CN113603776B - IL17 antibodies and uses thereof - Google Patents

IL17 antibodies and uses thereof Download PDF

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CN113603776B
CN113603776B CN202111069984.5A CN202111069984A CN113603776B CN 113603776 B CN113603776 B CN 113603776B CN 202111069984 A CN202111069984 A CN 202111069984A CN 113603776 B CN113603776 B CN 113603776B
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antibody
amino acid
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acid sequence
antigen
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CN113603776A (en
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王华菁
李博华
何晓文
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Oricell Therapeutics Co Ltd
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    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
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    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The present application provides an antibody, antigen binding fragment thereof, which is expressed as 6 × 10 ‑9 K of M or less D Values bind to IL17 protein. The antibody and the antigen binding fragment thereof have strong specific recognition binding capacity on the IL17 protein, and can inhibit the binding of the IL17 protein and an IL17 receptor and/or inhibit the secretion of cytokines by immune cells. The application also provides the use of the antibodies, antigen-binding fragments thereof, in the treatment or prevention of diseases associated with inappropriate or excessive production of IL 17.

Description

IL17 antibodies and uses thereof
The application is a divisional application of Chinese patent application with application date of 2019, 2 and 11 months, application number of 201980012724.9 and invention name of 'IL 17 antibody and application thereof'.
Technical Field
The application relates to the field of biomedicine, in particular to an IL17 antibody and application thereof.
Background
Interleukin 17 (IL 17), also known as CTLA-8, is a pro-inflammatory cytokine produced by T cells and stimulates a variety of other cytokines to be secreted by various non-immune cells. IL17 is produced primarily by activated memory T cells and acts by binding to ubiquitous cell surface receptors (IL 17R) (Yao et al, cytokine,9, 794-800 (1997)).
IL17 acts as a pro-inflammatory cytokine whose inappropriate or excessive production can lead to a variety of diseases. For example, rheumatoid Arthritis (Witowski et al, cell Mol Life Sci, 61.
Studies have shown that antibodies targeting IL-17 (e.g., secukinumab, ixekizumab) can be used to prevent and/or treat psoriasis (e.g., plaque psoriasis), psoriatic arthritis, ankylosing spondylitis, and other diseases or disorders associated with inappropriate or excessive production of IL 17. However, there is still a need for novel anti-IL 17 antibodies that specifically recognize and bind to IL17 protein, inhibit the binding of IL17 protein to IL17 receptors, and/or inhibit cytokine secretion by immune cells.
Disclosure of Invention
The present application provides an IL17 antibody, antigen-binding fragment thereof, having one or more of the following properties: 1) Can bind to IL17 protein with higher affinity and specificity; 2) Capable of inhibiting the binding of an IL17 protein to an IL17 receptor, such as an IL17A protein or an IL17F protein; 3) Capable of inhibiting the expression of cytokines, in particular chemokines (e.g. CXCL 1); and 4) can be used to treat autoimmune diseases. The application also provides a preparation method and application of the IL17 antibody and the antigen binding fragment thereof.
In one aspect, the present application provides an antibody or antigen-binding fragment thereof at 6 x10 -9 M or less K D Values bind to IL17 protein.
In certain embodiments, the antibody or antigen-binding fragment thereof inhibits binding of an IL17 protein to an IL17 receptor.
In certain embodiments, the antibody or antigen-binding fragment thereof inhibits the expression of a cytokine.
In certain embodiments, the antibody or antigen-binding fragment thereof, the cytokine comprises: interleukins and chemokines.
In certain embodiments, the antibody or antigen-binding fragment thereof, the chemokine comprises: CXCL1.
In certain embodiments, the antibody or antigen-binding fragment thereof can ameliorate or treat a disease or disorder associated with inappropriate or excessive production of IL 17. In certain embodiments, the disease or disorder comprises: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, respiratory inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises: monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises: fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scFv and/or VhH.
In certain embodiments, the antibody, or antigen-binding fragment thereof, that competes for binding to the IL17 protein with a reference antibody, wherein the reference antibody comprises a light chain variable region comprising LCDR1, LCDR2 and LCDR3 and a heavy chain variable region, the LCDR1 comprising the amino acid sequence set forth in any one of seq id nos: 126, 22 and 40; the LCDR2 comprises an amino acid sequence shown in any one of the following items: 5, 23 and 41; and the LCDR3 comprises an amino acid sequence shown in any one of the following items: 6, 24 and 131; and, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in any one of the following: 123, 19 and 37; the HCDR2 comprises an amino acid sequence shown in any one of the following items: 124, 20 and 38; and, the HCDR3 comprises an amino acid sequence set forth in any one of seq id nos: 125, 21 and 130.
In certain embodiments, the LCDR1 of the reference antibody comprises an amino acid sequence set forth in any one of seq id no: SEQ ID NO.4 and SEQ ID NO. 96. In certain embodiments, the LCDR3 of the reference antibody comprises the amino acid sequence set forth in any one of seq id no:42 and 102 SEQ ID NO.
In certain embodiments, the HCDR1 of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 1, 91 and 92 SEQ ID NO. In certain embodiments, the HCDR2 of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 2 and 93 SEQ ID NO; in certain embodiments, the HCDR3 of the reference antibody comprises an amino acid sequence as set forth in any one of seq id nos: 3, 94, 95, 39, 100 and 101.
In certain embodiments, the light chain variable region of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 16, 134, 34, 36, 52 and 132; and, the heavy chain variable region of the reference antibody comprises an amino acid sequence set forth in any one of: 15, 135, 33, 35, 51 and 133.
In certain embodiments, the light chain variable region of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 54, 103, 18 and 109.
In certain embodiments, the heavy chain variable region of the reference antibody comprises an amino acid sequence set forth in any one of seq id nos: 53, 105, 107, 17, 111, 114, 117 and 120.
In certain embodiments, the light chain of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 64, 68, 72, 138, 84 and 136; and, the heavy chain of the reference antibody comprises an amino acid sequence set forth in any one of seq id nos: 62, 66, 70, 139, 82 and 137.
In certain embodiments, the light chain of the reference antibody comprises an amino acid sequence set forth in any one of seq id nos: 90, 104, 78 and 110. In certain embodiments, the heavy chain of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 88, 106, 108, 76, 112, 115, 118 and 121.
In certain embodiments, the antibody comprises an antibody light chain or fragment thereof comprising LCDR1, and the LCDR1 comprises an amino acid sequence set forth in any one of seq id no:126, 22 and 40.
In certain embodiments, the amino acid sequence of the LCDR1 in the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in any one of seq id nos: SEQ ID NO.4 and SEQ ID NO. 96. In certain embodiments, the antibody light chain or fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in any one of seq id nos: 5, 23 and 41. In certain embodiments, the antibody light chain or fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in any one of seq id nos: 6, 24 and 131.
In certain embodiments, the LCDR3 comprises the amino acid sequence set forth in any one of seq id nos: 42 and 102 SEQ ID NO.
In certain embodiments, the antibody light chain or fragment thereof further comprises the framework regions L-FR1, L-FR2, L-FR3 and L-FR4. In certain embodiments, the framework regions L-FR1, L-FR2, L-FR3 and L-FR4 are selected from any one of the following groups: human consensus framework sequences and human germline sequences.
In certain embodiments, the C-terminus of the L-FR1 is linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 comprises an amino acid sequence set forth in any one of: 127, 29 and 47.
In certain embodiments, the L-FR1 comprises an amino acid sequence set forth in any one of: SEQ ID NO 11 and SEQ ID NO 97. In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, the L-FR2 comprising the amino acid sequence set forth in any one of: 128, 30 and 48 SEQ ID NO.
In certain embodiments, the L-FR2 comprises an amino acid sequence set forth in any one of: SEQ ID NO 12 and SEQ ID NO 98. In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, the L-FR3 comprising an amino acid sequence set forth in any one of: 13, 31 and 49.
In certain embodiments, the N-terminus of the L-FR4 is linked directly or indirectly to the C-terminus of the LCDR3, the L-FR4 comprising the amino acid sequence set forth in any one of: 14, 32 and 50.
In certain embodiments, the antibody light chain or fragment thereof comprises a light chain variable region VL, and the light chain variable region VL comprises the amino acid sequence set forth in any one of: 16, 134, 34, 36, 52 and 132.
In some embodiments, the light chain variable region comprises an amino acid sequence set forth in any one of: 54, 103, 18 and 109.
In certain embodiments, the antibody light chain or fragment thereof further comprises a human constant region or a murine constant region. In certain embodiments, the antibody light chain or fragment thereof comprises a human constant region, and the human constant region comprises a human Ig κ or Ig λ constant region. In certain embodiments, the antibody light chain or fragment thereof comprises an amino acid sequence set forth in any one of seq id nos: 64, 68, 72, 138, 84 and 136 SEQ ID NO. In certain embodiments, the antibody light chain or fragment thereof comprises an amino acid sequence set forth in any one of seq id nos: SEQ ID NO 90, SEQ ID NO 104, SEQ ID NO 78 and SEQ ID NO 110.
In certain embodiments, the antibody comprises an antibody heavy chain or fragment thereof. In certain embodiments, the antibody heavy chain or fragment thereof comprises HCDR1 and the HCDR1 comprises the amino acid sequence set forth in any one of seq id nos: 123, 19 and 37. In certain embodiments, the HCDR1 comprises an amino acid sequence as set forth in any one of seq id nos: 1, 91 and 92 SEQ ID NO. In certain embodiments, the antibody heavy chain or fragment thereof comprises HCDR2 and the HCDR2 comprises the amino acid sequence of any one of seq id no:124, 20 and 38. In certain embodiments, the HCDR2 comprises an amino acid sequence as set forth in any one of seq id nos: SEQ ID NO 2 and SEQ ID NO 93.
In certain embodiments, the antibody heavy chain or fragment thereof comprises an HCDR3 and the HCDR3 comprises an amino acid sequence as set forth in any one of seq id nos: 125, 21 and 130.
In certain embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of seq id nos: 3, 94, 95, 39, 100 and 101.
In certain embodiments, the antibody heavy chain or fragment thereof further comprises the framework regions H-FR1, H-FR2 and H-FR4. In certain embodiments, the framework regions are selected from any one of the following groups: human consensus framework sequences and human germline sequences. In certain embodiments, the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, the H-FR1 comprising an amino acid sequence set forth in any one of: 7, 25 and 43. In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR2 comprising an amino acid sequence set forth in any one of: SEQ ID NO 8, SEQ ID NO 26 and SEQ ID NO 44. In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, the H-FR3 comprising an amino acid sequence set forth in any one of: 129, 27 and 45. In certain embodiments, the H-FR3 comprises an amino acid sequence set forth in any one of seq id nos: SEQ ID NO 9 and SEQ ID NO 99. In certain embodiments, the N-terminus of the H-FR4 is linked, directly or indirectly, to the C-terminus of the HCDR3, the H-FR4 comprising an amino acid sequence set forth in any one of seq id no:10, 28 and 46.
In certain embodiments, the antibody heavy chain or fragment thereof comprises a heavy chain variable region VH comprising the amino acid sequence set forth in any one of: 15, 135, 33, 35, 51 and 133.
In certain embodiments, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in any one of: 53, 105, 107, 17, 111, 114, 117 and 120.
In certain embodiments, the antibody heavy chain or fragment thereof further comprises a human constant region or a murine constant region.
In certain embodiments, the antibody heavy chain or fragment thereof comprises a human constant region, and the human constant region comprises a human IgG constant region. In certain embodiments, the IgG constant region comprises a human IgG1 constant region.
In certain embodiments, the antibody heavy chain or fragment thereof comprises an amino acid sequence set forth in any one of seq id nos: 62, 66, 70, 139, 82 and 137.
In some embodiments, the heavy chain comprises the amino acid sequence set forth in any one of seq id nos: 88, 106, 108, 76, 112, 115, 118 and 121.
In certain embodiments, the IL7 protein is a human IL17 protein. In certain embodiments, the IL7 protein comprises an IL17A protein and/or an IL17F protein.
In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding an antibody or antigen-binding fragment thereof described herein.
In another aspect, the present application provides a vector comprising a nucleic acid molecule as described herein.
In another aspect, the present application provides a host cell comprising a nucleic acid molecule as described herein or a vector as described herein.
In another aspect, the present application provides a method of making the antibody or antigen-binding fragment thereof, comprising culturing the host cell described herein under conditions such that the antibody or antigen-binding fragment thereof described herein is expressed. In certain embodiments, it optionally comprises harvesting the antibody or antigen-binding fragment thereof.
In another aspect, the present application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein, a nucleic acid molecule described herein, a vector described herein, and/or a host cell described herein, and optionally a pharmaceutically acceptable adjuvant.
In another aspect, the application provides the use of the antibody or antigen-binding fragment thereof in the manufacture of a medicament for the prevention or treatment of a disease or disorder. In certain embodiments, the disease or disorder comprises: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, respiratory inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
In certain embodiments, it is used for the prevention or treatment of a disease or disorder. In certain embodiments, the disease or disorder comprises: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, airway inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
In another aspect, the present application provides a method of preventing or treating a disease comprising administering an antibody or antigen-binding fragment thereof described herein, a nucleic acid molecule described herein, a vector described herein, a host cell described herein, and/or a pharmaceutical composition described herein.
In certain embodiments, the disease or disorder comprises: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, respiratory inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
In another aspect, the present application provides a method of inhibiting binding of an IL17 protein and an IL17 ligand comprising administering an antibody or antigen-binding fragment thereof described herein, a nucleic acid molecule described herein, a vector described herein, a host cell described herein, and/or a pharmaceutical composition described herein.
In another aspect, the present application provides a method of inhibiting a biological activity of an IL17 protein comprising administering an antibody or antigen-binding fragment thereof described herein, a nucleic acid molecule described herein, a vector described herein, a host cell described herein, and/or a pharmaceutical composition described herein.
In another aspect, the present application provides a method of inhibiting chemokine expression comprising the antibody or antigen-binding fragment thereof described herein, the nucleic acid molecule described herein, the vector described herein, the host cell described herein, and/or the pharmaceutical composition described herein. In certain embodiments, the chemokine comprises CXCL1.
Other aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present disclosure have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as claimed in the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.
Drawings
The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:
FIGS. 1A-1C show amino acid sequence alignments of the heavy and light chain variable regions of the IL17 antibodies of the present application with related germline sequences.
FIGS. 2A-2C show that IL17 antibodies of the present application inhibit the binding of IL17A protein to the IL17 receptor.
FIG. 3 shows that IL17 antibodies of the present application inhibit IL17A protein from inducing HT-29 cells to produce the chemokine CXCL1.
FIG. 4 shows the binding activity of IL17 antibodies to IL17F protein in the present application.
FIG. 5 shows that IL17 antibodies of the present application inhibit the binding of IL17A protein to the IL17 protein receptor.
FIGS. 6A-6C show that IL17 antibodies of the present application inhibit IL17A protein from inducing CXCL1 production by HT-29 cells.
FIGS. 7A-7C show that IL17 antibodies of the present application inhibit IL17A protein from inducing IL-8 production in HS-27 cells.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.
In the present application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., fab', and (Fab) 2 Fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. The term "variable region" or "variable domain" refers to the amino-terminal domain of a heavy or light chain of an antibody. Of heavy and light chainsThe variable domains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain an antigen binding site. The VH and VL regions can be further distinguished as hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).
The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein. The basic four-chain antibody unit is a heterotetrameric glycoprotein, consisting of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 elementary heterotetramer units together with an additional polypeptide called the J chain and contain 10 antigen binding sites, while IgA antibodies comprise 2-5 elementary four chain units that can be polymerized to form multivalent assemblies in combination with the J chain. In the case of IgG, the four chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at the N-terminus, followed by three (for each alpha and gamma chain) or four (for the mu and epsilon isotypes) constant domains (CH). Human IgG has four subtypes: igG1, igG2, igG3, and IgG4.
In the present application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. <xnotran> , , (, Kohler Milstein, nature,256:495-97 (1975); hongo , hybridoma,14 (3): 253-260 (1995), harlow , antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 1988); hammerling , monoclonal Antibodies and T-Cell Hybridomas,563-681, (Elsevier, N.Y., 1981)), DNA (, US4, 816, 567), (, Clackson , nature,352:624-628 (1991); marks , J.Mol.Biol.,222:581-597 (1992); sidhu , J.Mol.Biol.338 (2): 299-310 (2004); lee , J.Mol.Biol.340 (5): 1073-1093 (2004); fellouse, proc.Natl.Acad.Sci.USA101 (34): 12467-12472 (2004); Lee J.Immunol.Methods284 (1-2): 119-132 (2004)), (, WO 1998/24893;WO 1996/34096;WO1996/33735;WO1991/10741;Jakobovits , proc.Natl.Acad.Sci.USA,90:2551 (1993); jakobovits , nature,362:255-258 (1993); bruggemann , year in Immunol,7:33 (1993); US5, 545, 807;US5, 545, 806;US5, 569, 825;US5, </xnotran> 625 126; US5, 633, 425 and US5, 661, 016,marks et al, bio/Technology,10:779-783 (1992); lonberg et al, nature,368:856-859 (1994); morrison, nature,368:812-813 (1994); fishwild et al, nature Biotechnol.,14:845-851 (1996); neuberger, nature Biotechnol,14:826 (1996); and Lonberg and huskzar, lntern. Rev Immunol,13:65-93 (1995)).
In the present application, the term "framework region" (FR) generally refers to those variable domain residues other than CDR residues.
In the present application, the term "human consensus framework" generally refers to the amino acid residues that are most frequently present in human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, a subtype of this sequence is that described in the sequence of the protein of Interest for immunization as indicated by Kabat et al (see Sequences of Proteins of Immunological Interest, 5 th edition, public health service, national institute of health, bethesda, md. (1991)). Examples include, for VL, this subtype may be the subtype kappa I, kappa II, kappa III or kappa IV as described in Kabat et al. In addition, the subtype for VH may be subtype I, subtype II or subtype III as described in Kabat et al. Alternatively, a human consensus framework can be obtained as described above, wherein particular residues, such as human framework residues, are selected based on their homology to the donor framework by aligning the donor framework to a collection of multiple human framework sequences. The acceptor human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence thereof, or it may comprise pre-existing amino acid sequence variations. In some embodiments, the number of pre-existing amino acid changes can be 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less (e.g., 1). In certain embodiments, the alteration of the amino acid sequence may comprise a substitution or deletion of a specified residue, or an insertion of at least one amino acid residue in the vicinity of a specified residue. An insertion "near" a specified residue means an insertion within one to two residues adjacent thereto. Insertions may be at the N-terminus or C-terminus of the designated residues. In the present application, the amino acid modification may be a substitution.
In the present application, the term "single chain antibody" (scFv) generally refers to a molecule in which the variable region of the heavy chain and the variable region of the light chain of an antibody are linked by a short peptide linker (linker).
In the present application, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to a corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while the remaining segments of the chain are homologous to corresponding sequences in another species. For example, the variable regions of both the light and heavy chains are derived from the variable region of an antibody from one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human). For example, to obtain a chimeric antibody, B cells or hybridoma cells of non-human origin can be used to produce the variable region, in combination with a constant region from a human. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region with which it is combined. Also, since the constant region of a chimeric antibody may be of human origin, chimerism may be less likely to elicit an immune response from the antibody upon injection than if the constant region were of non-human origin.
In the present application, the term "humanized antibody" generally refers to engineered antibodies derived from antibodies of non-human species (e.g., mouse or rat), immunoglobulin-binding proteins, and polypeptides, which have reduced immunogenicity to humans using genetic engineering techniques, while still retaining the antigen-binding properties of the original antibody. For example, CDR grafting (Jones et al, nature 321 (1986)); including "remodeling" (remodeling), (Verhoeyen, et al, 1988science 239 1534-1536, riechmann, et al, 1988Nature 332-337 Tempest, et al, bio/Technol 1991 9), "hyperaddition" (hyperpolymerization), (Queen, et al, 1989Proc Natl Acad Sci USA 86. Other regions, such as the hinge region and constant region domains, may also be humanized if they are also derived from non-human sources.
In this application, the term "fully human antibody" generally means that all of the antibody (including the constant region portion of the antibody, the CH and CL regions) are encoded by genes of human origin. The fully human antibody can greatly reduce the immune side reaction of the heterologous antibody to the human body.
In the present application, the term "antigen-binding fragment" generally refers to a portion of an intact antibody, e.g., the antigen-binding and/or variable region of an intact antibody. In certain embodiments, the antigen binding fragment may comprise a Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, and/or VhH. In certain embodiments, the antibodies described herein can include diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Digestion of an antibody with intact structure by papain (e.g., removal of the Fc region and hinge region) results in two identical antigen binding fragments, referred to as "Fab" fragments. The Fab fragment consists of the entire light chain, the variable region of the heavy chain (VH) and the first constant domain of the heavy chain (CH 1). Each Fab fragment is monovalent with respect to antigen binding, i.e. it has a single antigen binding site. F (ab) 2 Antibody fragments were originally produced as pairs of Fab fragments with a cysteine linkage between them. Digestion of antibodies with intact structure by pepsin results in a single large F (ab') 2 Fragments which roughly correspond to two Fab fragments with different antigen binding activity linked by a disulfide bond and which are still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. The Fv fragment consists of the VL and VH domains of a single arm of an antibody.
In the present application, the term "IL17 protein" generally refers to interleukin 17. To date, six IL17 family members have been discovered, IL17A, IL17B, IL17C, IL17D, IL17E (also named IL-25), and IL17F. IL17A is the prototype of the IL17 family, and IL17F has the highest homology (about 50%) with IL17A, and its coding gene maps to the same segment of the chromosome, 6p12. For example, genBank accession number for human IL17A may be NP _002181. For example, the IL17 protein may comprise the amino acid sequence shown in UniProtKB database under Accession No. q16552. As another example, the IL17F protein may comprise the amino acid sequence shown in NCBI database accession No. NP _ 443104.1. Members of the IL17 family function as homodimers or heterodimers. IL17A is a proinflammatory factor, and like IL-1Rs and TLRs receptors, IL17A also induces downstream genes by activating NF-. Kappa.B, MAPK. IL17 can mediate the phosphorylation cascade to inactivate C/EBPb. IL17 protein can activate Erk via the SEFIR-TILL domain of IL17 RA. IL17 proteins are associated with autoimmune diseases such as multiple/systemic sclerosis (MS), rheumatoid Arthritis (RA), systemic Lupus Erythematosus (SLE), inflammatory Bowel Disease (IBD), psoriasis, osteoarthritis, osteo-implant loosening, ankylosing spondylitis. IL17 proteins are also involved in host defense, particularly at the site of the mucosa of the lung, gut, oral cavity, etc. (e.g., stroke, asthma, acute transplant rejection, airway inflammation, inflammatory bowel disease, sepsis, septicemia, allergy, ischemia, endotoxic shock, etc.). IL-17 protein can induce proinflammatory factors such as IL-6, IL-1b, TNF alpha and the like which mediate defense responses. IL-17 protein also induces cytokines (G-CSF) and chemokines (CXCL 1, CXCL2, CXCL 5) that promote polymorphonuclear leukocyte proliferation and survival to regulate neutrophils. In addition, IL-17 protein also targets lymphocytes, dendritic cells and other immune cells to mucosal surfaces by inducing proteins that are chemotactic for these cells (CXCL 9, CXCL10, CCL 20); monocytes were recruited by inducing CCL2 and CCL 7.
In the present application, the term "IL17 receptor" (IL 17R) refers generally to a member of the IL17 receptor family, including IL17RA to IL17RE. For example, genBank accession number for human IL17R may be CAJ86450.1. All family members are single-pass transmembrane proteins with conserved structural motifs, including an extracellular fibronectin III-like domain and an intracellular SEF/IL17R (SEFIR) domain. Members of the IL17 receptor family can combine into distinct receptor complexes, with IL17RA being the largest molecule to date in this family, the signaling universal subunit of at least four ligands. Recent studies have shown that blocking the PI3K pathway can upregulate IL17RA, which is likely to enhance IL17 signaling.
In the present application, the term "cytokine" generally refers to a class of small molecules proteins with a wide range of biological activities that are synthesized and secreted by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. The cytokine can have various functions of regulating innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells, damaged tissue repair and the like. In the present application, the cytokines may include interleukins and chemokines. For example, the cytokine is a chemokine.
In this application, the term "CXCL" generally refers to a CXC chemokine family ligand. It includes CXCL1, CXCL2, CXCL3, CXCL12, CXCL14 and the like. Wherein CXCL1 (chemokine (C-X-C motif) ligand 1) is expressed by macrophages, neutrophils and epithelial cells, has neutrophil chemotactic activity, and CXCL1 plays a role in spinal cord development by inhibiting the migration of oligodendrocyte precursors and is involved in angiogenesis, arteriogenesis, inflammation, wound healing and tumorigenic processes.
In this application, the term "K D "used interchangeably with" KD "generally refers to the dissociation equilibrium constant, in M (mol/L), of a particular antibody-antigen interaction. K D The concentration of substance AB and substance a and substance B resulting from dissociation thereof can be calculated as: k is D = c (a) × c (B)/c (AB). From this equation, K D The larger the value, the more dissociation indicates the weaker the affinity between the substances A, B; otherwise, K D Smaller values indicate less dissociation and stronger affinity between the substances A and B.
In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, isolated from their natural environment, or artificially synthesized.
In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector for primarily inserting DNA or RNA into a cell, a vector for primarily replicating DNA or RNA, and a vector for primarily expressing transcription and/or translation of DNA or RNA. The vector also includes vectors having a plurality of the above-described functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell containing the vector.
In the present application, the term "host cell" generally refers to an individual cell, cell line or cell culture that may or may already contain a plasmid or vector comprising a nucleic acid molecule as described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof as described herein. The host cell may comprise progeny of a single host cell. Due to natural, accidental, or deliberate mutation, the progeny cells may not be identical in morphology or in genome to the original parent cell, but are capable of expressing the antibody or antigen-binding fragment thereof described herein. The host cell may be obtained by transfecting cells in vitro with the vector described herein. The host cell may be a prokaryotic cell (e.g., E.coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell). In some embodiments, the host cell is a mammalian cell. For example, the mammalian cell can be a CHO-K1 cell. In the present application, the term "recombinant host cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific cell but also a progeny of such a cell.
In the present application, the term "tumor" generally refers to a neoplasm formed by local tissue cell proliferation in a body (e.g., cells or components thereof) of a mammal under the action of various tumorigenic factors. In the present application, the tumor may comprise a rectal adenocarcinoma.
In this application, the term "between" \8230; \8230 ";" generally means that the C-terminus of a certain amino acid fragment is directly or indirectly linked to the N-terminus of a first amino acid fragment and the N-terminus thereof is directly or indirectly linked to the C-terminus of a second amino acid fragment. In the light chain, for example, the N-terminus of the L-FR2 is linked directly or indirectly to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is linked directly or indirectly to the N-terminus of the LCDR 2. For another example, the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR3. In the heavy chain, for example, the N-terminus of the H-FR2 is linked directly or indirectly to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is linked directly or indirectly to the N-terminus of the HCDR 2. For another example, the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2, and the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR 3.
In the present application, when describing the substitution of amino acid residues in a sequence, the expression "XnY" generally denotes the substitution of residue X with residue Y at position n in the sequence. For example, the amino acid substitution "R175L" indicates that residue R at position 175 in the sequence is substituted with residue L.
In this application, the term "comprising" is used in a generic sense to mean including, summarizing, containing or encompassing. In some cases, the meaning of "is", "consisting of 8230 \8230;" 8230 ";" is also indicated.
In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.
Antibodies, antigen binding fragments thereof
Competitive binding to a reference antibody
In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can compete with a reference antibody for binding to the IL17 protein, wherein the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the light chain variable region of the reference antibody can comprise LCDR1, LCDR2, and LCDR3.
In the present application, the LCDR1 may comprise an amino acid sequence as set forth in any one of: 126, 22 and 40; the LCDR2 may comprise an amino acid sequence as set forth in any one of: 5, 23 and 41; and, the LCDR3 may comprise an amino acid sequence set forth in any one of seq id nos: 6, 24 and 131; and, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence shown in any one of: 123, 19 and 37; the HCDR2 may comprise an amino acid sequence as set forth in any one of: 124, 20 and 38; and, the HCDR3 may comprise an amino acid sequence as set forth in any one of: 125, 21 and 130.
For example, the LCDR1 of the reference antibody can comprise an amino acid sequence set forth in any one of seq id no: SEQ ID NO.4 and SEQ ID NO. 96.
For example, the LCDR3 of the reference antibody can comprise the amino acid sequence set forth in any one of seq id nos: 42 and 102 SEQ ID NO.
For example, the HCDR1 of the reference antibody can comprise the amino acid sequence set forth in any one of seq id nos: 1, 91 and 92 SEQ ID NO.
For example, the HCDR2 of the reference antibody can comprise the amino acid sequence set forth in any one of seq id nos: SEQ ID NO 2 and SEQ ID NO 93.
For example, the HCDR3 of the reference antibody can comprise the amino acid sequence set forth in any one of seq id nos: 3, 94, 95, 39, 100 and 101.
For example, the variable region of the light chain of the reference antibody may comprise an amino acid sequence set forth in any one of: 16, 134, 34, 36, 52 and 132; and, the heavy chain variable region of the reference antibody may comprise an amino acid sequence set forth in any one of: 15, 135, 33, 35, 51 and 133.
For example, the light chain variable region of the reference antibody may comprise an amino acid sequence set forth in any one of seq id nos: 54, 103, 18 and 109.
For example, the heavy chain variable region of the reference antibody may comprise an amino acid sequence set forth in any one of seq id nos: 53, 105, 107, 17, 111, 114, 117 and 120.
For example, the light chain of the reference antibody may comprise an amino acid sequence set forth in any one of: 64, 68, 72, 138, 84 and 136; and, the heavy chain of the reference antibody may comprise an amino acid sequence set forth in any one of: 62, 66, 70, 139, 82 and 137.
For example, the light chain of the reference antibody can comprise an amino acid sequence set forth in any one of: SEQ ID NO 90, SEQ ID NO 104, SEQ ID NO 78 and SEQ ID NO 110.
For example, the heavy chain of the reference antibody may comprise an amino acid sequence set forth in any one of: 88, 106, 108, 76, 112, 115, 118 and 121.
For example, an antibody, antigen-binding fragment thereof described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 can comprise SEQ ID No. 4; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; the amino acid sequence of the HCDR1 can comprise SEQ ID NO 1; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 3. For example, the reference antibody can include antibody YN-008 or an antibody having the same LCDR1-3 and HCDR1-3. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region can include SEQ ID NO 16; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 15. For example, the reference antibody can comprise antibody YN-008 or an antibody having the same light chain variable region and heavy chain variable region as the reference antibody. For example, the reference antibody may comprise a light chain and a heavy chain, the amino acid sequence of the light chain may be as set forth in SEQ ID NO.64 and the amino acid sequence of the heavy chain may be as set forth in SEQ ID NO. 62. For example, the reference antibody can comprise antibody YN-008 or have the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof, described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 can comprise SEQ ID NO 22; the amino acid sequence of LCDR2 can comprise SEQ ID NO 23, the amino acid sequence of LCDR3 can comprise SEQ ID NO 24; the amino acid sequence of HCDR1 can include SEQ ID NO 19; the amino acid sequence of HCDR2 can include SEQ ID NO 20; the amino acid sequence of HCDR3 can include SEQ ID NO 21. For example, the reference antibody can include antibody YN-009 or an antibody having the same LCDR1-3 and HCDR1-3 as it does. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the amino acid sequence of the light chain variable region can comprise SEQ ID NO:34; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 33. For example, the reference antibody can comprise antibody YN-009 or an antibody having the same light chain variable region and heavy chain variable region as it. For example, the reference antibody may comprise a light chain and a heavy chain, the amino acid sequence of the light chain may be as set forth in SEQ ID NO 68 and the amino acid sequence of the heavy chain may be as set forth in SEQ ID NO 66. For example, the reference antibody can include antibody YN-009 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof, described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the LCDR1 amino acid sequence thereof can comprise SEQ ID NO 40; the amino acid sequence of LCDR2 can include SEQ ID NO 41; the amino acid sequence of LCDR3 can include SEQ ID NO 42; the amino acid sequence of HCDR1 can include SEQ ID NO 37; the amino acid sequence of HCDR2 can include SEQ ID NO 38; the amino acid sequence of HCDR3 can include SEQ ID NO:39. For example, the reference antibody can include antibody YN-010 or an antibody having the same LCDR1-3 and HCDR1-3. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the amino acid sequence of the light chain variable region can comprise SEQ ID NO 52; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 51. For example, the reference antibody can comprise antibody YN-010 or an antibody having the same light chain variable region and heavy chain variable region as it. For example, the reference antibody may comprise a light chain and a heavy chain, the amino acid sequence of the light chain may be as set forth in SEQ ID NO.72 and the amino acid sequence of the heavy chain may be as set forth in SEQ ID NO. 70. For example, the reference antibody can comprise antibody YN-010 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof, described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 can comprise SEQ ID No. 4; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; the amino acid sequence of the HCDR1 can comprise SEQ ID NO 1; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 3. For example, the reference antibody can include antibody YN-011 or an antibody having the same LCDR1-3 and HCDR1-3 as it does. For example, the reference antibody comprises L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 can include SEQ ID NO 11; the amino acid sequence of L-FR2 can include SEQ ID NO 12; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 9; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the antibody or antigen-binding fragment thereof can comprise antibody YN-011 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4 as it does. For example, the reference antibody can comprise a light chain variable region whose amino acid sequence can include SEQ ID NO:18; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 17. For example, the reference antibody can comprise antibody YN-011 or an antibody having the same light chain variable region and heavy chain variable region as it. For example, the reference antibody may comprise a light chain and a heavy chain, the amino acid sequence of the light chain may be as set forth in SEQ ID NO:78 and the amino acid sequence of the heavy chain may be as set forth in SEQ ID NO: 76. For example, the reference antibody can comprise antibody YN-011 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 can comprise SEQ ID NO 22; the amino acid sequence of LCDR2 can include SEQ ID NO 23; the amino acid sequence of LCDR3 can include SEQ ID NO 24; the amino acid sequence of HCDR1 can include SEQ ID NO 19; the amino acid sequence of HCDR2 can include SEQ ID NO 20; the amino acid sequence of HCDR3 can include SEQ ID NO 21. For example, the reference antibody can include antibody YN-012 or an antibody having the same LCDR1-3 and HCDR1-3 as it does. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the amino acid sequence of the light chain variable region can include SEQ ID NO:36; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 35. For example, the reference antibody can comprise antibody YN-012 or an antibody having the same light chain variable region and heavy chain variable region as it does. For example, the reference antibody can comprise a light chain and a heavy chain, the amino acid sequence of the light chain can be as set forth in SEQ ID NO.84 and the amino acid sequence of the heavy chain can be as set forth in SEQ ID NO. 82. For example, the reference antibody can include antibody YN-012 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof, described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the LCDR1 amino acid sequence thereof can comprise SEQ ID NO 40; the amino acid sequence of LCDR2 can include SEQ ID NO 41; the amino acid sequence of LCDR3 can include SEQ ID NO 42; the amino acid sequence of HCDR1 can include SEQ ID NO 37; the amino acid sequence of HCDR2 can include SEQ ID NO 38; the amino acid sequence of HCDR3 can include SEQ ID NO:39. For example, the reference antibody can include antibody YN-013 or an antibody having the same LCDR1-3 and HCDR1-3. For example, the reference antibody can comprise L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 can comprise SEQ ID NO 47; the amino acid sequence of L-FR2 can include SEQ ID NO 48; the amino acid sequence of L-FR3 can include SEQ ID NO 49; the amino acid sequence of L-FR4 may include SEQ ID NO 50; and the amino acid sequence of H-FR1 may include SEQ ID NO 43; the amino acid sequence of H-FR2 can include SEQ ID NO 44; the amino acid sequence of H-FR3 can include SEQ ID NO 45; the amino acid sequence of H-FR4 can include SEQ ID NO 46. For example, the reference antibody can comprise antibody YN-013 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the amino acid sequence of the light chain variable region can include SEQ ID NO:54; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 53. For example, the reference antibody can comprise antibody YN-013 or an antibody having the same light chain variable region and heavy chain variable region as the reference antibody. For example, the reference antibody may comprise a light chain and a heavy chain, the amino acid sequence of the light chain may be as set forth in SEQ ID NO.90 and the amino acid sequence of the heavy chain may be as set forth in SEQ ID NO. 88. For example, the reference antibody can comprise antibody YN-013 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof, described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 can comprise SEQ ID NO:40; the amino acid sequence of LCDR2 can include SEQ ID NO 41; the amino acid sequence of LCDR3 can include SEQ ID NO 102; the amino acid sequence of HCDR1 can include SEQ ID NO 37; the amino acid sequence of HCDR2 can include SEQ ID NO 38; the amino acid sequence of HCDR3 can include SEQ ID NO 100. For example, the reference antibody can include antibody YN-065 or an antibody having LCDR1-3 and HCDR1-3 identical thereto. For example, the reference antibody can comprise L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 can comprise SEQ ID NO 47; the amino acid sequence of L-FR2 can include SEQ ID NO 48; the amino acid sequence of L-FR3 can include SEQ ID NO 49; the amino acid sequence of L-FR4 may include SEQ ID NO 50; and the amino acid sequence of H-FR1 may comprise SEQ ID NO 43; the amino acid sequence of H-FR2 can include SEQ ID NO 44; the amino acid sequence of H-FR3 can include SEQ ID NO 45; the amino acid sequence of H-FR4 can include SEQ ID NO 46. For example, the reference antibody can include antibody YN-065 or an antibody having LCDR1-3, HCDR1-3, L-FR1-4, and H-FR1-4 identical thereto. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region can comprise SEQ ID NO 103; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 105. For example, the reference antibody can comprise antibody YN-065 or an antibody having the same light chain variable region and heavy chain variable region. For example, the reference antibody can comprise a light chain and a heavy chain, the amino acid sequence of the light chain can be as set forth in SEQ ID NO 104 and the amino acid sequence of the heavy chain can be as set forth in SEQ ID NO 106. For example, the reference antibody can include antibody YN-065 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof, described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the LCDR1 amino acid sequence thereof can comprise SEQ ID NO 40; the amino acid sequence of LCDR2 can include SEQ ID NO 41; the amino acid sequence of LCDR3 can include SEQ ID NO 102; the amino acid sequence of HCDR1 can include SEQ ID NO 37; the amino acid sequence of HCDR2 can include SEQ ID NO 38; the amino acid sequence of HCDR3 can include SEQ ID NO 101. For example, the reference antibody can include antibody YN-066 or an antibody having the same LCDR1-3 and HCDR1-3 as it. For example, the reference antibody can comprise L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 can comprise SEQ ID NO 47; the amino acid sequence of L-FR2 can include SEQ ID NO 48; the amino acid sequence of L-FR3 can include SEQ ID NO 49; the amino acid sequence of L-FR4 may include SEQ ID NO 50; and the amino acid sequence of H-FR1 may include SEQ ID NO 43; the amino acid sequence of H-FR2 can include SEQ ID NO 44; the amino acid sequence of H-FR3 can include SEQ ID NO 45; the amino acid sequence of H-FR4 can include SEQ ID NO 46. For example, the reference antibody can include antibody YN-066 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4, and H-FR1-4. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region can comprise SEQ ID NO 103; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 107. For example, the reference antibody can comprise antibody YN-066 or an antibody having the same light chain variable region and heavy chain variable region. For example, the reference antibody can comprise a light chain and a heavy chain, the amino acid sequence of the light chain can be as set forth in SEQ ID NO.104 and the amino acid sequence of the heavy chain can be as set forth in SEQ ID NO. 108. For example, the reference antibody can include antibody YN-066 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 can comprise SEQ ID NO:96; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; the amino acid sequence of HCDR1 can include SEQ ID NO 91; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 94. For example, the reference antibody can include antibody YN-067 or an antibody having the same LCDR1-3 and HCDR1-3. For example, the reference antibody can comprise L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 can comprise SEQ ID NO 97; the amino acid sequence of L-FR2 can include SEQ ID NO 98; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 9; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the reference antibody can include antibody YN-067 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4, and H-FR1-4. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the amino acid sequence of the light chain variable region can comprise SEQ ID NO:109; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 111. For example, the reference antibody can comprise antibody YN-067 or an antibody having the same light chain variable region and heavy chain variable region. For example, the reference antibody may comprise a light chain and a heavy chain, the amino acid sequence of the light chain may be as set forth in SEQ ID NO.110 and the amino acid sequence of the heavy chain may be as set forth in SEQ ID NO. 112. For example, the reference antibody can include antibody YN-067 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 can comprise SEQ ID NO:96; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; the amino acid sequence of the HCDR1 can comprise SEQ ID NO 1; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 95. For example, the reference antibody can include antibody YN-068 or an antibody having the same LCDR1-3 and HCDR1-3. For example, the reference antibody can comprise L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 can include SEQ ID NO 97; the amino acid sequence of L-FR2 can include SEQ ID NO 98; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 9; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the reference antibody can include antibody YN-068 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4, and H-FR1-4. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region can comprise SEQ ID NO:109; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 114. For example, the reference antibody can comprise antibody YN-068 or an antibody having the same light chain variable region and heavy chain variable region. For example, the reference antibody can comprise a light chain and a heavy chain, the amino acid sequence of the light chain can be as set forth in SEQ ID NO.110 and the amino acid sequence of the heavy chain can be as set forth in SEQ ID NO. 115. For example, the reference antibody can include antibody YN-068 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof, described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 thereof can include SEQ ID NO:96; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; the amino acid sequence of HCDR1 can include SEQ ID NO 92; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 94. For example, the reference antibody can include antibody YN-069 or an antibody having the same LCDR1-3 and HCDR1-3. For example, the reference antibody can comprise L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 can comprise SEQ ID NO 97; the amino acid sequence of L-FR2 can include SEQ ID NO 98; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 9; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the reference antibody can include antibody YN-069 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4, and H-FR1-4. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the amino acid sequence of the light chain variable region can comprise SEQ ID NO:109; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 117. For example, the reference antibody can comprise antibody YN-069 or an antibody having the same light chain variable region and heavy chain variable region as it. For example, the reference antibody may comprise a light chain and a heavy chain, the amino acid sequence of the light chain may be as set forth in SEQ ID NO.110 and the amino acid sequence of the heavy chain may be as set forth in SEQ ID NO. 118. For example, the reference antibody can include antibody YN-069 or an antibody having the same light and heavy chains as it.
For example, an antibody, antigen-binding fragment thereof described herein can compete with a reference antibody for binding to an IL17 protein (e.g., a human IL17 protein). The reference antibody can comprise LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 thereof can include SEQ ID NO:96; the amino acid sequence of LCDR2 can include SEQ ID No. 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; the amino acid sequence of HCDR1 can include SEQ ID NO 91; the amino acid sequence of HCDR2 can comprise SEQ ID NO 93; the amino acid sequence of HCDR3 can include SEQ ID NO 94. For example, the reference antibody can include antibody YN-070 or an antibody having the same LCDR1-3 and HCDR1-3 as it does. For example, the reference antibody can comprise L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 can comprise SEQ ID NO 97; the amino acid sequence of L-FR2 can include SEQ ID NO 98; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 99; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the reference antibody can include antibody YN-070 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4, and H-FR1-4 as it does. For example, the reference antibody can comprise a light chain variable region and a heavy chain variable region, and the amino acid sequence of the light chain variable region can comprise SEQ ID NO:109; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 120. For example, the reference antibody can comprise antibody YN-069 or an antibody having the same light chain variable region and heavy chain variable region as it. For example, the reference antibody may comprise a light chain and a heavy chain, the amino acid sequence of the light chain may be as set forth in SEQ ID NO.110 and the amino acid sequence of the heavy chain may be as set forth in SEQ ID NO. 121. For example, the reference antibody can comprise antibody YN-070, or an antibody having the same light and heavy chains as it.
Antibodies, antigen binding fragments thereof, as described herein
In one aspect, the present application provides an antibody, antigen-binding fragment thereof, which is expressed as 6 x10 -9 M or less (e.g., the K D May be no higher than about 6X 10 -9 M, not higher than about 1X 10 -9 M, not higher than about 9X 10 -10 M, not higher than about 8X 10 -10 M, not higher than about 7X 10 -10 M, not higher than about 6X 10 -10 M, not higher than about 5X 10 -10 M, not higher than about 410 -10 M, not higher than about 3X 10 -10 M, not higher than about 2X 10 -10 M, not higher than about 1X 10 -10 M is not higher than about 1X 10 -11 M or below) of D Values bind to IL17 protein.
In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can compete with IL17 ligand for binding to IL17 protein. In certain instances, the antibodies, antigen-binding fragments thereof, described herein can inhibit the expression of cytokines. For example, the cytokine may be selected from the group consisting of: interleukins and chemokines. For example, the chemokine can comprise CXCL1.
The antibodies, antigen-binding fragments thereof, described herein can ameliorate or treat a disease or disorder associated with inappropriate or excessive production of IL 17. For example, the disease or disorder may be selected from the group consisting of: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, respiratory inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
The IL17 proteins described herein may include human IL17A proteins. For example, the IL17 protein may comprise an amino acid sequence as shown in UniProtKB database under Accession No. q16552.
In certain embodiments, an IL17 protein described herein can include an IL17F protein (e.g., a human IL17F protein). For example, the IL17F protein may comprise the amino acid sequence shown in NCBI database accession number NP _ 443104.1.
The antigen binding fragments described herein may be selected from the group consisting of: fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scfv and/or VhH.
The antibodies, antigen-binding fragments thereof, described herein can comprise an antibody light chain or fragment thereof. In certain embodiments, the antibody comprises: monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and/or fully human antibodies. For example, the antibody light chain or fragment thereof may comprise a human constant region, which may comprise a human Ig κ or Ig λ constant region.
The antibodies, antigen-binding fragments thereof, described herein can comprise an antibody light chain or fragment thereof, which can also include LCDR1-3.
In the present application, the antibody light chain or fragment thereof may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence set forth in any one of: 126, 22 and 40.
In the present application, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 126:
RSSQSLLHSNGYNYLX 16 (SEQ ID NO: 126); wherein, X 16 = D or E.
In certain embodiments, the LCDR1 comprises at least one amino acid substitution as compared to the LCDR1 of the antibody of SEQ ID No.4 selected from the group consisting of: at X 16 Amino acid substitution(s) of (b).
In certain embodiments, the antibody LCDR1 is at least contained at X, as compared to LCDR1 of the antibody of SEQ ID NO.4 16 Amino acid substitution of (b), wherein, X 16 The amino acid at (A) may be substituted with D or E.
For example, the LCDR1 may comprise the amino acid sequence shown below: SEQ ID NO.4 and SEQ ID NO. 96.
In the present application, the antibody light chain or fragment thereof may comprise LCDR2, and the LCDR2 may comprise an amino acid sequence selected from the group consisting of seq id no:5, 23 and 41.
In the present application, the antibody light chain or fragment thereof may comprise an LCDR3, said LCDR3 may comprise an amino acid sequence as set forth in any one of: 6, 24 and 131.
In the present application, the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 131:
QTWDX 5 X 6 TSKYV (SEQ ID NO: 131); wherein X 5 = H or Y; x 6 = G or W.
In certain embodiments, the LCDR3 is at least contained within the LCDR3 of the antibody of SEQ ID NO.42, as compared to the LCDR3 of the antibodyAn amino acid substitution selected from the group consisting of: at X 5 And/or X 6 Amino acid substitution(s) of (c).
In certain embodiments, the LCDR3 is comprised within at least the antibody comprised at X, as compared to the LCDR3 of the antibody represented by SEQ ID NO.42 5 And/or X 6 Amino acid substitution of (a), wherein, X 5 The amino acid at (A) may be substituted by H or Y, X 6 The amino acid at (A) may be substituted with G or W.
For example, the LCDR3 may comprise the amino acid sequence shown below: 42 and 102 SEQ ID NO.
In the antibodies, antigen-binding fragments thereof, described herein, the antibody light chain or fragment thereof may further comprise the framework region L-FR1-4. In the present application, the framework region L-FR1-4 may be selected from the group consisting of: human consensus framework sequences and human germline sequences.
In the present application, the C-terminus of the L-FR1 may be linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 comprises an amino acid sequence represented by any one of: 127, 29 and 47.
In the present application, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 127:
DIVMTQSPLSLX 12 VTPGEPASISC (SEQ ID NO: 127); wherein, X 12 = P or H.
In certain embodiments, the L-FR1 comprises at least the amino acid substitution selected from the group consisting of: at X 12 Amino acid substitution(s) of (c).
In certain embodiments, the L-FR1 is at least contained at X as compared to the L-FR1 of the antibody represented by SEQ ID NO.11 12 Amino acid substitution of (a), wherein, X 12 The amino acid at (A) may be substituted with P or H.
For example, the L-FR1 may comprise an amino acid sequence represented by any one of: SEQ ID NO 11 and SEQ ID NO 97.
In the present application, the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may comprise an amino acid sequence represented by any one of: 128, 30 and 48.
In the present application, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 128:
WYLQKPGQSPQX 12 LIY (SEQ ID NO: 128); wherein X 12 = L or P.
In certain embodiments, the L-FR2 comprises at least the amino acid substitution selected from the group consisting of: at X 12 Amino acid substitution(s) of (b).
In certain embodiments, the L-FR2 is at least contained at X as compared to the L-FR2 of the antibody represented by SEQ ID NO.12 12 Amino acid substitution of (a), wherein, X 12 The amino acid at (A) may be substituted with L or P.
For example, the L-FR2 may comprise an amino acid sequence set forth in any one of: SEQ ID NO 12 and SEQ ID NO 98.
In the present application, the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 may comprise an amino acid sequence represented by any one of: 13, 31 and 49.
In the present application, the N-terminus of the L-FR4 may be linked directly or indirectly to the C-terminus of the LCDR3, and the L-FR4 may comprise an amino acid sequence selected from the group consisting of: 14, 32 and 50.
In the present application, the antibody light chain or the fragment thereof in the antibody, the antigen binding fragment thereof may comprise a light chain variable region VL, and the light chain variable region VL may comprise an amino acid sequence represented by any one of the following: 16, 134, 34, 36, 52 and 132.
In certain embodiments, the light chain variable region may comprise an amino acid sequence set forth in any one of: 54, 103, 18 and 109.
In the present application, the antibody light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 132:
SYELTQPPSVSVSPGQTASITCSGDDLGSKYASWYQQKPGQSPVLVIYQDDQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQTWDX 92 X 93 TSKYVFGPGTKLTVLG (SEQ ID NO: 132); wherein X 92 = H or Y; x 93 = G or W.
In some embodiments, the antibody light chain variable region or fragment thereof comprises at least an amino acid substitution selected from any one of the following: at X 92 And/or X 93 Amino acid substitution(s) of (c).
In some embodiments, the antibody light chain variable region or fragment thereof is comprised at least at X as compared to the light chain variable region of the antibody of SEQ ID NO:54 92 And/or X 93 Amino acid substitution of (a), wherein, X 92 The amino acid at (A) may be substituted with H or Y; x 93 The amino acid at (A) may be substituted with G or W.
For example, the antibody light chain variable region may comprise an amino acid sequence set forth in any one of: SEQ ID NO:54 and SEQ ID NO:103.
In the present application, the antibody light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 134:
DIVMTQSPLSLX 12 VTPGEPASISCRSSQSLLHSNGYNYLX 39 WYLQKPGQSPQX 51 LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPTFGGGTKLEIKR；
wherein X 12 = P or H; x 39 = D or E; x 51 = L or P.
In some embodiments, the antibody light chain variable region or fragment thereof may comprise at least amino acid substitutions selected from any of the following groups, as compared to the light chain variable region of the antibody set forth in SEQ ID NO: 18: at X 12 、X 39 And/or X 51 Amino acid substitution(s) of (c).
In some embodiments, the antibody light chain or fragment thereof can be comprised at least at X as compared to the light chain variable region of the antibody of SEQ ID NO.18 12 、X 39 And/or X 51 Amino acid substitution of (a), wherein, X 12 The amino acid at (b) may be substituted with P or H; x 39 The amino acid at (b) may be substituted with D or E; x 51 The amino acid at (A) may be substituted by L orP。
For example, the antibody light chain variable region may comprise an amino acid sequence set forth in any one of: 18 and 109.
In the present application, the antibody light chain or fragment thereof may further comprise a human constant region or a murine constant region. In the present application, the human constant region may comprise a human Ig κ or Ig λ constant region.
In the present application, the antibody light chain or fragment thereof may comprise an amino acid sequence set forth in any one of: 64, 68, 72, 138, 84 and 136 SEQ ID NO. In certain embodiments, the antibody light chain or fragment thereof may comprise an amino acid sequence set forth in any one of seq id nos: 90, 104, 78 and 110. In certain embodiments, the antibody light chain binding fragment (e.g., the antigen binding fragment can be a Fab) can comprise an amino acid sequence selected from the group consisting of: 110 in SEQ ID NO.
In the present application, the antibody light chain or fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 136:
SYELTQPPSVSVSPGQTASITCSGDDLGSKYASWYQQKPGQSPVLVIYQDDQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQTWDX 92 X 93 TSKYVFGPGTKLTVLGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:136)。
wherein X 92 = H or Y; x 93 = G or W.
In some embodiments, the antibody light chain or fragment thereof comprises at least amino acid substitutions in any one of the groups selected from: at X 92 And/or X 93 Substitution of the amino acid at (a).
In some embodiments, the antibody light chain or fragment thereof is comprised at least at X as compared to the light chain of the antibody represented by SEQ ID NO.90 92 And/or X 93 Amino acid substitution of (a), wherein, X 92 The amino acid at (a) may be substituted with H or Y; x 93 The amino acid at (A) may be substituted with G or W.
For example, the antibody light chain or fragment thereof may comprise an amino acid sequence set forth in any one of seq id nos: 90 and 104.
In the present application, the antibody light chain or fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 138:
DIVMTQSPLSLX 12 VTPGEPASISCRSSQSLLHSNGYNYLX 39 WYLQKPGQSPQX 51 LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:138)。
wherein X 12 = P or H; x 39 = D or E; x 51 = L or P.
In some embodiments, the antibody light chain or fragment thereof comprises at least an amino acid substitution selected from any one of the following groups: at X 12 、X 39 And/or X 51 Substitution of the amino acid at (a).
In some embodiments, the antibody light chain or fragment thereof is at least as contained at X as compared to the light chain of the antibody represented by SEQ ID NO.78 12 、X 39 And/or X 51 Amino acid substitution of (a), wherein, X 12 The amino acid at (b) may be substituted with P or H; x 39 The amino acid at (b) may be substituted with D or E; x 51 The amino acid at (A) may be substituted with L or P.
For example, the antibody light chain or fragment thereof may comprise an amino acid sequence set forth in any one of seq id no:78 and 110.
In the present application, the antibodies, antigen-binding fragments thereof, described herein can comprise an antibody heavy chain or fragment thereof, which can include HCDR1-3.
In the present application, the antibody heavy chain or fragment thereof may comprise HCDR1, said HCDR1 comprising the amino acid sequence set forth in any one of: 123, 19 and 37. In certain embodiments, the HCDR1 comprises an amino acid sequence set forth in any one of seq id nos: 1, 91 and 92 SEQ ID NO.
In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 123:
DYAX 4 X 5 (SEQ ID NO: 123); wherein, X 4 = M or I; x 5 And = S or N.
In some embodiments, the HCDR1 comprises at least one amino acid substitution as compared to the HCDR1 of the antibody of SEQ ID No.1 selected from the group consisting of:
(1) At X 5 Substitution of the amino acid at (a);
(2) At X 4 And/or X 5 Substitution of the amino acid at (a).
In some embodiments, the HCDR1 is at least contained at X as compared to HCDR1 of an antibody represented by SEQ ID NO.1 5 Amino acid substitution of (a), wherein, X 92 The amino acid at (A) may be substituted with M or I.
In some embodiments, the HCDR1 is at least contained at X as compared to HCDR1 of an antibody represented by SEQ ID NO.1 4 And/or X 5 Amino acid substitution of (a), wherein, X 92 The amino acid at (A) may be substituted by M or I, X 5 The amino acid at (A) may be substituted with S or N.
For example, the HCDR1 may comprise an amino acid sequence as set forth in any one of: 1, 91 and 92 SEQ ID NO.
In the present application, the antibody heavy chain or fragment thereof may comprise an HCDR2, the HCDR2 comprising an amino acid sequence as set forth in any one of seq id no:124, 20 and 38. In certain embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of seq id nos: SEQ ID NO.2 and SEQ ID NO. 93.
In the present application, the antibody heavy chain or fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 124:
FIRSKAYGGTTEYAX 15 SVKG (SEQ ID NO: 124); wherein, X 15 And (= T or A).
In some embodiments, the HCDR2 comprises at least one amino acid substitution as compared to the HCDR2 of an antibody as set forth in SEQ ID No.2 selected from the group consisting of: at X 15 Substitution of the amino acid at (a).
In some embodiments, the HCDR2 is at least contained at X as compared to the HCDR2 of the antibody of SEQ ID NO.2 15 Amino acid substitution of (a), wherein, X 15 The amino acid at (A) may be substituted with T or A.
For example, the HCDR2 may comprise an amino acid sequence as set forth in any one of: SEQ ID NO 2 and SEQ ID NO 93.
In the present application, the antibody heavy chain or fragment thereof comprises HCDR3 and the HCDR3 may comprise the amino acid sequence set forth in any one of: 125, 21 and 130. In the present application, the HCDR3 may comprise an amino acid sequence as set forth in any one of: 3, 94, 95, 39, 100 and 101.
In the present application, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 125:
DGSDX 5 IRYX 9 DWLFNYFWDL (SEQ ID NO: 125); wherein, X 5 = G or E, X 9 = F or L.
In some embodiments, the HCDR3 can comprise at least one amino acid substitution as compared to the HCDR3 of an antibody as set forth in SEQ ID No.3 selected from the group consisting of:
(1) At X 5 Substitution of the amino acid at (a);
(2) At X 5 And/or X 9 Substitution of the amino acid at (a).
In some embodiments, the HCDR3 can be comprised at least at X as compared to HCDR3 of an antibody represented by SEQ ID NO.3 5 Amino acid substitution of (a), wherein, X 5 The amino acid at (A) may be substituted with G or E.
In some embodiments, the HCDR3 can be comprised at least at X as compared to HCDR3 of an antibody represented by SEQ ID NO.3 5 And/or X 9 Amino acid substitution of (a), wherein, X 5 The amino acid at (A) may be substituted by G or E, X 9 The amino acid at (A) may be substituted with F or L.
For example, the HCDR3 may comprise the amino acid sequence set forth in any one of seq id nos: 3, 94 and 95.
In the present application, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 130:
GX 2 X 3 SX 5 X 6 X 7 FDY(SEQ ID NO:130)。
wherein X 2 = R or G; x 3 = Y or N; x 5 = G or D; x 6 = W or F; x 7 = Y or N.
In some embodiments, the HCDR3 can comprise at least one amino acid substitution as compared to the heavy chain of the antibody of SEQ ID No.39 selected from the group consisting of:
(1) At X 2 、X 3 、X 5 And/or X 7 Substitution of the amino acid at (a);
(2) At X 3 、X 6 And/or X 7 Substitution of the amino acid at (a).
In some embodiments, the HCDR3 can be comprised at least at X as compared to the HCDR3 of the antibody of SEQ ID NO:39 5 Amino acid substitution of (a), wherein, X 2 The amino acid at (A) may be substituted by R or G, X 3 The amino acid at (A) may be substituted by Y or N, X 5 The amino acid at (A) may be substituted by G or D, X 7 The amino acid at (A) may be substituted with Y or N.
In some embodiments, the antibody HCDR3 may be comprised at least at X as compared to HCDR3 of the antibody of SEQ ID NO:39 3 、X 6 And/or X 7 Amino acid substitution of (a), wherein, X 3 The amino acid at (A) may be substituted by Y or N, X 6 The amino acid at (A) may be substituted by W or F, X 7 The amino acid at (A) may be substituted with Y or N.
For example, the HCDR3 may comprise the amino acid sequence set forth in any one of seq id nos: 39, 100 and 101, respectively.
The antibody heavy chain or fragment thereof of the antibodies, antigen binding fragments thereof described herein may also include the framework region H-FR1-4. In the present application, the framework regions may be selected from the group consisting of: human consensus framework sequences and human germline sequences.
In certain embodiments, the C-terminus of the H-FR1 can be linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 can comprise an amino acid sequence selected from the group consisting of: SEQ ID NO 7, SEQ ID NO 25 and SEQ ID NO 43.
In certain embodiments, the H-FR2 can be located between the HCDR1 and the HCDR2, and the H-FR2 can comprise an amino acid sequence selected from the group consisting of: SEQ ID NO 8, SEQ ID NO 26 and SEQ ID NO 44.
In certain embodiments, the H-FR3 can be located between the HCDR2 and the HCDR3, and the H-FR3 can comprise an amino acid sequence set forth in any one of: 129, 27 and 45. For example, the H-FR3 may comprise an amino acid sequence set forth in any one of: SEQ ID NO 9 and SEQ ID NO 99.
In the present application, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 129:
RFTISRDDSKSIAYLQMNSLKTX 23 DTAVYYCAR (SEQ ID NO: 129); wherein X 23 = E or Q.
In some embodiments, the H-FR3 comprises at least the amino acid substitution selected from the group consisting of: at X 23 Substitution of the amino acid at (a).
In some embodiments, the H-FR3 is at least contained at X as compared to the H-FR3 of the antibody represented by SEQ ID NO.9 5 Amino acid substitution of (a), wherein, X 23 The amino acid at (A) may be substituted with E or Q.
For example, the H-FR3 may comprise an amino acid sequence set forth in any one of: 9 and 99 SEQ ID NO.
In certain embodiments, the N-terminus of the H-FR4 can be linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 can comprise an amino acid sequence selected from the group consisting of: 10, 28 and 46.
In the present application, the antibody heavy chain or fragment thereof may comprise a heavy chain variable region VH, and the heavy chain variable region VH may comprise an amino acid sequence set forth in any one of: 15, 135, 33, 35, 51 and 133. In certain embodiments, the heavy chain variable region VH may comprise an amino acid sequence set forth in any one of: 53, 105, 107, 17, 111, 114, 117 and 120.
In the present application, the antibody heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO. 133:
QVTLKESGGGLIQPGGSLRLSCAASGFTVSSNHMSWVRQAPGKGLEWVSIIYSDGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGX 99 X 100 SX 102 X 103 X 104 FDYWGQGTLVTVSS (SEQ ID NO: 133); wherein X 99 = R or G; x 100 = Y or N; x 102 = G or D; x 103 = W or F; x 104 = Y, N or S.
In some embodiments, the heavy chain variable region may comprise at least one amino acid substitution selected from at least one of:
(1) At X 99 ，X 100 ，X 102 And/or X 104 Substitution of the amino acid at (a);
(2) At X 100 ，X 103 And/or X 104 Substitution of the amino acid at (a).
In some embodiments, the heavy chain variable region may be comprised at least at X as compared to the heavy chain variable region of the antibody of SEQ ID NO:53 99 ，X 100 ，X 102 And/or X 104 Amino acid substitution of (b), wherein, X 99 The amino acid at (A) may be substituted by R or G, X 100 The amino acid at (A) may be substituted by Y or N, X 102 The amino acid at (A) may be substituted by G or D, X 103 The amino acid at (A) may be substituted by W or F, X 104 The amino acid at (A) may be substituted with Y or N.
In some embodiments, the heavy chain variable region may be comprised at least at X as compared to the heavy chain variable region of the antibody of SEQ ID NO:53 100 ，X 103 And/or X 104 Amino acid substitution of (b), wherein, X 100 The amino acid at (A) may be substituted by Y or N, X 103 The amino acid at (A) may be substituted by W or F, X 104 The amino acid at (A) may be substituted with Y or S.
For example, the antibody heavy chain variable region may comprise an amino acid sequence set forth in any one of: 53, 105 and 107 SEQ ID NO.
In the present application, the antibody heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 135:
QVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAX 34 X 35 WFRQAPGKGLEWVGFIRSKAYGGTTEYAX 54 SVKGRFTISRDDSKSIAYLQMNSLKTX 91 DTAVYYCARDGSDX 105 IRYX 109 DWLFNYWYFLDLWGRGTLVTVSS (SEQ ID NO: 135), wherein X34= M or I; x35= S or N; x54= a or T; x91= E or Q; x105= G or E; x109= F or L.
In some embodiments, the heavy chain variable region comprises at least one amino acid substitution selected from at least one of:
(1) At X 35 And/or X 105 Substitution of the amino acid at (a);
(2) At X 105 And/or X 109 Substitution of the amino acid at (a);
(3) At X 34 、X 35 And/or X 105 Substitution of the amino acid at (a);
(4) At X 35 、X 91 And/or X 105 Substitution of the amino acid at (a).
In some embodiments, the heavy chain variable region may be comprised at least at X as compared to the heavy chain variable region of the antibody of SEQ ID NO 17 35 And/or X 105 Amino acid substitution of (b), wherein, X 35 The amino acid at (A) may be substituted by S or N, X 105 The amino acid at (A) may be substituted with G or E.
In some embodiments, the heavy chain variable region may be comprised at least at X as compared to the heavy chain variable region of the antibody of SEQ ID NO 17 105 And/or X 109 Amino acid substitution of (a), wherein, X 105 Amino acids of (A) can be extractedSubstituted by G or E, X 109 The amino acid at (A) may be substituted with F or L.
In some embodiments, the heavy chain variable region may be comprised at least at X as compared to the heavy chain variable region of the antibody of SEQ ID NO 17 34 、X 35 And/or X 105 Amino acid substitution of (b), wherein, X 34 The amino acid at (A) may be substituted by M or I, X 35 The amino acid at (A) may be substituted by S or N, X 105 The amino acid at (A) may be substituted with G or E.
In some embodiments, the heavy chain variable region may be comprised at least at X as compared to the heavy chain variable region of the antibody of SEQ ID NO 17 34 、X 35 And/or X 105 Amino acid substitution of (a), wherein, X 35 The amino acid at (A) may be substituted by S or N, X 91 The amino acid at (A) may be substituted by E or Q, X 105 The amino acid at (A) may be substituted with G or E.
For example, the antibody heavy chain variable region may comprise an amino acid sequence set forth in any one of: SEQ ID NO 17, 111, 114, 117 and 120.
In the antibodies, antigen-binding fragments thereof, described herein, the antibody heavy chain or fragment thereof may further comprise a human constant region, and the human constant region may comprise a human IgG constant region. For example, the human IgG constant region can comprise a human IgG1 constant region.
In the present application, the antibody heavy chain or fragment thereof may comprise an amino acid sequence set forth in any one of: 62, 66, 70, 139, 82 and 137.
In certain embodiments, the heavy chain may comprise an amino acid sequence set forth in any one of: 88, 106, 108, 76, 112, 115, 118 and 121. In certain embodiments, the antigen-binding fragment of the antibody heavy chain (e.g., the antigen-binding fragment can be a Fab) can comprise an amino acid sequence selected from the group consisting of: 113, 116, 119 and 122.
In the present application, the antibody heavy chain or fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 137:
QVTLKESGGGLIQPGGSLRLSCAASGFTVSSNHMSWVRQAPGKGLEWVSIIYSDGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGX 99 X 100 SX 102 X 103 X 104 FDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:137)。
wherein X 99 = R or G; x 100 = Y or N; x 102 = G or D; x 103 = W or F; x 104 = Y, N or S. In some embodiments, the antibody heavy chain or fragment thereof may comprise at least the amino acid substitutions selected from at least one of:
(1) At X 99 ，X 100 ，X 102 And/or X 104 Substitution of the amino acid at (a);
(2) At X 100 ，X 103 And/or X 104 Substitution of the amino acid at (a).
In some embodiments, the heavy chain of the antibody or fragment thereof can be comprised at least at X as compared to the heavy chain of the antibody represented by SEQ ID NO:88 99 ，X 100 ，X 102 And/or X 104 Amino acid substitution of (b), wherein, X 99 The amino acid at (A) may be substituted by R or G, X 100 The amino acid at (A) may be substituted by Y or N, X 102 The amino acid at (A) may be substituted by G or D, X 103 The amino acid at (A) may be substituted by W or F, X 104 The amino acid at (A) may be substituted with Y or N.
In some embodiments, the heavy chain of the antibody or fragment thereof can be comprised at least at X as compared to the heavy chain of the antibody represented by SEQ ID NO:88 100 ，X 103 And/or X 104 Amino acid substitution of (a), whichIn, X 100 The amino acid at (A) may be substituted by Y or N, X 103 The amino acid at (A) may be substituted by W or F, X 104 The amino acid at (A) may be substituted with Y or S.
For example, the antibody heavy chain or fragment thereof may comprise an amino acid sequence set forth in any one of seq id nos: 88, 106 and 108.
In the present application, the antibody heavy chain or fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 139:
QVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAX 34 X 35 WFRQAPGKGLEWVGFIRSKAYGGTTEYAX 54 SVKGRFTISRDDSKSIAYLQMNSLKTX 91 DTAVYYCARDGSDX 105 IRYX 109 DWLFNYWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:139)。
wherein X 34 = M or I; x 35 = S or N; x 54 = A or T; x 91 = E or Q; x 105 = G or E; x 109 = F or L. In some embodiments, the antibody heavy chain or fragment thereof may comprise at least an amino acid substitution at least one selected from the group consisting of:
(1) At X 35 And/or X 105 Substitution of the amino acid at (a);
(2) At X 105 And/or X 109 Substitution of the amino acid at (a);
(3) At X 34 、X 35 And/or X 105 Substitution of the amino acid at (a);
(4) At X 35 、X 91 And/or X 105 Substitution of the amino acid at (a).
In some embodiments, the antibody heavy chain or fragment thereof may be comprised at least at X as compared to the heavy chain of the antibody of SEQ ID NO.76 35 And/or X 105 Amino acid substitution of (a), wherein, X 35 The amino acid at (A) may be substituted by S or N, X 105 The amino acid at (A) may be substituted with G or E.
In some embodiments, the heavy chain of the antibody or fragment thereof can be comprised at least at X as compared to the heavy chain of the antibody represented by SEQ ID NO:76 105 And/or X 109 Amino acid substitution of (b), wherein, X 105 The amino acid at (A) may be substituted by G or E, X 109 The amino acid at (A) may be substituted with F or L.
In some embodiments, the heavy chain of the antibody or fragment thereof can be comprised at least at X as compared to the heavy chain of the antibody represented by SEQ ID NO:76 34 、X 35 And/or X 105 Amino acid substitution of (a), wherein, X 34 The amino acid at (A) may be substituted by M or I, X 35 The amino acid at (A) may be substituted by S or N, X 105 The amino acid at (A) may be substituted with G or E.
In some embodiments, the heavy chain of the antibody or fragment thereof can be comprised at least at X as compared to the heavy chain of the antibody represented by SEQ ID NO:76 34 、X 35 And/or X 105 Amino acid substitution of (a), wherein, X 35 The amino acid at (A) may be substituted by S or N, X 91 The amino acid at (A) may be substituted by E or Q, X 105 The amino acid at (A) may be substituted with G or E.
For example, the antibody heavy chain or fragment thereof may comprise an amino acid sequence set forth in any one of seq id nos: 76, 112, 115, 118 and 121.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof described herein can comprise SEQ ID No. 4; the amino acid sequence of LCDR2 can include SEQ ID No. 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; and the amino acid sequence of HCDR1 can include SEQ ID NO 1; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 3. For example, the antibody, antigen-binding fragment thereof, can comprise antibody YN-008 or an antibody having the same LCDR1-3 and HCDR1-3. In certain embodiments, the amino acid sequence of the light chain variable region of an antibody, antigen-binding fragment thereof, described herein can comprise SEQ ID NO 16; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 15. For example, the antibody, antigen-binding fragment thereof, can comprise antibody YN-008, or an antibody having the same light chain variable region and heavy chain variable region as the antibody. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain and a heavy chain, the amino acid sequence of the light chain can be as set forth in SEQ ID NO:64 and the amino acid sequence of the heavy chain can be as set forth in SEQ ID NO: 62. For example, the antibody, antigen binding fragment thereof, can include antibody YN-008 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof, described herein can comprise SEQ ID No. 22; the amino acid sequence of LCDR2 can include SEQ ID NO 23; the amino acid sequence of LCDR3 can include SEQ ID NO 24; and the amino acid sequence of HCDR1 can comprise SEQ ID NO 19; the amino acid sequence of HCDR2 can include SEQ ID NO 20; the amino acid sequence of HCDR3 can include SEQ ID NO 21. For example, the antibody, antigen binding fragment thereof, can include antibody YN-009 or an antibody having the same LCDR1-3 and HCDR1-3 as it. In certain embodiments, the amino acid sequence of the light chain variable region of an antibody, antigen-binding fragment thereof, described herein can comprise SEQ ID No. 34; and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO 33. For example, the antibody, antigen binding fragment thereof, can comprise antibody YN-009 or an antibody having the same light chain variable region and heavy chain variable region thereof. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO.68 and a heavy chain having the amino acid sequence set forth in SEQ ID NO. 66. For example, the antibody, antigen binding fragment thereof, can include antibody YN-009 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof, described herein can comprise SEQ ID No. 40; the amino acid sequence of LCDR2 can include SEQ ID NO 41; the amino acid sequence of LCDR3 can include SEQ ID NO 42; and the amino acid sequence of HCDR1 can include SEQ ID NO 37; the amino acid sequence of HCDR2 can include SEQ ID NO 38; the amino acid sequence of HCDR3 can include SEQ ID NO:39. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-010 or an antibody having the same LCDR1-3 and HCDR1-3. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain variable region whose amino acid sequence can include SEQ ID NO 52; and it may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 51. For example, the antibody, antigen binding fragment thereof, can comprise antibody YN-010 or an antibody having the same light chain variable region and heavy chain variable region thereof. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO.72 and a heavy chain having the amino acid sequence set forth in SEQ ID NO. 70. For example, the antibody, antigen-binding fragment thereof can comprise antibody YN-010 or an antibody having the same light and heavy chains thereof.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof described herein can comprise SEQ ID No. 4; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; and the amino acid sequence of HCDR1 can comprise SEQ ID NO 1; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 3. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-011 or an antibody having the same LCDR1-3 and HCDR1-3. In some cases, the amino acid sequence of L-FR1 in the antibody, antigen-binding fragments thereof, can include SEQ ID NO 11; the amino acid sequence of L-FR2 can include SEQ ID NO 12; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 9; and the amino acid sequence of H-FR4 may include SEQ ID NO 10. For example, the antibody, antigen-binding fragment thereof, can comprise antibody YN-011 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4 as it does. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can include SEQ ID No. 18; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 17. For example, the antibody, antigen-binding fragment thereof, can comprise antibody YN-011 or an antibody having the same light chain variable region and heavy chain variable region. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO:78 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 76. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-011 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof, described herein can comprise SEQ ID No. 22; the amino acid sequence of LCDR2 can include SEQ ID NO 23; the amino acid sequence of LCDR3 can include SEQ ID NO 24; and the amino acid sequence of HCDR1 can include SEQ ID NO 19; the amino acid sequence of HCDR2 can include SEQ ID NO 20; the amino acid sequence of HCDR3 can include SEQ ID NO 21. For example, the antibody, antigen binding fragment thereof, can include antibody YN-012 or an antibody having the same LCDR1-3 and HCDR1-3. The amino acid sequence of L-FR1 in the antibodies, antigen-binding fragments thereof, described herein can include SEQ ID NO 29; the amino acid sequence of L-FR2 can include SEQ ID NO 30; the amino acid sequence of L-FR3 may include SEQ ID NO 31; the amino acid sequence of L-FR4 can include SEQ ID NO 32; and the amino acid sequence of H-FR1 may comprise SEQ ID NO 25; the amino acid sequence of H-FR2 can include SEQ ID NO 26; the amino acid sequence of H-FR3 can include SEQ ID NO 27; the amino acid sequence of H-FR4 can include SEQ ID NO 28. For example, the antibody, antigen-binding fragment thereof, may include antibody YN-012 or an antibody having LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4 identical thereto. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can include SEQ ID NO 36; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 35. For example, the antibody, antigen binding fragment thereof can include antibody YN-012 or an antibody having the same light chain variable region and heavy chain variable region. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO:84 and a heavy chain having the amino acid sequence set forth in SEQ ID NO: 82. For example, the antibody, antigen binding fragment thereof can include antibody YN-012 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof described herein can comprise SEQ ID No. 40; the amino acid sequence of LCDR2 can include SEQ ID NO 41; the amino acid sequence of LCDR3 can include SEQ ID NO 42; and the amino acid sequence of HCDR1 can comprise SEQ ID NO 37; the amino acid sequence of HCDR2 can include SEQ ID NO 38; the amino acid sequence of HCDR3 can include SEQ ID NO:39. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-013 or an antibody having the same LCDR1-3 and HCDR1-3. The amino acid sequence of L-FR1 in the antibodies, antigen-binding fragments thereof, described herein can include SEQ ID NO 47; the amino acid sequence of L-FR2 can include SEQ ID NO 48; the amino acid sequence of L-FR3 can include SEQ ID NO 49; the amino acid sequence of L-FR4 may include SEQ ID NO 50; and the amino acid sequence of H-FR1 may include SEQ ID NO 43; the amino acid sequence of H-FR2 can include SEQ ID NO 44; the amino acid sequence of H-FR3 can include SEQ ID NO 45; the amino acid sequence of H-FR4 can include SEQ ID NO 46. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-013 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4 as it was. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can include SEQ ID NO 54; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 53. For example, the antibody, antigen-binding fragment thereof, can comprise antibody YN-013 or an antibody having the same light chain variable region and heavy chain variable region. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO.90 and a heavy chain having the amino acid sequence set forth in SEQ ID NO. 88. For example, the antibody, antigen-binding fragment thereof can comprise antibody YN-013 or an antibody having the same light and heavy chains as the same.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof, described herein can comprise SEQ ID No. 40; the amino acid sequence of LCDR2 can include SEQ ID NO 41; the amino acid sequence of LCDR3 can include SEQ ID NO 102; and the amino acid sequence of HCDR1 can include SEQ ID NO 37; the amino acid sequence of HCDR2 can include SEQ ID NO 38; the amino acid sequence of HCDR3 can include SEQ ID NO 100. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-065 or an antibody having the same LCDR1-3 and HCDR1-3. The amino acid sequence of L-FR1 in the antibodies, antigen-binding fragments thereof, described herein can include SEQ ID NO 47; the amino acid sequence of L-FR2 can include SEQ ID NO 48; the amino acid sequence of L-FR3 can include SEQ ID NO 49; the amino acid sequence of L-FR4 can include SEQ ID NO 50; and the amino acid sequence of H-FR1 may include SEQ ID NO 43; the amino acid sequence of H-FR2 can include SEQ ID NO 44; the amino acid sequence of H-FR3 can include SEQ ID NO 45; the amino acid sequence of H-FR4 can include SEQ ID NO 46. For example, the antibody, antigen-binding fragment thereof can include antibody YN-065 or an antibody having LCDR1-3, HCDR1-3, L-FR1-4, and H-FR1-4 identical thereto. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can include SEQ ID NO 103; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 105. For example, the antibody, antigen binding fragment thereof, can include antibody YN-065 or an antibody having the same light chain variable region and heavy chain variable region. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO 104 and a heavy chain having the amino acid sequence set forth in SEQ ID NO 106. For example, the antibody, antigen binding fragment thereof can include antibody YN-065 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof, described herein can comprise SEQ ID No. 40; the amino acid sequence of LCDR2 can include SEQ ID NO 41; the amino acid sequence of LCDR3 can include SEQ ID NO 102; and the amino acid sequence of HCDR1 can comprise SEQ ID NO 37; the amino acid sequence of HCDR2 can include SEQ ID NO 38; the amino acid sequence of HCDR3 can include SEQ ID NO 101. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-066 or an antibody having the same LCDR1-3 and HCDR1-3 as it. The amino acid sequence of L-FR1 in the antibodies, antigen-binding fragments thereof, described herein can include SEQ ID NO 47; the amino acid sequence of L-FR2 can include SEQ ID NO 48; the amino acid sequence of L-FR3 can include SEQ ID NO 49; the amino acid sequence of L-FR4 may include SEQ ID NO 50; and the amino acid sequence of H-FR1 may comprise SEQ ID NO 43; the amino acid sequence of H-FR2 can include SEQ ID NO 44; the amino acid sequence of H-FR3 can include SEQ ID NO 45; the amino acid sequence of H-FR4 can include SEQ ID NO 46. For example, the antibody, antigen-binding fragment thereof can include antibody YN-066 or an antibody having LCDR1-3, HCDR1-3, L-FR1-4, and H-FR1-4 identical thereto. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can include SEQ ID NO 103; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 107. For example, the antibody, antigen-binding fragment thereof, can comprise antibody YN-066 or an antibody having the same light chain variable region and heavy chain variable region as it. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO.104 and a heavy chain having the amino acid sequence set forth in SEQ ID NO. 108. For example, the antibody, antigen binding fragment thereof can include antibody YN-066 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof described herein can comprise SEQ ID NO 96; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; and the amino acid sequence of HCDR1 can include SEQ ID NO 91; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 94. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-067 or an antibody having the same LCDR1-3 and HCDR1-3. The amino acid sequence of L-FR1 in the antibodies, antigen-binding fragments thereof, described herein can include SEQ ID NO 97; the amino acid sequence of L-FR2 can include SEQ ID NO 98; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 9; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the antibody, antigen-binding fragment thereof, may include antibody YN-067 or an antibody having LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4 identical thereto. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can comprise SEQ ID NO:109; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 111. For example, the antibody, antigen binding fragment thereof, can include antibody YN-067 or an antibody having the same light chain variable region and heavy chain variable region. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO.110 and a heavy chain having the amino acid sequence set forth in SEQ ID NO. 112. For example, the antibody, antigen binding fragment thereof can include antibody YN-067 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof described herein can comprise SEQ ID NO 96; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; and the amino acid sequence of HCDR1 can comprise SEQ ID NO 1; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 95. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-068 or an antibody having the same LCDR1-3 and HCDR1-3. The amino acid sequence of L-FR1 in the antibodies, antigen-binding fragments thereof, described herein can include SEQ ID NO 97; the amino acid sequence of L-FR2 can include SEQ ID NO 98; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 9; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the antibody, antigen-binding fragment thereof, may include antibody YN-068 or an antibody having LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4 identical thereto. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can include SEQ ID NO:109; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 114. For example, the antibody, antigen binding fragment thereof, can include antibody YN-068 or an antibody having the same light chain variable region and heavy chain variable region. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO.110 and a heavy chain having the amino acid sequence set forth in SEQ ID NO. 115. For example, the antibody, antigen-binding fragment thereof can include antibody YN-068 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof, described herein can comprise SEQ ID NO 96; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; and the amino acid sequence of HCDR1 can comprise SEQ ID NO 92; the amino acid sequence of HCDR2 can include SEQ ID NO 2; the amino acid sequence of HCDR3 can include SEQ ID NO 94. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-069 or an antibody having the same LCDR1-3 and HCDR1-3 as it. The amino acid sequence of L-FR1 in the antibodies, antigen-binding fragments thereof, described herein can include SEQ ID NO 97; the amino acid sequence of L-FR2 can include SEQ ID NO 98; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 9; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the antibody, antigen-binding fragment thereof, may include antibody YN-069 or an antibody having LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4 identical thereto. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can comprise SEQ ID NO:109; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 117. For example, the antibody, antigen binding fragment thereof, can include antibody YN-069 or an antibody having the same light chain variable region and heavy chain variable region. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO.110 and a heavy chain having the amino acid sequence set forth in SEQ ID NO. 118. For example, the antibody, antigen binding fragment thereof can include antibody YN-069 or an antibody having the same light and heavy chains as it.
In certain embodiments, the amino acid sequence of LCDR1 in the antibodies, antigen-binding fragments thereof, described herein can comprise SEQ ID NO 96; the amino acid sequence of LCDR2 can include SEQ ID NO 5; the amino acid sequence of LCDR3 can include SEQ ID NO 6; and the amino acid sequence of HCDR1 may comprise SEQ ID NO 91; the amino acid sequence of HCDR2 can include SEQ ID NO 93; the amino acid sequence of HCDR3 can include SEQ ID NO 94. For example, the antibody, antigen-binding fragment thereof, can include antibody YN-070 or an antibody having the same LCDR1-3 and HCDR1-3 as it. The amino acid sequence of L-FR1 in the antibodies, antigen-binding fragments thereof, described herein can include SEQ ID NO 97; the amino acid sequence of L-FR2 can include SEQ ID NO 98; the amino acid sequence of L-FR3 can include SEQ ID NO 13; the amino acid sequence of L-FR4 can include SEQ ID NO 14; and the amino acid sequence of H-FR1 may include SEQ ID NO 7; the amino acid sequence of H-FR2 can include SEQ ID NO 8; the amino acid sequence of H-FR3 can include SEQ ID NO 99; the amino acid sequence of H-FR4 can include SEQ ID NO 10. For example, the antibody, antigen-binding fragment thereof, may include antibody YN-070 or an antibody having the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4 as it does. In certain embodiments, the light chain of an antibody, antigen-binding fragment thereof, described herein can comprise a light chain variable region whose amino acid sequence can comprise SEQ ID NO:109; and wherein the heavy chain may comprise a heavy chain variable region whose amino acid sequence may comprise SEQ ID NO 120. For example, the antibody, antigen-binding fragment thereof, can comprise antibody YN-070, or an antibody having the same light chain variable region and heavy chain variable region as it. In certain embodiments, the antibodies, antigen-binding fragments thereof, described herein can comprise a light chain having the amino acid sequence set forth in SEQ ID NO.110 and a heavy chain having the amino acid sequence set forth in SEQ ID NO. 121. For example, the antibody, antigen-binding fragment thereof, can comprise antibody YN-070, or an antibody having the same light and heavy chains as it.
Reference in the present application to protein, polypeptide and/or amino acid sequences is also to be understood as including at least the following ranges: variants or homologues having the same or similar function as said protein or polypeptide.
In the present application, the variant may be a protein or polypeptide having substitution, deletion or addition of one or more amino acids in the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or a fragment thereof that specifically binds to CD38 protein). For example, the functional variant may comprise a protein or polypeptide that has been altered by at least 1, such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%,70%,80%,90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to the alteration. For example, the substitution may be a conservative substitution.
In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to CD38 protein).
In the present application, homology generally refers to similarity, similarity or relatedness between two or more sequences. The "percentage of sequence homology" can be calculated by: the two sequences to be aligned are compared over a comparison window, the number of positions in the two sequences at which the same nucleic acid base (e.g., a, T, C, G, I) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gin, cys, and Met) is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percent sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared or over a region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. Description of FASTA algorithm can be found in "improved tools for biological sequence comparison" by w.r.pearson and d.j.lipman, proceedings of the american national academy of sciences (proc.natl.acad.sci.), 85:2444-2448, 1988; and "rapid and sensitive protein similarity search" by d.j.lipman and w.r.pearson, science,227:1435-1441, 1989. See "an essential local alignment (alignment) search tool" by s.altschul, w.gish, w.miller, e.w.myers, and d.lipman, journal of molecular biology, 215:403-410, 1990.
Nucleic acids, vectors, host cells and methods of making
In another aspect, the present application also provides isolated one or more nucleic acid molecules encoding the antibodies, antigen-binding fragments thereof, described herein. For example, each of the one or more nucleic acid molecules may encode the entire antibody, an antigen-binding fragment thereof, or a portion thereof (e.g., one or more of an HCDR1-3, an LCDR1-3, a VL, a VH, a light chain, or a heavy chain).
The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (ii) produced by clonal recombination, (iii) purified, e.g., fractionated by enzymatic cleavage and gel electrophoresis, or (iv) synthesized, e.g., by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques.
In the present application, nucleic acids encoding the antibodies, antigen-binding fragments thereof, can be prepared by a variety of methods known in the art, including, but not limited to, overlap extension PCR using restriction fragment procedures or using synthetic oligonucleotides, as described in Sambrook et al, molecular Cloning, A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y.,1989; and Ausube et al Current Protocols in Molecular Biology, greene Publishing and Wiley-Interscience, new York N.Y.,1993.
In another aspect, the present application provides one or more vectors comprising one or more of the nucleic acid molecules described herein. One or more of the nucleic acid molecules may be included in each vector. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may contain expression control elements that allow for the proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements that regulate gene transcription or mRNA translation, among others. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but typically comprises 5' non-transcribed sequences and 5' and 3' non-translated sequences, such as TATA box, capping sequences, CAAT sequences, etc., which are involved in initiation of transcription and translation, respectively. For example, the 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional linkage nucleic acid. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerase, the human U6RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (e.g., CMV), wherein a portion of the promoter may be fused with a portion of the promoter of other cellular proteins (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not include additional introns. One or more of the nucleic acid molecules described herein can be operably linked to the expression control element.
The vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.
In another aspect, the present application provides a host cell that may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein. In certain embodiments, each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cell, e.g., prokaryotic cells (e.g., bacterial cells), CHO cells, NS/0 cells, HEK293T cells, or HEK293A cells, or other eukaryotic cells, such as plant-derived cells, fungal or yeast cells, and the like. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like. For example, the host cell may be COS, CHO, NSO, sf9, sf21, DH5a, BL21 (DE 3) or TG1.
In another aspect, the present application provides methods of making the antibodies, antigen-binding fragments thereof, described herein. The method may comprise culturing a host cell described herein under conditions such that the antibody, antigen-binding fragment thereof, is expressed. For example, these methods can be performed by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like, which are known to those of ordinary skill in the art.
In certain instances, the methods can further comprise the step of harvesting (e.g., isolating and/or purifying), the antibodies, antigen-binding fragments thereof, described herein. For example, protein G-Sepharose or protein A-Sepharose may be used for affinity chromatography, and the antibody, antigen-binding fragment thereof, described herein may be purified and isolated by gel electrophoresis and/or high performance liquid chromatography, or the like. For example, the fusion protein polypeptide bound to the affinity column can also be eluted by using a high salt buffer, changing the pH, or the like.
Pharmaceutical composition and application
In another aspect, the present application provides a pharmaceutical composition that can comprise an antibody, antigen-binding fragment thereof, as described herein, and optionally a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes, and/or nonionic surfactants, and the like.
In the present application, the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration or administration via subcutaneous depot.
The pharmaceutical compositions may be used to inhibit or delay the development or progression of a disease or disorder, and/or may alleviate and/or stabilize the state of a disease or disorder.
The pharmaceutical compositions described herein may comprise a prophylactically and/or therapeutically effective amount of the antibody, antigen-binding fragment thereof. The prophylactically and/or therapeutically effective amount is the amount required to be able to prevent and/or treat (at least partially treat) a disease or disorder and/or any complications thereof in a subject suffering from or at risk of developing the same.
In another aspect, the application provides the use of the antibody, antigen-binding fragment thereof, in the manufacture of a medicament for the prevention or treatment of a disease or disorder.
In another aspect, the antibodies, antigen-binding fragments thereof, provided herein are useful for preventing or treating a disease or disorder. In another aspect, the present application provides a method of preventing or treating a disease or disorder comprising administering (e.g., administering to a subject in need thereof) an antibody, antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, and/or the pharmaceutical composition described herein.
In the present application, the disease or disorder may be selected from the group consisting of: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, respiratory inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
In another aspect, the present application provides a method of inhibiting binding of an IL17 protein to an IL17 ligand comprising administering an antibody, antigen-binding fragment thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition described herein. For example, the method may be an in vitro or ex vivo method.
In another aspect, the present application provides a method of inhibiting a biological activity of an IL17 protein comprising administering an antibody, antigen-binding fragment thereof, nucleic acid molecule, vector, host cell and/or pharmaceutical composition described herein. For example, the method may be an in vitro or ex vivo method.
In another aspect, the present application provides a method of inhibiting chemokine expression comprising administering said antibody, antigen-binding fragment thereof, said nucleic acid molecule, said vector, said host cell and/or said pharmaceutical composition. For example, the chemokine can comprise CXCL1. For example, the method may be an in vitro or ex vivo method.
In another aspect, the present application provides the following embodiments:
1. an antibody or antigen-binding fragment thereof at 6 x10 -9 K of M or less D Values bind to IL17 protein.
2. The antibody or antigen binding fragment thereof according to embodiment 1, which inhibits binding of an IL17 protein to an IL17 receptor.
3. The antibody or antigen binding fragment thereof according to any one of embodiments 1-2, which inhibits the expression of a cytokine.
4. The antibody or antigen-binding fragment thereof of embodiment 3, wherein the cytokine comprises: interleukins and chemokines.
5. The antibody or antigen-binding fragment thereof of embodiment 4, wherein the chemokine comprises CXCL1.
6. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-5, which can ameliorate or treat a disease or disorder associated with inappropriate or excessive production of IL 17.
7. The antibody or antigen-binding fragment thereof according to embodiment 6, the disease or disorder comprising: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, respiratory inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
8. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-7, the antibody comprising: monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies.
9. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-8, the antigen-binding fragment comprising: fab, fab ', F (ab) 2, fv fragments, F (ab') 2, scFv, di-scFv and/or VhH.
10. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-9, which competes for binding to the IL17 protein with a reference antibody, wherein the reference antibody comprises a light chain variable region comprising LCDR1, LCDR2 and LCDR3 and a heavy chain variable region, the LCDR1 comprising the amino acid sequence set forth in any one of seq id no:126, 22 and 40; the LCDR2 comprises an amino acid sequence shown in any one of the following items: 5, 23 and 41; and the LCDR3 comprises an amino acid sequence shown in any one of the following items: 6, 24 and 131; and the number of the first and second groups is,
the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises an amino acid sequence shown in any one of the following items: 123, 19 and 37; the HCDR2 comprises an amino acid sequence shown in any one of the following items: 124, 20 and 38; and, the HCDR3 comprises an amino acid sequence set forth in any one of seq id nos: 125, 21 and 130.
11. The antibody, antigen-binding fragment thereof, of embodiment 10, wherein the LCDR1 of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: SEQ ID NO.4 and SEQ ID NO. 96.
12. The antibody or antigen-binding fragment thereof according to any one of embodiments 10-11, wherein the LCDR3 of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 42 and 102 SEQ ID NO.
13. The antibody or antigen-binding fragment thereof according to any one of embodiments 10-12, wherein the HCDR1 of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 1, 91 and 92 SEQ ID NO.
14. The antibody or antigen-binding fragment thereof according to any one of embodiments 10-13, wherein the HCDR2 of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 2 and 93 SEQ ID NO;
15. the antibody or antigen-binding fragment thereof according to any one of embodiments 10-14, wherein the HCDR3 of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 3, 94, 95, 39, 100 and 101.
16. The antibody or antigen-binding fragment thereof according to any one of embodiments 10-15, wherein the light chain variable region of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 16, 134, 34, 36, 52 and 132; and the number of the first and second electrodes,
the heavy chain variable region of the reference antibody comprises an amino acid sequence set forth in any one of: 15, 135, 33, 35, 51 and 133.
17. The antibody or antigen-binding fragment thereof according to embodiment 16, wherein the light chain variable region of the reference antibody comprises the amino acid sequence set forth in any one of: 54, 103, 18 and 109.
18. The antibody or antigen-binding fragment thereof according to any one of embodiments 16-17, wherein the heavy chain variable region of the reference antibody comprises the amino acid sequence set forth in any one of seq id nos: 53, 105, 107, 17, 111, 114, 117 and 120.
19. The antibody or antigen-binding fragment thereof according to any one of embodiments 10-18, wherein the light chain of the reference antibody comprises the amino acid sequence set forth in any one of: 64, 68, 72, 138, 84 and 136; and, the heavy chain of the reference antibody comprises an amino acid sequence set forth in any one of seq id nos: 62, 66, 70, 139, 82 and 137.
20. The antibody or antigen-binding fragment thereof of embodiment 19, wherein the light chain of the reference antibody comprises an amino acid sequence set forth in any one of seq id nos: 90, 104, 78 and 110.
21. The antibody or antigen-binding fragment thereof according to any one of embodiments 19-20, wherein the heavy chain of the reference antibody comprises the amino acid sequence set forth in any one of: 88, 106, 108, 76, 112, 115, 118 and 121.
22. The antibody or antigen-binding fragment thereof of any one of embodiments 1-21, wherein the antibody comprises an antibody light chain or fragment thereof.
23. The antibody or antigen-binding fragment thereof of embodiment 22, the antibody light chain or fragment thereof comprising LCDR1, and the LCDR1 comprising the amino acid sequence set forth in any one of seq id nos: 126, 22 and 40.
24. The antibody or antigen-binding fragment thereof according to embodiment 23, wherein the amino acid sequence of LCDR1 comprises the amino acid sequence of any one of seq id no: SEQ ID NO.4 and SEQ ID NO. 96.
25. The antibody or antigen-binding fragment thereof of any one of embodiments 22-24, the antibody light chain or fragment thereof comprising LCDR2, and the LCDR2 comprising the amino acid sequence set forth in any one of seq id nos: 5, 23 and 41.
26. The antibody or antigen-binding fragment thereof of any one of embodiments 22-25, the antibody light chain or fragment thereof comprising an LCDR3, and the LCDR3 comprising the amino acid sequence set forth in any one of seq id nos: 6, 24 and 131.
27. The antibody or antigen-binding fragment thereof of embodiment 26, wherein the LCDR3 comprises the amino acid sequence set forth in any one of seq id no:42 and 102 SEQ ID NO.
28. The antibody or antigen-binding fragment thereof of any one of embodiments 22-27, wherein the antibody light chain or fragment thereof further comprises the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
29. The antibody or antigen binding fragment thereof of embodiment 28, wherein the framework regions L-FR1, L-FR2, L-FR3 and L-FR4 are selected from any one of the following groups: human consensus framework sequences and human germline sequences.
30. The antibody or antigen-binding fragment thereof of any one of embodiments 28-29, wherein the C-terminus of L-FR1 is linked, directly or indirectly, to the N-terminus of the LCDR1, and wherein the L-FR1 comprises the amino acid sequence set forth in any one of seq id nos: 127, 29 and 47.
31. The antibody or antigen-binding fragment thereof of embodiment 30, wherein the L-FR1 comprises an amino acid sequence set forth in any one of: 11 and 97, respectively.
32. The antibody or antigen-binding fragment thereof of any one of embodiments 28-31, wherein said L-FR2 is located between said LCDR1 and said LCDR2, said L-FR2 comprising the amino acid sequence set forth in any one of seq id nos: 128, 30 and 48 SEQ ID NO.
33. The antibody or antigen-binding fragment thereof of embodiment 32, wherein the L-FR2 comprises an amino acid sequence set forth in any one of: 12 and 98 in SEQ ID NO.
34. The antibody or antigen-binding fragment thereof of any one of embodiments 28-33, wherein the L-FR3 is located between the LCDR2 and the LCDR3, the L-FR3 comprising the amino acid sequence set forth in any one of seq id nos: 13, 31 and 49.
35. The antibody or antigen-binding fragment thereof of any one of embodiments 28-34, wherein the N-terminus of the L-FR4 is linked, directly or indirectly, to the C-terminus of the LCDR3, and wherein the L-FR4 comprises the amino acid sequence set forth in any one of seq id nos: 14, 32 and 50.
36. The antibody or antigen-binding fragment thereof of any one of embodiments 22-35, the antibody light chain or fragment thereof comprising a light chain variable region VL, and the light chain variable region VL comprises the amino acid sequence set forth in any one of: 16, 134, 34, 36, 52 and 132.
37. The antibody or antigen binding fragment thereof of embodiment 36, wherein the light chain variable region comprises the amino acid sequence set forth in any one of: 54, 103, 18 and 109.
38. The antibody or antigen-binding fragment thereof of any one of embodiments 22-37, wherein the antibody light chain or fragment thereof further comprises a human constant region or a murine constant region.
39. The antibody or antigen-binding fragment thereof of embodiment 38, the antibody light chain or fragment thereof comprising a human constant region, and the human constant region comprising a human Ig κ or Ig λ constant region.
40. The antibody or antigen-binding fragment thereof according to any one of embodiments 22-39, wherein the antibody light chain or fragment thereof comprises an amino acid sequence set forth in any one of seq id nos: 64, 68, 72, 138, 84 and 136 SEQ ID NO.
41. The antibody or antigen-binding fragment thereof of embodiment 40, wherein the antibody light chain or fragment thereof comprises an amino acid sequence set forth in any one of seq id nos: 90, 104, 78 and 110.
42. The antibody or antigen binding fragment thereof according to any one of embodiments 1-41, said antibody comprising an antibody heavy chain or fragment thereof.
43. The antibody or antigen-binding fragment thereof of embodiment 42, the antibody heavy chain or fragment thereof comprising an HCDR1, and the HCDR1 comprising the amino acid sequence set forth in any one of seq id nos: 123, 19 and 37.
44. The antibody or antigen-binding fragment thereof of embodiment 43, wherein the HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 91 and 92 SEQ ID NO.
45. The antibody or antigen-binding fragment thereof of any one of embodiments 42-44, the antibody heavy chain or fragment thereof comprising HCDR2, and the HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NO:124, 20 and 38 SEQ ID NO.
46. The antibody or antigen-binding fragment thereof of embodiment 45, wherein the HCDR2 comprises an amino acid sequence of any one of SEQ ID NO: SEQ ID NO.2 and SEQ ID NO. 93.
47. The antibody or antigen-binding fragment thereof of any one of embodiments 42-46, the antibody heavy chain or fragment thereof comprising an HCDR3, and the HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NO:125, 21 and 130.
48. The antibody or antigen-binding fragment thereof of embodiment 47, wherein said HCDR3 comprises an amino acid sequence of any one of SEQ ID NO:3, 94, 95, 39, 100 and 101.
49. The antibody or antigen binding fragment thereof according to any one of embodiments 42-48, wherein the antibody heavy chain or fragment thereof further comprises framework regions H-FR1, H-FR2 and H-FR4.
50. The antibody or antigen binding fragment thereof according to embodiment 49, wherein the framework region is selected from any one of the following groups: human consensus framework sequences and human germline sequences.
51. The antibody or antigen-binding fragment thereof of any one of embodiments 49-50, wherein the C-terminus of H-FR1 is linked, directly or indirectly, to the N-terminus of the HCDR1, and wherein the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: SEQ ID NO 7, SEQ ID NO 25 and SEQ ID NO 43.
52. The antibody or antigen-binding fragment thereof of any one of embodiments 49-51, wherein said H-FR2 is located between said HCDR1 and said HCDR2, said H-FR2 comprising an amino acid sequence set forth in any one of SEQ ID NO:8, 26 and 44.
53. The antibody or antigen-binding fragment thereof of any one of embodiments 49-52, wherein said H-FR3 is located between said HCDR2 and said HCDR3, said H-FR3 comprising an amino acid sequence set forth in any one of SEQ ID NO:129, 27 and 45.
54. The antibody or antigen-binding fragment thereof of embodiment 53, wherein said H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO: SEQ ID NO 9 and SEQ ID NO 99.
55. The antibody or antigen-binding fragment thereof of any one of embodiments 49-54, wherein the N-terminus of H-FR4 is linked, directly or indirectly, to the C-terminus of the HCDR3, and wherein the H-FR4 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10, 28 and 46 SEQ ID NO.
56. The antibody or antigen-binding fragment thereof of any one of embodiments 49-55, the antibody heavy chain or fragment thereof comprising a heavy chain variable region VH, and the heavy chain variable region VH comprises the amino acid sequence set forth in any one of: 15, 135, 33, 35, 51 and 133.
57. The antibody or antigen-binding fragment thereof of embodiment 56, wherein the heavy chain variable region VH comprises the amino acid sequence set forth in any one of: 53, 105, 107, 17, 111, 114, 117 and 120.
58. The antibody or antigen-binding fragment thereof of any one of embodiments 42-57, wherein the antibody heavy chain or fragment thereof further comprises a human constant region or a murine constant region.
59. The antibody or antigen-binding fragment thereof of embodiment 58, wherein the antibody heavy chain or fragment thereof comprises a human constant region, and the human constant region comprises a human IgG constant region.
60. The antibody or antigen binding fragment thereof of any one of embodiments 58-59, the IgG constant region comprises a human IgG1 constant region.
61. The antibody or antigen-binding fragment thereof according to any one of embodiments 42-60, wherein the antibody heavy chain or fragment thereof comprises an amino acid sequence set forth in any one of: 62, 66, 70, 139, 82 and 137.
62. The antibody or antigen-binding fragment thereof of embodiment 61, wherein the heavy chain comprises the amino acid sequence set forth in any one of seq id nos: 88, 106, 108, 76, 112, 115, 118 and 121.
63. The antibody or antigen-binding fragment thereof of any one of embodiments 1-62, wherein the IL7 protein is a human IL17 protein.
64. The antibody or antigen-binding fragment thereof of any one of embodiments 1-62, the IL7 protein comprises an IL17A protein and/or an IL17F protein.
65. An isolated one or more nucleic acid molecules encoding the antibody or antigen-binding fragment thereof of any one of embodiments 1-62.
66. A vector comprising the nucleic acid molecule of embodiment 65.
67. A host cell comprising the nucleic acid molecule of embodiment 65 or the vector of embodiment 66.
68. A method of making the antibody or antigen-binding fragment thereof, the method comprising culturing the host cell of embodiment 67 under conditions such that the antibody or antigen-binding fragment thereof of any one of embodiments 1-62 is expressed.
69. The method of embodiment 68, optionally comprising harvesting the antibody or antigen-binding fragment thereof.
70. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in embodiments 1-62, a nucleic acid molecule as described in embodiment 65, a vector as described in embodiment 66, and/or a host cell as described in embodiment 67, and optionally a pharmaceutically acceptable adjuvant.
71. Use of an antibody or antigen-binding fragment thereof according to any one of embodiments 1-62 in the manufacture of a medicament for the prevention or treatment of a disease or disorder.
72. The use according to embodiment 71, the disease or condition comprising: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, airway inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
73. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-62 for use in the prevention or treatment of a disease or disorder.
74. The antibody or antigen-binding fragment thereof of embodiment 73, the disease or disorder comprising: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, respiratory inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
75. A method of preventing or treating a disease comprising administering the antibody or antigen-binding fragment thereof of any one of embodiments 1-62, the nucleic acid molecule of embodiment 65, the vector of embodiment 66, the host cell of embodiment 67, and/or the pharmaceutical composition of embodiment 70.
76. The method of embodiment 75, the disease or condition comprising: ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, osteoimplant loosening, acute transplant rejection, respiratory inflammation, inflammatory bowel disease, sepsis, septic or endotoxic shock, bone loss, psoriasis, allergies, ischemia, systemic sclerosis, stroke, and asthma.
77. A method of inhibiting binding of an IL17 protein and an IL17 ligand comprising administering an antibody or antigen-binding fragment thereof of any one of embodiments 1-62, a nucleic acid molecule of embodiment 65, a vector of embodiment 66, a host cell of embodiment 67, and/or a pharmaceutical composition of embodiment 70.
78. A method of inhibiting a biological activity of an IL17 protein comprising administering the antibody or antigen-binding fragment thereof of any one of embodiments 1-62, the nucleic acid molecule of embodiment 65, the vector of embodiment 66, the host cell of embodiment 67, and/or the pharmaceutical composition of embodiment 70.
79. A method of inhibiting chemokine expression comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-62, the nucleic acid molecule of embodiment 65, the vector of embodiment 66, the host cell of embodiment 67, and/or the pharmaceutical composition of embodiment 70.
80. The method of embodiment 79, wherein the chemokine comprises CXCL1.
Without wishing to be bound by any theory, the following examples are intended only to illustrate the working of the apparatus, method and system of the present application and are not intended to limit the scope of the invention of the present application.
Examples
The examples shown below are intended to illustrate specific embodiments of the invention and are not intended to limit the scope of the specification or claims in any way. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. A Laboratory Manual,2nd edition, cold spring Harbor Laboratory Press.
Example 1 screening of anti-IL 17 antibody Using phage antibody library
Recombinant human IL17A protein (Beijing Yiqiao Shenzhou biotechnology, inc.) is used as antigen for sorting phage natural humanized antibody library (Protocyte medicine technology, inc.). Antigen IL17A protein 1ml is coated in an enzyme-linked immune tube by using CBS buffer solution, the concentration of IL17A protein is 20 mu g/ml (first round and second round) or 10 mu g/ml (third round and fourth round), and the temperature is kept overnight at 4 ℃; the next day, 2ml of PBS buffer solution containing 10% of skimmed milk powder is used for sealing the immune tube; adding 1ml of the blocked phage into an immune tube, and incubating for 1h at room temperature; PBST wash 10 times (first, second) or 15 times (third, fourth); adding 800 μ L of Gly-HCl buffer solution with pH2.2 for elution, and immediately adding 400 μ L of Tris-HCl buffer solution with pH8.0 for neutralization; adding into 20ml E.coli SS320 with logarithmic growth phase OD about 0.8, mixing, standing at 37 deg.C for 1 hr; taking out 500 mu L of the suspension for determining the titer of the phage and preserving the glycerol; coating the rest bacterial liquid on a flat plate, and culturing overnight in an incubator at 37 ℃; scraping off the flat bacteria on the next day, inoculating into 80ml of 2YT-Amp culture medium according to a certain proportion to enable OD to be equal to 0.2, culturing for several hours until OD reaches 0.8, adding 160 mu L of helper phage, uniformly mixing, and standing for 1h at 37 ℃; adding IPTG and kanamycin, performing shaking culture at 250rpm and 30 ℃ overnight; collecting supernatant, precipitating phage with PEG/NaCl solution, and suspending in 1.5ml PBS buffer solution; resuspended phage were used for the next round of enrichment screening, and after 4 rounds of panning, significant enrichment was observed. And (3) identifying the phage antibody clone obtained by panning by ELISA: human IL17A protein (12047-HNAS) was coated on 96-well ELISA plates at a concentration of 1. Mu.g/ml and overnight at 4 ℃. Then, non-specific binding sites were blocked with 10% skim milk powder, washed thoroughly, and the monoclonal phage supernatant was added to a 96-well plate and incubated at 37 ℃ for 2 hours. After washing, adding (GE Healtcare, 27-9421-01) to act for 45min at 37 ℃, adding TMB to develop color after washing, acting for 5-10min at room temperature, finally stopping reaction by using sulfuric acid, and measuring OD value of each hole at 450 nm.
3 phage antibody clones 1F10, 1B6, 1F3 which specifically bind to human IL17A were identified by ELISA.
Example 2 expression and purification of anti-IL 17A fully human intact antibody
The phage antibody clone 1F10 was redesigned to be a complete IgG1, kappa antibody: designing a primer to perform PCR amplification on VH of the phage antibody, and cloning a PCR product to a pCMV-IgG1NEM vector subjected to double enzyme digestion by AgeI and SalI through recombination; primers were designed to perform PCR amplification on VL of phage antibodies, and PCR products were cloned by recombination into AgeI and BsiWI double digested pCMV-kappa vector. After the sequencing is correct, the heavy and light chain expression vectors are co-transfected into 293F cells for transient expression, and purified by a ProteinA column, and the complete IgG1 and kappa antibody of 1F10 is named YN-008.
Phage antibodies 1B6 and 1F3 are respectively redesigned into complete IgG1 and kappa antibodies, and expression and purification are carried out, wherein the expression and purification method is the same as that of 1F 10. The intact IgG1, kappa antibody of 1B6 was designated YN-009; the intact IgG1, kappa antibody of 1F3 was designated YN-010.
The VH sequences and VL sequences of YN-008, YN-009 and YN-010 were analyzed using the Kabat system for determining CDR regions, thereby determining the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 and L-CDR1, L-CDR2 and L-CDR 3.
The sequencing result shows that the amino acid sequences of LCDR1-3 of the anti-IL 17A fully human antibody YN-008 are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the amino acid sequence of VL is shown in SEQ ID NO. 16; the corresponding nucleotide sequence is shown as SEQ ID NO. 56; the amino acid sequence of the light chain is shown as SEQ ID NO. 64; the corresponding nucleotide sequence is shown as SEQ ID NO. 63; the amino acid sequences of HCDR1-3 of the antibody YN-008 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; the amino acid sequence of VH is shown in SEQ ID NO. 15; the corresponding nucleotide sequence is shown as SEQ ID NO. 55; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 62; the corresponding nucleotide sequence is shown in SEQ ID NO. 61.
The amino acid sequences of LCDR1-3 of the anti-IL 17A fully human antibody YN-009 are respectively shown as SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO. 24; the amino acid sequence of VL is shown in SEQ ID NO. 34; the corresponding nucleotide sequence is shown as SEQ ID NO. 58; the amino acid sequence of the light chain is shown as SEQ ID NO. 68; the corresponding nucleotide sequence is shown as SEQ ID NO. 67; the amino acid sequences of HCDR1-3 of antibody YN-009 are shown in SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, respectively; the amino acid sequence of VH is shown in SEQ ID NO. 33; the corresponding nucleotide sequence is shown as SEQ ID NO. 57; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 66; the corresponding nucleotide sequence is shown in SEQ ID NO. 65.
The amino acid sequences of LCDR1-3 of the anti-IL 17A fully human antibody YN-010 are respectively shown as SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO. 42; the amino acid sequence of VL is shown in SEQ ID NO. 52; the corresponding nucleotide sequence is shown as SEQ ID NO. 60; the amino acid sequence of the light chain is shown as SEQ ID NO. 72; the corresponding nucleotide sequence is shown as SEQ ID NO. 71; the amino acid sequences of HCDR1-3 of antibody YN-010 are respectively shown as SEQ ID NO.37, SEQ ID NO.38 and SEQ ID NO. 39; the amino acid sequence of VH is shown in SEQ ID NO. 51; the corresponding nucleotide sequence is shown as SEQ ID NO. 59; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 70; the corresponding nucleotide sequence is shown in SEQ ID NO. 69.
Example 3: germline forms of fully human anti-IL 17A antibodies YN-008, YN-009, YN-010 (germlined version)
Comparing the anti-IL 17A fully human antibody YN-008 heavy chain immunoglobulin sequence to the known human germline immunoglobulin heavy chain sequence, it was confirmed that the heavy chain of YN-008 utilized VH segments from human germline IGHV3-49 x 03, D segments from human germline IGHD3-9 x 01, and JH segments from human germline IGHJ2 x 01.
Alignment of the YN-008 light chain immunoglobulin sequence with the known human germline immunoglobulin light chain sequence confirmed that the YN-008 light chain utilized VL segments from human germline IGKV2-28 x 01, JL segments from human germline IGKJ4 x 01.
Alignment of the heavy chain immunoglobulin sequence of fully human anti-IL 17A antibody YN-009 with the known human germline immunoglobulin heavy chain sequence confirmed that the YN-009 heavy chain utilized VH segments from human germline IGHV1-46 x 01, D segments from human germline IGHD3-22 x 01, and JH segments from human germline IGHJ3 x 02.
Alignment of the YN-009 light chain immunoglobulin sequence with the known human germline immunoglobulin light chain sequence confirmed that the YN-009 light chain utilized VL segments from human germline IGKV3-1 x 01, JL segments from human germline IGLJ1 x 01.
Comparison of the anti-IL 17A fully human antibody YN-010 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences confirmed that the YN-010 heavy chain utilizes a VH segment from human germline IGHV3-53 x 01, a D segment from human germline IGHD6-19 x 01, and a JH segment from human germline IGHJ4 x 02.
Alignment of the YN-010 light chain immunoglobulin sequence with a known human germline immunoglobulin light chain sequence confirmed that the YN-010 light chain utilized VL segments from human germline IGLV3-1 x 01, JL segments from human germline IGLJ1 x 01.
(1) To minimize the immunogenicity of YN-008, some amino acid residues can be mutated back to the germline sequence. YN-011, a germline form of YN-008 (germlined version), was prepared by returning 3 amino acids in the FR1 region of the YN-008 heavy chain variable region to the germline sequence and reverting 1 amino acid in the FR1 region of the YN-008 light chain variable region to the germline sequence (see FIG. 1A). The heavy chain expression vector of the anti-IL 17A fully human antibody YN-011 is obtained by site-specific mutagenesis using a mutagenesis kit (Tiangen point mutagenesis kit, KM 101) on the basis of the heavy chain expression plasmid of YN-008 constructed as described above. The YN-011 light chain expression vector is obtained by utilizing a mutation kit (Tiangen point mutation kit, KM 101) to perform site-specific mutation on the basis of a YN-008 light chain expression plasmid.
The result showed that the amino acid sequence of VL of YN-011 is shown in SEQ ID NO. 18; the corresponding nucleotide sequence is shown as SEQ ID NO. 74; the amino acid sequence of the light chain is shown as SEQ ID NO. 78; the corresponding nucleotide sequence is shown as SEQ ID NO. 77; the amino acid sequence of VH of YN-011 is shown in SEQ ID NO. 17; the corresponding nucleotide sequence is shown as SEQ ID NO. 73; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 76; the corresponding nucleotide sequence is shown in SEQ ID NO. 75.
Reference is made to YN-008 antibody as method procedure for expression and purification of YN-011 antibody.
(2) To minimize immunogenicity of YN-009, some amino acid residues may be mutated back to the germline sequence. YN-012 is a germline version of YN-009, prepared by returning 2 amino acids in the FR1 region of the YN-009 heavy chain variable region to the germline sequence and 1 amino acid in the FR2 region of the YN-009 light chain variable region to the germline sequence (see FIG. 1B). The heavy chain expression vector of anti-IL 17A fully human antibody YN-012 was obtained by site-directed mutagenesis using a mutagenesis kit (Tiangen point mutagenesis kit, KM 101) on the basis of the heavy chain expression plasmid of YN-009 constructed as described above. YN-012 light chain expression vector was obtained by site-directed mutagenesis using a mutagenesis kit (Tiangen point mutagenesis kit, KM 101) based on YN-009 light chain expression plasmid.
The result showed that the amino acid sequence of VL of YN-012 is shown in SEQ ID NO. 36; the corresponding nucleotide sequence is shown as SEQ ID NO. 80; the amino acid sequence of the light chain is shown as SEQ ID NO. 84; the corresponding nucleotide sequence is shown as SEQ ID NO. 83; the amino acid sequence of VH of YN-012 is shown in SEQ ID NO. 35; the corresponding nucleotide sequence is shown as SEQ ID NO. 79; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 82; the corresponding nucleotide sequence is shown in SEQ ID NO. 81.
As for the method for expression and purification of the YN-012 antibody, reference was made to the YN-008 antibody.
(3) To minimize the immunogenicity of YN-010, some amino acid residues may be mutated back to the germline sequence. YN-013 is a germline form of YN-010 (germlined version) prepared by returning 1 amino acid in the FR1 region and 1 amino acid in the FR3 region of the YN-010 heavy chain variable region to the germline sequence and 1 amino acid in the FR1 region, 3 amino acids in the FR2 region and 1 amino acid in the FR3 region of the YN-010 light chain variable region to the germline sequence (see FIG. 1C). The heavy chain expression vector of anti-IL 17A fully human antibody YN-013 was obtained by site-directed mutagenesis using a mutagenesis kit (Tiangen point mutagenesis kit, KM 101) on the basis of the heavy chain expression plasmid of YN-010 constructed as described above. The YN-013 light chain expression vector is obtained by site-directed mutagenesis using a mutagenesis kit (Tiangen point mutagenesis kit, KM 101) on the basis of an YN-010 light chain expression plasmid.
The result showed that the amino acid sequence of VL of YN-013 was shown in SEQ ID NO. 54; the corresponding nucleotide sequence is shown as SEQ ID NO. 86; the amino acid sequence of the light chain is shown as SEQ ID NO. 90; the corresponding nucleotide sequence is shown as SEQ ID NO. 89; the amino acid sequence of VH of YN-013 is shown in SEQ ID NO. 53; the corresponding nucleotide sequence is shown as SEQ ID NO. 85; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 88; the corresponding nucleotide sequence is shown as SEQ ID NO. 87;
as for the method for expressing and purifying the YN-013 antibody, reference was made to the YN-008 antibody.
Example 4: binding affinity detection of IL17 antibodies
The binding affinity of IL17 antibodies YN-008, YN-009, YN-010, YN-011, YN-012 and YN-013 to the recombinant human IL17A protein was measured by using an Octet RED384 instrument (Pall ForteBio). First, the human IL17A protein was labeled with Biotin (EZ-Link S. Mu. Lfo-NHS-LC-Biotin, pierce, 21327). Analysis of binding kinetics between antigen and antibody was performed by biofilm interference (BLI) technique using a fortebio octet RED384 instrument (PALL) molecular interaction analyzer (both antigen and antibody dilutions used 0.1% bsa and 0.02% tween20 in PBS buffer).The antigen with the concentration of 50nM and coupled by biotin is fixed with the SA sensor at 1500rpm/min for 10 minutes; then, it was combined with a double-diluted antibody solution (100 nM to 0 nM) for 10min,1500rpm/min. Finally, dissociation was performed for 10 minutes at 1500rpm/min. The remaining antibody will be regenerated by glycine pulses. The obtained results were subjected to Data Analysis using the octet Data Analysis 9.0 software (fortebio) to calculate the binding strength between the antigen and the antibody, and K was obtained D Value, ka (1/Ms) value and Kd (1/s) value (R2 value)>0.95 is a trusted result).
The results are shown in Table 1.
TABLE 1 affinity of IL17 antibody to human IL17A
Figure BDA0003259822830000531
Example 5: IL17 antibodies inhibit binding between human IL17A protein and human IL17R
IL17 antibodies YN-008, YN-009, YN-010, YN-011, YN-012, YN-013, were evaluated for their ability to block the binding of human IL17A to the human IL17 receptor using an Octet RED384 instrument (Pall ForteBio). First, human IL17RA/Fc protein was labeled with Biotin (EZ-Link S. Mu. Lfo-NHS-LC-Biotin, pierce, 21327) (Beijing Bethes Biotech Co., ltd., H5257). The kinetics of binding between IL17 antibody-inhibited human IL17A protein and the human IL17 receptor was analyzed by the biofilm interference (BLI) technique using a fortebio octet RED384 instrument (PALL) molecular interaction analyzer (antigen, antibody dilutions were each 0.1% BSA and 0.02% tween PBS buffer). The recombinant human IL17RA with the concentration of 100nM and coupled by biotin is fixed with the SA sensor at 1500rpm/min, combined for 10min; human IL17A protein at a final concentration of 100nM was mixed with a three-fold dilution of the antibody solution (2000 nM-0 nM), incubated for 60 minutes and then machine-bound for 10 minutes at 1500rpm/min. Finally, dissociation was carried out for 10 minutes at 1500rpm/min. The remaining antigen-antibody complex will be regenerated by glycine pulses. The results obtained were subjected to Data Analysis using the octet Data Analysis 9.0 software (fortebio) to calculate the inhibitory intensity of the antibody against the antigen and the ligand. Results see FIG. 2, wherein FIGS. 2A-2C show the results of inhibition of binding of human IL17A protein and human IL17 receptor by IL17 antibodies YN-011, YN-012, YN-013, in sequence, with the concentration of the antibody (in Log nM) on the abscissa and RU values on the ordinate. The results in FIG. 2 show that IL17 antibodies YN-011, YN-012 and YN-013 are effective in inhibiting the binding of human IL17A protein to human IL17 receptor.
Example 6: IL17 antibody inhibits IL17A protein from inducing HT-29 cells to produce chemokine CXCL1
HT-29 cells are human colorectal adenocarcinoma epithelial cells (Shanghai cell Bank of Chinese academy) that naturally express the IL17 receptor. Incubation of HT-29 cells with human IL17A protein results in the production of the chemokine CXCL1, which can be detected using ELISA.
2×10 5 HT-29 cells were added to 96-well plates at a concentration of one ml and cultured at 37 ℃ for 12 hours. A mixture of 2nM IL17A and a gradient of IL17 antibody (YN-011, YN-012 or YN-013) or control antibody (IgG Fc) was incubated at 37 ℃ for 1 hour, added to the above 96-well plate, incubated with HT-29 cells at 37 ℃ for 48 hours, and then treated with CXCL1 ELISA kit (R-ELISA kit)&Company D) the level of CXCL1 in the cell culture supernatant was measured. The results are shown in FIG. 3, with antibody concentration on the abscissa and CXCL1 concentration on the ordinate. The results in FIG. 3 show that the IL17 antibodies YN-011, YN-012 and YN-013 can effectively inhibit the production of chemokine CXCL1 by HT-29 cells.
Example 7: cross-binding Activity with IL-17F protein
IL-17F protein (product of ACRObiosystems) was diluted to 2. Mu.g/ml with PBS buffer (pH 7.2), 50. Mu.L/well, and coated overnight at 4 ℃. After 5% skim milk was blocked at room temperature for 2 hours, different concentrations of each IL-17 antibody were added at 50. Mu.L/well and incubated at room temperature for 2 hours. The supernatant was discarded, washed 3 times with PBST, and then diluted HRP-labeled goat anti-human IgG monoclonal antibody (product of KPL Co.) was added thereto at a concentration of 50. Mu.L/well and incubated at room temperature for 45min. After PBST was sufficiently washed, color was developed with TMD, and absorbance (OD 450) at 450nm was measured with an enzyme-labeled instrument (Elx type automatic enzyme-labeled colorimeter, BIO-TEK, USA). The results of the experiment are shown in FIG. 4, and the results show that the antibodies YN008 and YN011 can be cross-bound with IL17F protein.
Example 8: affinity maturation of IL-17 antibody YN013
Design of primers A single chain antibody (scFv) gene for IL-17 antibody YN-013 was constructed by overlap PCR and cloned into a pDF phagemid vector, which was designated pDF-YN-013scFv. Design of degenerate primers with pDF-YN-013scFv as template six CDR regions of antibody YN-013 (FIG. 1C) were randomized separately by overlap PCR.
The scFv gene fragment obtained by overlapping PCR after CDR region randomization is cut by BssHII and NheI, then connected with a pDF phagemid carrier cut by BssHII and NheI, 1 mu g of the connection product is electroporated and transferred into TG1 electroporation competent cells, coated with 2YT + AG plate after dilution by multiple times, the bacterial lawn is scraped from the plate the next day, expanded to 300ml of 2YT amp culture medium, cultured at 37 ℃ to about OD0.8, added with helper phage and mixed evenly, then kept stand for 1 hour, added with kanamycin with final concentration of 1IPTG, 50 mu g/ml and shaken at 30 ℃ overnight. Centrifuging the next day, collecting supernatant, filtering with 0.45 filter membrane for sterilization, adding 1/5 volume of PEG-NaCl to precipitate phage, centrifuging to obtain precipitate, resuspending the precipitate with 1/10 volume of PBS, measuring OD260 to calculate phage pfu, and storing at 4 deg.C, wherein the phage antibody library can be directly used for later-stage panning.
Recombinant human IL17A protein (Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) was used as an antigen for the above phage antibody library sorting. Coating 1ml of antigen in an enzyme-linked immune tube by using CBS buffer solution, wherein the concentration of the antigen is 100nM (first round, second round) or 5nM (third round), and keeping the temperature at 4 ℃ overnight; the next day, the immune tube is sealed by 2ml of PBS buffer solution containing 10 percent of skimmed milk powder; adding 1ml of the blocked phase into an immune tube, and incubating for 1h at room temperature; PBST wash 20 times (first round), 50 times (second round), or 100 times (third round); adding 800 μ l of Gly-HCl buffer solution with pH2.2 for elution, and immediately adding 400 μ l of Tris-HCl buffer solution with pH8.0 for neutralization; adding into 20ml E.coli TG1 with logarithmic growth phase OD about 0.8, mixing, standing at 37 deg.C for 1 hr; taking out 500 μ l for determining bacteriophage titer and preserving glycerol; coating the rest bacterial liquid on a flat plate, and culturing overnight in an incubator at 37 ℃; scraping off the flat bacteria on the next day, inoculating into 80ml of 2YT-Amp culture medium according to a certain proportion to enable OD to be equal to 0.2, culturing for several hours until OD reaches 0.8, adding 160 mu l of helper phase, uniformly mixing, and standing for 1h at 37 ℃; adding IPTG and Kan antibiotics, shaking and culturing at 250rpm and 30 ℃ overnight; collecting supernatant, precipitating phage with PEG/NaCl solution, and suspending in 1.5ml PBS buffer solution; the resuspended phage were used for the next round of enrichment screening, and after 3 rounds of panning, significant enrichment was observed. And (3) identifying the phage antibody clone obtained by panning by ELISA: human IL17A protein was coated on 96-well ELISA plates at a concentration of 1. Mu.g/ml overnight at 4 ℃. Non-specific binding sites were then blocked with 10% skim milk powder, washed thoroughly, and the monoclonal phage supernatant was added to a 96-well plate and incubated for 2 hours at 37 ℃. After fully washing, adding HRP labeled anti-M13 antibody (GE health care, 27-9421-01), acting at 37 ℃ for 45min, fully washing, adding TMB for color development, acting at room temperature for 5-10min, finally stopping reaction by using sulfuric acid, measuring OD value of each hole at 450nm, and selecting phage antibody clone with high OD450 value for sequencing. After obtaining the heavy chain and light chain variable region gene sequences of each phage antibody clone, redesigning each phage antibody into a full-length IgG1, kappa antibody: designing primers to perform PCR amplification on the VH cloned by each phage antibody, and cloning the PCR product to a pCMV-IgG1NDL antibody heavy chain expression vector subjected to AgeI and SalI double enzyme digestion; primers were designed to perform PCR amplification on VL of each phage antibody clone, and PCR products were cloned by recombination into a pCMV-kappa antibody light chain expression vector digested by AgeI and BsiWI. After the sequencing is correct, the heavy and light chain expression vectors of each antibody are co-transfected into 293F cells for transient expression, cell culture supernatants are collected after 7 days of culture in a serum-free medium and purified by a ProteinA column to obtain antibody proteins, the purified antibodies are dialyzed by PBS, and finally the antibody proteins are quantified by a BCA Protein Assay Kit (Pierce, 23225).
The binding affinity of IL17 antibodies to recombinant human IL17A protein was measured by using Octet RED384 instrument (Pall ForteBio). First, human IL17A-Fc protein (Shanghai Protocyte medicine Co., ltd.) was labeled with Biotin (EZ-Link S. Mu. Lfo-NHS-LC-Biotin, pierce, 21327). Analysis of the binding kinetics between antigen and antibody was performed by the biofilm interference (BLI) technique using a fortebio octet RED384 instrument (PALL) molecular interaction Analyzer (antigen, antibody dilutions all used 0.1% BSA and 0.02% tween PBS buffer). Biotin-coupled antigens with a concentration of 50nMFixation with SA sensor, 1500rpm/min, binding for 5 minutes; then combined with a double diluted antibody solution (100, 50, 25, 12.5,6.25,3.125, 1.56nM) for 5min,1500rpm/min. Finally, dissociation was carried out for 10 minutes at 1500rpm/min. The results obtained were subjected to Data Analysis by Octet Data Analysis 9.0 software (Pall ForteBio Co.) to obtain K D Value, K on (1/Ms) value and K off (1/s) value. After multiple rounds of screening and combination, 2 YN-013 antibody mutant clones with the highest affinity were named YN-065 antibody and YN-066 antibody, respectively. The affinity values obtained for these 2 antibodies determined by the above method are shown in table 2. The affinity data show that YN-065 and YN-066 have 14-fold and 12-fold affinity, respectively, for secukinumab.
TABLE 2 affinity of IL-17 antibodies to human IL-17A
Figure BDA0003259822830000561
The amino acid sequence of VL of the YN-065 antibody is shown in SEQ ID NO.103, and the amino acid sequence of the light chain is shown in SEQ ID NO. 104; the amino acid sequence of VH of the YN-065 antibody is shown as SEQ ID NO.105, and the amino acid sequence of heavy chain is shown as SEQ ID NO. 106;
the amino acid sequence of VL of the YN-066 antibody is shown in SEQ ID NO.103, and the amino acid sequence of the light chain is shown in SEQ ID NO. 104; the amino acid sequence of VH of the YN-066 antibody is shown as SEQ ID NO.107, and the amino acid sequence of heavy chain is shown as SEQ ID NO. 108;
example 9: affinity maturation of IL-17 antibody YN011
Design of primers Single chain antibody (scFv) gene of IL-17 antibody YN-011 was constructed by overlap PCR and cloned into pDF phagemid vector, denoted pDF-YN-011scFv. The scFv sequence was randomized by random mutagenesis kit (Agilent, cat. 200552) using pDF-YN-011scFv as template to design primers.
The randomized scFv gene fragment is subjected to double enzyme digestion by BssHII and NheI, then is connected with a pDF phagemid vector subjected to double enzyme digestion by BssHII and NheI, 1 mu g of the connection product is electroporated and transferred into TG1 electroporation competent cells, the cells are coated on 2YT + AG plates after being diluted in multiple proportions, the lawn is scraped from the plates the next day, the cells are expanded to 300YT + Amp culture medium, the cells are cultured at 37 ℃ to about OD0.8, the cells are added with helper phage, are uniformly mixed and then are kept stand for 1 hour, kanamycin with the final concentration of 1mM IPTG,50 mu g/ml is added, and the cells are shaken at 30 ℃ overnight. Centrifuging the next day, collecting supernatant, filtering with 0.45 filter membrane for sterilization, adding 1/5 volume of PEG-NaCl to precipitate phage, centrifuging to obtain precipitate, resuspending the precipitate with 1/10 volume of PBS, measuring OD260 to calculate phage pfu, and storing at 4 deg.C, wherein the phage antibody library can be directly used for later-stage panning.
Recombinant human IL17A protein (Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) was used as an antigen for the above phage antibody library sorting. 1ml of antigen is coated in an enzyme-linked immune tube by CBS buffer solution, the concentration of the antigen is 100nM (first round, second round) or 5nM (third round), and the temperature is 4 ℃ overnight; the next day, 2ml of PBS buffer solution containing 10% of skimmed milk powder is used for sealing the immune tube; adding 1ml of the blocked phase into an immune tube, and incubating for 1h at room temperature; PBST wash 20 times (first round), 50 times (second round), or 100 times (third round); adding Gly-HCl buffer solution with pH of 2.2 to elute with 800 μ l, immediately adding 400 μ l Tris-HCl buffer solution with pH of 8.0 to neutralize; adding into 20ml E.coli TG1 with logarithmic growth phase OD about 0.8, mixing, standing at 37 deg.C for 1 hr; taking out 500 μ l of the suspension for determining the titer of the phage and preserving the glycerol; coating the residual bacterial liquid on a flat plate, and culturing overnight in an incubator at 37 ℃; scraping off the flat bacteria on the next day, inoculating into 80ml of 2YT-Amp culture medium according to a certain proportion to enable OD to be equal to 0.2, culturing for several hours until OD reaches 0.8, adding 160 mu l of helper phase, uniformly mixing, and standing for 1h at 37 ℃; adding IPTG and Kan antibiotics, performing shaking culture at 250rpm and 30 ℃ overnight; collecting supernatant, precipitating phage with PEG/NaCl solution, and suspending in 1.5ml PBS buffer solution; the resuspended phage were used for the next round of enrichment screening, and after 3 rounds of panning, significant enrichment was observed. And (3) identifying the phage antibody clone obtained by panning by ELISA: human IL17A protein was coated on 96-well ELISA plates at a concentration of 1. Mu.g/ml overnight at 4 ℃. Then, non-specific binding sites were blocked with 10% skim milk powder, washed thoroughly, and the monoclonal phage supernatant was added to a 96-well plate and incubated at 37 ℃ for 2 hours. After washing well, HRP-labeled anti-M13 antibody (GE healthcare, 27-9421-01) was added thereto and allowed to act at 37 ℃Washing for 45min, adding TMB for color development, reacting at room temperature for 5-10min, stopping reaction with sulfuric acid, measuring OD value of each well at 450nm, and selecting phage antibody clone with high OD450 value for sequencing. After obtaining the heavy and light chain variable region gene sequences of each phage antibody clone, we redesign each phage antibody into Fab fragments: designing primers to perform PCR amplification on VH of phage antibody clone and PCR amplification on a first antibody heavy chain constant region on an antibody heavy chain expression vector pCMV-IgG1NDL respectively, constructing a VHCH1 gene segment through overlapped PCR, and cloning the VHCH1 gene segment to a pCMV-IgG1NDL antibody heavy chain expression vector subjected to double enzyme digestion by AgeI and BamH 1; primers were designed to perform PCR amplification on VL of each phage antibody clone, and PCR products were cloned by recombination into a pCMV-kappa antibody light chain expression vector digested by AgeI and BsiWI. After the sequencing is correct, the heavy and light chain expression vectors of each Fab fragment are co-transfected into 293F cells for transient expression, cell culture supernatants are collected after 7 days of culture in a serum-free medium and purified by a Protein G column to obtain antibody Protein, the purified antibody is dialyzed by PBS, and finally the BCA Protein Assay Kit is used for quantification (Pierce, 23225). The binding affinity of the IL17 antibody Fab fragments to the recombinant human IL17A protein was measured by Octet RED384 instrument (Pall ForteBio). First, human IL17A-Fc protein (Shanghai Protocyte medicine Co., ltd.) was labeled with Biotin (EZ-Link S. Mu. Lfo-NHS-LC-Biotin, pierce, 21327). Analysis of the binding kinetics between antigen and antibody was performed by the biofilm interference (BLI) technique using a fortebio octet RED384 instrument (PALL) molecular interaction Analyzer (antigen, antibody dilutions all used 0.1% BSA and 0.02% tween PBS buffer). The antigen coupled with biotin at a concentration of 50nM was immobilized with SA sensor at 1500rpm/min for 5 min; then combined with a double diluted antibody solution (100, 50, 25, 12.5,6.25,3.125, 1.56nM) for 5min,1500rpm/min. Finally, dissociation was performed for 10 minutes at 1500rpm/min. The obtained results were subjected to Data Analysis using the octet Data Analysis 9.0 software (fortebio) to calculate the binding strength between the antigen and the antibody, and K was obtained D Values, ka (1/Ms) values and Kd (1/s) values. After multiple rounds of screening and combination, the 4 YN-011 antibodies with highest affinity are mutatedThe variant Fab was named YN-067Fab, YN-068Fab, YN-069Fab and YN-070Fab, respectively. The affinity values obtained for these 4 Fab fragments determined by the above method are shown in table 3.
The light chain amino acid sequence of the YN-067Fab is shown as SEQ ID NO. 110; the heavy chain amino acid sequence of the YN-067Fab antibody is shown in SEQ ID NO. 113.
The light chain amino acid sequence of the YN-068Fab is shown as SEQ ID NO. 110; the heavy chain amino acid sequence of the YN-068Fab antibody is shown in SEQ ID NO. 116.
The light chain amino acid sequence of the YN-069Fab is shown as SEQ ID NO. 110; the heavy chain amino acid sequence of the YN-069Fab antibody is shown in SEQ ID NO. 119.
The light chain amino acid sequence of the YN-070Fab is shown as SEQ ID NO. 110; the heavy chain amino acid sequence of the YN-070Fab antibody is shown in SEQ ID NO. 122.
We also constructed, expressed and purified the Fab fragment of the IL-17 antibody secukinumab, named secukinumab-Fab, by reference to the above method, and determined the affinity of secukinumab-Fab by the above affinity determination method, with the affinity values as shown in Table 3. The affinity data show that the affinity of YN-067Fab, YN-068Fab, YN-069Fab and YN-070Fab is respectively 8.8 times, 3.0 times, 8.4 times and 27 times that of secukumab-Fab.
TABLE 3 affinity of IL-17 antibody Fab fragments for human IL-17A
Figure BDA0003259822830000581
The method for redesigning phage antibodies to full-length IgG1, kappa antibodies is as follows: designing primers to perform PCR amplification on the VH cloned by each phage antibody, and cloning the PCR product to a pCMV-IgG1NDL antibody heavy chain expression vector subjected to AgeI and SalI double enzyme digestion; primers were designed to perform PCR amplification on VL of each phage antibody clone, and PCR products were cloned by recombination into a pCMV-kappa antibody light chain expression vector digested by AgeI and BsiWI. After the sequencing is correct, the heavy and light chain expression vectors of each antibody are co-transfected into 293F cells for transient expression, cell culture supernatants are collected after 7 days of culture in a serum-free medium and purified by a Protein A column to obtain antibody proteins, the purified antibodies are dialyzed by PBS, and finally the antibody proteins are quantified by a BCA Protein Assay Kit (Pierce, 23225). Full-length IgG1 and kappa antibodies corresponding to YN-067-Fab, YN-068-Fab, YN-069-Fab and YN-070-Fab are respectively named YN-067, YN-068, YN-069 and YN-070.
The amino acid sequence of VL of the YN-067 antibody is shown in SEQ ID NO.109, and the amino acid sequence of the light chain is shown in SEQ ID NO. 110; the amino acid sequence of VH of YN-067 antibody is shown in SEQ ID NO.111, and the amino acid sequence of heavy chain is shown in SEQ ID NO. 112.
The amino acid sequence of VL of the YN-068 antibody is shown in SEQ ID NO.109, and the amino acid sequence of the light chain is shown in SEQ ID NO. 110; the amino acid sequence of VH of YN-068 antibody is shown in SEQ ID NO.114, and the amino acid sequence of heavy chain is shown in SEQ ID NO. 115.
The amino acid sequence of VL of the YN-069 antibody is shown in SEQ ID NO.109, and the amino acid sequence of the light chain is shown in SEQ ID NO. 110; the amino acid sequence of VH of YN-069 antibody is shown in SEQ ID NO.117, and the amino acid sequence of heavy chain is shown in SEQ ID NO. 118.
The VL amino acid sequence of the YN-070 antibody is shown as SEQ ID NO.109, and the amino acid sequence of the light chain is shown as SEQ ID NO. 110; the amino acid sequence of the VH of the YN-070 antibody is shown as SEQ ID NO.120, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 121.
Example 10: IL17 antibodies inhibit binding between human IL17A protein and human IL17R
IL17 antibodies YN-065, YN-066, YN-067, YN-068, YN-069, YN-070 were evaluated for their ability to block the binding of human IL17A to the human IL17 receptor using an Octet RED384 instrument (Pall ForteBio). First, human IL17RA/Fc protein was labeled with Biotin (EZ-Link S. Mu. Lfo-NHS-LC-Biotin, pierce, 21327) (Beijing Bethes Biotech Co., ltd., H5257). Inhibition of binding between human IL17A protein and human IL17 receptor by IL17 antibody was analyzed by biofilm interference (BLI) technique using a fortebio octet RED384 instrument (PALL) molecular interaction analyzer (antigen, antibody dilutions were 0.1% bsa and 0.02% tween20 in PBS buffer). The recombinant human IL17RA with the concentration of 100nM and coupled by biotin is fixed with the SA sensor at 1500rpm/min, combined for 10min; human IL17A protein at a final concentration of 50nM was mixed with a quadruplicate dilution of the antibody solution (1000, 250, 62.5, 15.625,3.9,0.97,0 nM), incubated for 60 minutes and then bound on the machine for 10 minutes at 1500rpm/min. Finally, dissociation was performed for 1 min at 1500rpm/min. The obtained results were subjected to Data Analysis by the octet Data Analysis 9.0 software (fortebio) to calculate the inhibitory intensity of the antibody against the antigen and the ligand. FIG. 5 shows the results of inhibition of binding between human IL17A protein and human IL17 receptor by IL17 antibodies YN-065, YN-066, YN-067, YN-068, YN-069 and YN070, wherein the abscissa represents the concentration of the antibody and the ordinate represents the RU value. The results in FIG. 5 show that IL17 antibodies YN-065, YN-066, YN-067, YN-068, YN-069 and YN070 are effective in inhibiting the binding of human IL17A protein to the human IL17 receptor.
Example 11: IL17 antibody inhibits IL17A protein from inducing HT-29 cells to produce chemokine CXCL1
HT-29 cells are human colorectal adenocarcinoma epithelial cells (Shanghai cell Bank of Chinese academy) that naturally express the IL17 receptor. Incubation of HT-29 cells with human IL17A protein results in the production of the chemokine CXCL1, which can be detected using ELISA.
2×10 5 Each/ml of HT-29 cells was added to a 96-well plate and incubated at 37 ℃ for 12 hours. A mixture of 2nM IL17A (Beijing-Cassia Biotechnology Ltd.) and a gradient of IL17 antibody or control antibody (IgG Fc) was incubated at 37 ℃ for 1 hour, added to the above 96-well plate, incubated with HT-29 cells at 37 ℃ for 48 hours, and then treated with CXCL1 ELISA kit (R)&Company D) the level of CXCL1 in the cell culture supernatant was measured. Results referring to fig. 6, the abscissa is the concentration of the antibody and the ordinate is the concentration of CXCL1. The results of fig. 6A and 6B show that: IL17 antibodies YN-065, YN-066, YN-067 and YN-068 can effectively inhibit HT-29 cells from producing a chemokine CXCL1, and the inhibition capability of the antibodies is obviously stronger than that of Secukinumab. The results in FIG. 6C show that YN069 and YN070 inhibit CXCL1 production by HT-29 cells similarly to YN 068.
Example 12: IL17 antibody inhibits IL17A protein from inducing HS-27 cells to produce interleukin 8 (IL-8)
IL-17 induces IL-8 secretion by human HS-27 cells (ATCC # CRL-1634), and IL-8 can be detected using ELISA.
2×10 5 HS-27 cells were added to a 96-well plate at one/ml and cultured at 37 ℃ for 12 hours. A mixture of 100ng/ml IL17A (Beijing Quizhou Biotechnology Co., ltd.) and gradient IL17 antibody or control antibody (IgG Fc) was incubated at 37 ℃ for 1 hour, added to the above 96-well plate, incubated with HS-27 cells at 37 ℃ for 48 hours, and then treated with IL-8 ELISA kit (R)&Company D) the level of IL-8 in the cell culture supernatant was determined. The results are shown in FIG. 7, with antibody concentration on the abscissa and IL-8 concentration on the ordinate. The results of fig. 7A and 7B show that: IL17 antibodies YN-065, YN-066, YN-067 and YN-068 can effectively inhibit the IL-8 production of HS-27 cells, and the inhibition capability of the antibodies is obviously stronger than that of Secukinumab, and the results of FIG. 7C show that YN069 and YN070 have similar inhibition capability of HS-27 cells on the IL-8 production as that of YN 068.
The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.
Sequence listing
<110> original Biotechnology (Shanghai) Limited liability company
<120> IL17 antibodies and uses thereof
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<223> YN-008 VL
<400> 16
Glu Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 17
<211> 131
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011 VH
<400> 17
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Gly Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser
130
<210> 18
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011 VL
<400> 18
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 19
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009/012 HCDR1
<400> 19
Ser Tyr Ala Met Asn
1 5
<210> 20
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009/012 HCDR2
<400> 20
Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln
1 5 10 15
Gly
<210> 21
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009/012 HCDR3
<400> 21
Asp Gly Ser Gly Tyr Tyr Gly Gly Ser Gly Ala Phe Asp Ile
1 5 10
<210> 22
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009/012 LCDR1
<400> 22
Ser Gly Asp Asn Leu Gly Ser Lys Tyr Ala Ser
1 5 10
<210> 23
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009/012 LCDR2
<400> 23
Gln Asp Asn Lys Arg Pro Ser
1 5
<210> 24
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009/012 LCDR3
<400> 24
Gln Ala Trp Asp Ser Ser Thr Tyr Val
1 5
<210> 25
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 H-FR1
<400> 25
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
<210> 26
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 H-FR2
<400> 26
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
1 5 10
<210> 27
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 H-FR3
<400> 27
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu
1 5 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 28
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 H-FR4
<400> 28
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
1 5 10
<210> 29
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 L-FR1
<400> 29
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys
20
<210> 30
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 L-FR2
<400> 30
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
1 5 10 15
<210> 31
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 L-FR3
<400> 31
Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr
1 5 10 15
Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys
20 25 30
<210> 32
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 L-FR4
<400> 32
Phe Gly Ser Gly Thr Lys Leu Thr Val Leu Gly
1 5 10
<210> 33
<211> 123
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009 VH
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Ser Gly Tyr Tyr Gly Gly Ser Gly Ala Phe Asp Ile
100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 34
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009 VL
<400> 34
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile His
35 40 45
Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Tyr Val
85 90 95
Phe Gly Ser Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 35
<211> 123
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 VH
<400> 35
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Ser Gly Tyr Tyr Gly Gly Ser Gly Ala Phe Asp Ile
100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 36
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 VL
<400> 36
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Tyr Val
85 90 95
Phe Gly Ser Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 37
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010/013 HCDR1
<400> 37
Ser Asn His Met Ser
1 5
<210> 38
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010/013 HCDR2
<400> 38
Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 39
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010/013 HCDR3
<400> 39
Gly Arg Tyr Ser Gly Trp Tyr Phe Asp Tyr
1 5 10
<210> 40
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010/013 LCDR1
<400> 40
Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala Ser
1 5 10
<210> 41
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010/013 LCDR2
<400> 41
Gln Asp Asp Gln Arg Pro Ser
1 5
<210> 42
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010/013 LCDR3
<400> 42
Gln Thr Trp Asp His Gly Thr Ser Lys Tyr Val
1 5 10
<210> 43
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 H-FR1
<400> 43
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser
20 25 30
<210> 44
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 H-FR2
<400> 44
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
1 5 10
<210> 45
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 H-FR3
<400> 45
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 46
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 H-FR4
<400> 46
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 47
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 L-FR1
<400> 47
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys
20
<210> 48
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 L-FR2
<400> 48
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
1 5 10 15
<210> 49
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 L-FR3
<400> 49
Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr
1 5 10 15
Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys
20 25 30
<210> 50
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 L-FR4
<400> 50
Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly
1 5 10
<210> 51
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010 VH
<400> 51
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Arg Tyr Ser Gly Trp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 52
<211> 109
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010 VL
<400> 52
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Thr Ile Thr Cys Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Met Arg
35 40 45
Gln Asp Asp Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Val Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp His Gly Thr Ser Lys
85 90 95
Tyr Val Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 53
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 VH
<400> 53
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Arg Tyr Ser Gly Trp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 54
<211> 109
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 VL
<400> 54
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asp Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp His Gly Thr Ser Lys
85 90 95
Tyr Val Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 55
<211> 393
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-008 VH (nucleotide)
<400> 55
cagctgcagc tgcaggagtc tgggggaggc ttggtacagc cagggcggtc cccgagactc 60
tcctgtacag cttctggatt cacctttggt gattatgcta tgagctggtt ccgccaggct 120
ccagggaagg ggctggagtg ggtaggtttc attagaagca aagcttatgg tgggacaaca 180
gaatacgccg cgtctgtgaa aggcagattc accatctcaa gagatgattc caaaagcatc 240
gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtgcgaga 300
gatggttctg atgggatacg atattttgac tggttattca actactggta cttcgatctc 360
tggggccgtg gcaccctggt cactgtctcc tca 393
<210> 56
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-008 VL (nucleotide)
<400> 56
gaaattgtga tgacacaatc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtca gagcctcctg catagtaatg ggtacaacta tttggattgg 120
tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180
tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240
agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300
actttcggcg gagggaccaa gctggagatc aaacgt 336
<210> 57
<211> 369
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009 VH (nucleotide)
<400> 57
caggtgcagc tggtacagtc tgggtctgag ttgaagaagc ctggggcctc agtgaaggtt 60
tcctgcaagg cttctggata caccttcact agctatgcta tgaattgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180
gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagatggt 300
agtggttatt acgggggcag cggtgctttt gatatctggg gccaagggac aatggtcacc 360
gtctcctca 369
<210> 58
<211> 321
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009 VL (nucleotides)
<400> 58
tcctatgagc tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60
acctgctctg gagataactt ggggagtaaa tatgcttcct ggtatcaaca aaagccaggc 120
cagtcccctg tgctggtcat ccatcaagat aacaagcggc cctcagggat ccctgagcga 180
ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240
gatgaggctg actattactg tcaggcgtgg gacagcagta cttatgtctt cggatctggg 300
accaagctga ccgtcctagg t 321
<210> 59
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010 VH (nucleotide)
<400> 59
caggtcacct tgaaggagtc tgggggaggc ttgatccagc ctggggggtc cctgagactc 60
tcctgtgcaa cctctgggtt caccgtcagt agcaaccaca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaatt atttatagcg atggtaccac atactacgca 180
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtctctt 240
caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agggcgatac 300
agtggctggt actttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354
<210> 60
<211> 327
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010 VL (nucleotide)
<400> 60
tcctatgagc tgactcagcc accctctgtg tctgtgtccc caggacagac agccacaatc 60
acctgctctg gagatgattt gggtagcaaa tatgcttcct ggtatcaaca gaggccaggc 120
cagtcccctg tcttggtcat gcgtcaggat gaccagcggc cctcagggat ccctgagcgg 180
ttctctggct ccaactctgg gaacactgcc accctgacca tcagcgggac tcaggttatg 240
gatgaggctg actattactg tcagacgtgg gaccacggca cctccaagta tgtcttcgga 300
cctgggacca agctcaccgt cctaggt 327
<210> 61
<211> 1383
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-008 heavy chain (nucleotide)
<400> 61
cagctgcagc tgcaggagtc tgggggaggc ttggtacagc cagggcggtc cccgagactc 60
tcctgtacag cttctggatt cacctttggt gattatgcta tgagctggtt ccgccaggct 120
ccagggaagg ggctggagtg ggtaggtttc attagaagca aagcttatgg tgggacaaca 180
gaatacgccg cgtctgtgaa aggcagattc accatctcaa gagatgattc caaaagcatc 240
gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtgcgaga 300
gatggttctg atgggatacg atattttgac tggttattca actactggta cttcgatctc 360
tggggccgtg gcaccctggt cactgtctcc tcagcgtcga ccaagggccc atcggtcttc 420
cccctggcac cctcctccaa gagcacctct gggggcacag cggccctggg ctgcctggtc 480
aaggactact tccccgaacc ggtgacggtg tcgtggaact caggcgccct gaccagcggc 540
gtgcacacct tcccggctgt cctacagtcc tcaggactct actccctcag cagcgtggtg 600
accgtgccct ccagcagctt gggcacccag acctacatct gcaacgtgaa tcacaagccc 660
agcaacacca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc 720
ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa 780
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 840
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 900
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 960
accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 1020
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1080
caggtgtaca ccctgccccc atcccgggaa gagatgacca agaaccaggt cagcctgacc 1140
tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1200
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1260
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1320
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1380
aaa 1383
<210> 62
<211> 461
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-008 heavy chain
<400> 62
Gln Leu Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Pro Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Gly Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 63
<211> 654
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-008 light chain (nucleotide)
<400> 63
gaaattgtga tgacacaatc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtca gagcctcctg catagtaatg ggtacaacta tttggattgg 120
tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180
tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240
agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300
actttcggcg gagggaccaa gctggagatc aaacgtacgg tggctgcacc atctgtcttc 360
atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420
aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 480
ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 540
agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 600
acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgc 654
<210> 64
<211> 218
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-008 light chain
<400> 64
Glu Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 65
<211> 1359
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009 heavy chain (nucleotide)
<400> 65
caggtgcagc tggtacagtc tgggtctgag ttgaagaagc ctggggcctc agtgaaggtt 60
tcctgcaagg cttctggata caccttcact agctatgcta tgaattgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180
gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagatggt 300
agtggttatt acgggggcag cggtgctttt gatatctggg gccaagggac aatggtcacc 360
gtctcctcag cgtcgaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420
acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 480
acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 540
cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600
acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660
gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720
ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggaagaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaa 1359
<210> 66
<211> 453
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009 heavy chain
<400> 66
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Ser Gly Tyr Tyr Gly Gly Ser Gly Ala Phe Asp Ile
100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Lys
450
<210> 67
<211> 639
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009 light chain (nucleotide)
<400> 67
tcctatgagc tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60
acctgctctg gagataactt ggggagtaaa tatgcttcct ggtatcaaca aaagccaggc 120
cagtcccctg tgctggtcat ccatcaagat aacaagcggc cctcagggat ccctgagcga 180
ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240
gatgaggctg actattactg tcaggcgtgg gacagcagta cttatgtctt cggatctggg 300
accaagctga ccgtcctagg tacggtggct gcaccatctg tcttcatctt cccgccatct 360
gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctacccc 420
agagaagcca aagtgcagtg gaaggtggac aacgccctgc agagcggaaa cagccaggaa 480
agcgtgacag agcaggattc caaggattcc acatacagcc tgagcagcac actgacactg 540
tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgacaca ccagggactg 600
tcctcccctg tgacaaagag cttcaacaga ggagaatgc 639
<210> 68
<211> 213
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-009 light chain
<400> 68
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile His
35 40 45
Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Tyr Val
85 90 95
Phe Gly Ser Gly Thr Lys Leu Thr Val Leu Gly Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 69
<211> 1344
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010 heavy chain (nucleotide)
<400> 69
caggtcacct tgaaggagtc tgggggaggc ttgatccagc ctggggggtc cctgagactc 60
tcctgtgcaa cctctgggtt caccgtcagt agcaaccaca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaatt atttatagcg atggtaccac atactacgca 180
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtctctt 240
caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agggcgatac 300
agtggctggt actttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcgtcg 360
accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420
gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480
tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540
tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600
tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660
tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780
acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840
gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900
taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960
aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga agagatgacc 1080
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200
tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320
agcctctccc tgtctccggg taaa 1344
<210> 70
<211> 448
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010 heavy chain
<400> 70
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Arg Tyr Ser Gly Trp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 71
<211> 645
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010 light chain (nucleotide)
<400> 71
tcctatgagc tgactcagcc accctctgtg tctgtgtccc caggacagac agccacaatc 60
acctgctctg gagatgattt gggtagcaaa tatgcttcct ggtatcaaca gaggccaggc 120
cagtcccctg tcttggtcat gcgtcaggat gaccagcggc cctcagggat ccctgagcgg 180
ttctctggct ccaactctgg gaacactgcc accctgacca tcagcgggac tcaggttatg 240
gatgaggctg actattactg tcagacgtgg gaccacggca cctccaagta tgtcttcgga 300
cctgggacca agctcaccgt cctaggtacg gtggctgcac catctgtctt catcttcccg 360
ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420
taccccagag aagccaaagt gcagtggaag gtggacaacg ccctgcagag cggaaacagc 480
caggaaagcg tgacagagca ggattccaag gattccacat acagcctgag cagcacactg 540
acactgtcca aggccgacta cgagaagcac aaggtgtacg cctgcgaagt gacacaccag 600
ggactgtcct cccctgtgac aaagagcttc aacagaggag aatgc 645
<210> 72
<211> 215
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-010 light chain
<400> 72
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Thr Ile Thr Cys Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Met Arg
35 40 45
Gln Asp Asp Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Val Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp His Gly Thr Ser Lys
85 90 95
Tyr Val Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 73
<211> 393
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011 VH (nucleotide)
<400> 73
caggtgcagc tggtggagtc tgggggaggc ttggtacagc cagggcggtc cctgagactc 60
tcctgtacag cttctggatt cacctttggt gattatgcta tgagctggtt ccgccaggct 120
ccagggaagg ggctggagtg ggtaggtttc attagaagca aagcttatgg tgggacaaca 180
gaatacgccg cgtctgtgaa aggcagattc accatctcaa gagatgattc caaaagcatc 240
gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtgcgaga 300
gatggttctg atgggatacg atattttgac tggttattca actactggta cttcgatctc 360
tggggccgtg gcaccctggt cactgtctcc tca 393
<210> 74
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011 VL (nucleotides)
<400> 74
gacattgtga tgacacaatc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtca gagcctcctg catagtaatg ggtacaacta tttggattgg 120
tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180
tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240
agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300
actttcggcg gagggaccaa gctggagatc aaacgt 336
<210> 75
<211> 1383
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011 heavy chain (nucleotide)
<400> 75
caggtgcagc tggtggagtc tgggggaggc ttggtacagc cagggcggtc cctgagactc 60
tcctgtacag cttctggatt cacctttggt gattatgcta tgagctggtt ccgccaggct 120
ccagggaagg ggctggagtg ggtaggtttc attagaagca aagcttatgg tgggacaaca 180
gaatacgccg cgtctgtgaa aggcagattc accatctcaa gagatgattc caaaagcatc 240
gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtgcgaga 300
gatggttctg atgggatacg atattttgac tggttattca actactggta cttcgatctc 360
tggggccgtg gcaccctggt cactgtctcc tcagcgtcga ccaagggccc atcggtcttc 420
cccctggcac cctcctccaa gagcacctct gggggcacag cggccctggg ctgcctggtc 480
aaggactact tccccgaacc ggtgacggtg tcgtggaact caggcgccct gaccagcggc 540
gtgcacacct tcccggctgt cctacagtcc tcaggactct actccctcag cagcgtggtg 600
accgtgccct ccagcagctt gggcacccag acctacatct gcaacgtgaa tcacaagccc 660
agcaacacca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc 720
ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa 780
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 840
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 900
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 960
accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 1020
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1080
caggtgtaca ccctgccccc atcccgggaa gagatgacca agaaccaggt cagcctgacc 1140
tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1200
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1260
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1320
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1380
aaa 1383
<210> 76
<211> 461
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011 heavy chain
<400> 76
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Gly Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 77
<211> 654
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011 light chain (nucleotide)
<400> 77
gacattgtga tgacacaatc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60
atctcctgca ggtctagtca gagcctcctg catagtaatg ggtacaacta tttggattgg 120
tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180
tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240
agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300
actttcggcg gagggaccaa gctggagatc aaacgtacgg tggctgcacc atctgtcttc 360
atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420
aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 480
ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 540
agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 600
acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgc 654
<210> 78
<211> 218
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011 light chain
<400> 78
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 79
<211> 369
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 VH (nucleotide)
<400> 79
caggtgcagc tggtacagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60
tcctgcaagg cttctggata caccttcact agctatgcta tgaattgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180
gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagatggt 300
agtggttatt acgggggcag cggtgctttt gatatctggg gccaagggac aatggtcacc 360
gtctcctca 369
<210> 80
<211> 321
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 VL (nucleotide)
<400> 80
tcctatgagc tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60
acctgctctg gagataactt ggggagtaaa tatgcttcct ggtatcaaca aaagccaggc 120
cagtcccctg tgctggtcat ctatcaagat aacaagcggc cctcagggat ccctgagcga 180
ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240
gatgaggctg actattactg tcaggcgtgg gacagcagta cttatgtctt cggatctggg 300
accaagctga ccgtcctagg t 321
<210> 81
<211> 1359
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 heavy chain (nucleotide)
<400> 81
caggtgcagc tggtacagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60
tcctgcaagg cttctggata caccttcact agctatgcta tgaattgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180
gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagatggt 300
agtggttatt acgggggcag cggtgctttt gatatctggg gccaagggac aatggtcacc 360
gtctcctcag cgtcgaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420
acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 480
acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 540
cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600
acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660
gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720
ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggaagaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaa 1359
<210> 82
<211> 453
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 heavy chain
<400> 82
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Ser Gly Tyr Tyr Gly Gly Ser Gly Ala Phe Asp Ile
100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445
Leu Ser Pro Gly Lys
450
<210> 83
<211> 639
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 light chain (nucleotide)
<400> 83
tcctatgagc tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60
acctgctctg gagataactt ggggagtaaa tatgcttcct ggtatcