is set forth in SEQ ID NO: 14 GAGATCGTCCTGACACAGAGTCCTGCTACCCTGAGCGCTTCCCCAGGAGA GAGGGCAACCATCTCCTGCCGCGCCTCTCAGaGCgTTAGCTCCTGGCTGG CTTGGTACCAGCAGAAGCCAGGCCAGGCACCCCGAAGCCTGATCTATTCC GCTTCTAaTCTGCAGAGCGGGATTCCCGCTAGATTCTCTGGCAGTGGGTC AGGAACAGACTTTACCCTGACAATCTCAAGCCTGGAGCCTGAAGATTTCG CCGTGTACTATTGCCAGCAGTACAACATCTACCCATATACATTTGGCCAG GGGACTCGGCTGGAGATCAAG The amino acid sequence of light chain variable region of H5L12 is set forth in SEQ ID NO: 15 EIVLTQSPATLSASPGERATISCRASQSVSSWLAWYQQKPGQAPRSLIYA ASNRQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPYTFGQ GTRLEIK The nucleotide sequence of light chain variable region of H5L12 is set forth in SEQ ID NO: 16 GAGATCGTCCTGACACAGAGTCCTGCTACCCTGAGCGCTTCCCCAGGAGA GAGGGCAACCATCTCCTGCCGCGCCTCTCAGaGCgTTAGCTCCTGGCTGG CTTGGTACCAGCAGAAGCCAGGCCAGGCACCCCGAAGCCTGATCTATGCC GCTTCTAaTCGGCAGAGCGGGATTCCCGCTAGATTCTCTGGCAGTGGGTC AGGAACAGACTTTACCCTGACAATCTCAAGCCTGGAGCCTGAAGATTTCG CCGTGTACTATTGCCAGCAGTACAACATCTACCCATATACATTTGGCCAG GGGACTCGGCTGGAGATCAAG The amino acid sequence of light chain variable region of H5L14 is set forth in SEQ ID NO: 17 EIVLTQSPATLSASPGERATISCRASQSVSSWLAWYQQKPGQAPRSLIYA ASNLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPFTFGQ GTRLEIK The nucleotide sequence of light chain variable region of H5L14 is set forth in SEQ ID NO: 18 GAGATCGTCCTGACACAGAGTCCTGCTACCCTGAGCGCTTCCCCAGGAGA GAGGGCAACCATCTCCTGCCGCGCCTCTCAGaGCgTTAGCTCCTGGCTGG CTTGGTACCAGCAGAAGCCAGGCCAGGCACCCCGAAGCCTGATCTATGCC GCTTCTAaTCTGCAGAGCGGGATTCCCGCTAGATTCTCTGGCAGTGGGTC AGGAACAGACTTTACCCTGACAATCTCAAGCCTGGAGCCTGAAGATTTCG CCGTGTACTATTGCCAGCAGTACAACATCTACCCATTTACATTTGGCCAG GGGACTCGGCTGGAGATCAAG LCDR1 of L10 is set forth in SEQ ID NO: 19 QSVGSW LCDR1 of L11, L12 and L14 is set forth in SEQ ID NO: 20 QSVSSW LCDR2 of L10, L11, L12 and L14 is set forth in SEQ ID NO: 21 ASN LCDR3 of L10, L11 and L12 is set forth in SEQ ID NO: 22 QQYNIYPYT LCDR3 of L14 is set forth in SEQ ID NO: 23 QQYNIYPFT The amino acid sequence of heavy chain variable region of H8L15 is set forth in SEQ ID NO: 24 EVQLVQSGAEVKKPGESLKISCQSSGYTFTSYWIGWVRQMPGQGLEWIG IMSPVDSDIRYNPMFRGQVTMSVDKSSSTAYLQWSSLKASDTAMYYCAR RRPGQGYFDFWGQGTMVTVSS The amino acid sequence of light chain variable region of H8L15 is set forth in SEQ ID NO: 25 EIVLTQSPATLSASPGERATISCRASQSVGTWVAWYQQKPGQAPRSLIY AASNLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPYTF GQGTRLEIK
[0346] For H8L15, HCDR1 is set forth in SEQ ID NO: 26, HCDR2 is set forth in SEQ ID NO: 4, HCDR3 is set forth in SEQ ID NO: 5, LCDR1 is set forth in SEQ ID NO: 27, LCDR2 is set forth in SEQ ID NO: 28, LCDR3 is set forth in SEQ ID NO: 22, FR-H1 is set forth in SEQ ID NO: 29, FR-H2 is set forth in SEQ ID NO: 30, FR-H3 is set forth in SEQ ID NO: 31, FR-H4 is set forth in SEQ ID NO: 32, FR-L1 is set forth in SEQ ID NO: 33, FR-L2 is set forth in SEQ ID NO: 34, FR-L3 is set forth in SEQ ID NO: 35, and FR-L4 is set forth in SEQ ID NO: 36
TABLE-US-00022 (SEQ ID NO: 26) HCDR1: GYTFTSYW (SEQ ID NO: 4) HCDR2: MSPVDSDI (SEQ ID NO: 5) HCDR3: ARRRPGQGYFDF (SEQ ID NO: 27) LCDR1: QSVGTW (SEQ ID NO: 28) LCDR2: AAS (SEQ ID NO: 22) LCDR3: QQYNIYPYT (SEQ ID NO: 29) FR-H1: EVQLVQSGAEVKKPGESLKISCQSS (SEQ ID NO: 30) FR-H2: IGWVRQMPGQGLEWIGI (SEQ ID NO: 31) FR-H3: RYNPMFRGQVTMSVDKSSSTAYLQWSSLKASDTAMYYC (SEQ ID NO: 32) FR-H4: WGQGTMVTVSS (SEQ ID NO: 33) FR-L1: EIVLTQSPATLSASPGERATISCRAS (SEQ ID NO: 34) FR-L2: VAWYQQKPGQAPRSLIY (SEQ ID NO: 35) FR-L3: NLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 36) FR-L4: FGQGTRLEIK The nucleotide sequence of heavy chain variable region of H8L15 is set forth in SEQ ID NO: 37 GAGGTGCAGCTGGTGCAGTCTGGGGCCGAAGTGAAGAAACCCGGGGAGAG TCTGAAGATCTCATGCCAGAGCTCCGGCTACACCTTCACCTCATATTGGA TCGGGTGGGTGAGACAGATGCCTGGCCAGGGGCTGGAATGGATCGGAATT ATGAGCCCAGTGGACTCCGATATTCGCTACAACCCCATGTTTCGAGGCCA GGTGACAATGAGCGTGGACAAGTCTAGTTCAACTGCTTATCTGCAGTGGA GCTCCCTGAAAGCCAGCGATACCGCTATGTACTATTGTGCCCGGAGAAGG CCTGGACAGGGCTACTTCGACTTTTGGGGGCAGGGAACTATGGTGACCGT CTCTAGT The nucleotide sequence of light chain variable region of H8L15 is set forth in SEQ ID NO: 38 GAGATCGTCCTGACACAGAGTCCTGCTACCCTGAGCGCTTCCCCAGGAGA GAGGGCAACCATCTCCTGCCGCGCCTCTCAGAGCGTTGGCACCTGGGTGG CTTGGTACCAGCAGAAGCCAGGCCAGGCACCCCGAAGCCTGATCTATGCC GCTTCTAATCTGCAGAGCGGGATTCCCGCTAGATTCTCTGGCAGTGGGTC AGGAACAGACTTTACCCTGACAATCTCAAGCCTGGAGCCTGAAGATTTCG CCGTGTACTATTGCCAGCAGTACAACATCTACCCATATACATTTGGCCAG GGGACTCGGCTGGAGATCAAG The heavy chain amino acid sequence of H8L15 is set forth in SEQ ID NO: 39 EVQLVQSGAEVKKPGESLKISCQSSGYTFTSYWIGWVRQMPGQGLEWIGI MSPVDSDIRYNPMFRGQVTMSVDKSSSTAYLQWSSLKASDTAMYYCARRR PGQGYFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The light chain amino acid sequence of H8L15 is set forth in SEQ ID NO: 40 EIVLTQSPATLSASPGERATISCRASQSVGTWVAWYQQKPGQAPRSLIYA ASNLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPYTFGQ GTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
[0347] For H5L9, the sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are the same as those of H8L15, the sequence of FR-L1 is set forth in SEQ ID NO: 41, the sequence of FR-L2 is set forth in SEQ ID NO: 42, FR-L3 is set forth in SEQ ID NO: 43, and FR-L4 is set forth in SEQ ID NO: 44
TABLE-US-00023 (SEQ ID NO: 41) FR-L1: DIQMTQSPSSLSASVGDRVTITCKAS (SEQ ID NO: 42) FR-L2: LAWYQQKPGKAPKSLIYS (SEQ ID NO: 43) FR-L3: RQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 44) FR-L4: FGQGTKLEIK
[0348] For H5L10, the sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are the same as those of H8L15, the sequence of FR-L1 is set forth in SEQ ID NO: 33, the sequence of FR-L2 is set forth in SEQ ID NO: 45, FR-L3 is set forth in SEQ ID NO: 46, and FR-L4 is set forth in SEQ ID NO: 36
TABLE-US-00024 (SEQ ID NO: 33) FR-L1: EIVLTQSPATLSASPGERATISCRAS (SEQ ID NO: 45) FR-L2: LAWYQQKPGQAPRSLIYA (SEQ ID NO: 46) FR-L3: LQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 36) FR-L4: FGQGTRLEIK
[0349] For H5L11, the sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are the same as those of H8L15, the sequence of FR-L1 is set forth in SEQ ID NO: 33, the sequence of FR-L2 is set forth in SEQ ID NO: 47, FR-L3 is set forth in SEQ ID NO: 46, and FR-L4 is set forth in SEQ ID NO: 36
TABLE-US-00025 (SEQ ID NO: 33) FR-L1: EIVLTQSPATLSASPGERATISCRAS (SEQ ID NO: 47) FR-L2: LAWYQQKPGQAPRSLIYS (SEQ ID NO: 46) FR-L3: LQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 36) FR-L4: FGQGTRLEIK
[0350] For H5L12, the sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are the same as those of H8L15, the sequence of FR-L1 is set forth in SEQ ID NO: 33, the sequence of FR-L2 is set forth in SEQ ID NO: 45, FR-L3 is set forth in SEQ ID NO: 48, and FR-L4 is set forth in SEQ ID NO: 36
TABLE-US-00026 (SEQ ID NO: 33) FR-L1: EIVLTQSPATLSASPGERATISCRAS (SEQ ID NO: 45) FR-L2: LAWYQQKPGQAPRSLIYA (SEQ ID NO: 48) FR-L3: RQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 36) FR-L4: FGQGTRLEIK
[0351] For H5L14, the sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are the same as those of H8L15, the sequence of FR-L1 is set forth in SEQ ID NO: 33, the sequence of FR-L2 is set forth in SEQ ID NO: 45, FR-L3 is set forth in SEQ ID NO: 46, and FR-L4 is set forth in SEQ ID NO: 36
TABLE-US-00027 (SEQ ID NO: 33) FR-L1: EIVLTQSPATLSASPGERATISCRAS (SEQ ID NO: 45) FR-L2: LAWYQQKPGQAPRSLIYA (SEQ ID NO: 46) FR-L3: LQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 36) FR-L4: FGQGTRLEIK The heavy chain amino acid sequence of H5L9, H5L10, H5L11, H5L12 and H5L14 is set forth in SEQ ID NO: 49 EVQLVQSGAEVKKPGESLKISCQSSGYSFTTYWIGWVRQMPGQGLEWIGI MSPVDSDIRYNPMFRGQVTMSVDKSSSTAYLQWSSLKASDTAMYYCARRR PGQGYFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The light chain amino acid sequence of H5L9 is set forth in SEQ ID NO: 50 DIQMTQSPSSLSASVGDRVTITCKASQNVGSWLAWYQQKPGKAPKSLIYS ASSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDIYPFTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC The light chain amino acid sequence of H5L10 is set forth in SEQ ID NO: 51 EIVLTQSPATLSASPGERATISCRASQSVGSWLAWYQQKPGQAPRSLIYA ASNLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPYTFGQ GTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC The light chain amino acid sequence of H5L11 is set forth in SEQ ID NO: 52 EIVLTQSPATLSASPGERATISCRASQSVSSWLAWYQQKPGQAPRSLIYS ASNLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPYTFGQ GTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC The light chain amino acid sequence of H5L12 is set forth in SEQ ID NO: 53 EIVLTQSPATLSASPGERATISCRASQSVSSWLAWYQQKPGQAPRSLIYA ASNRQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPYTFGQ GTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC The light chain amino acid sequence of H5L14 is set forth in SEQ ID NO: 54 EIVLTQSPATLSASPGERATISCRASQSVSSWLAWYQQKPGQAPRSLIYA ASNLQSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYNIYPFTFGQ GTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC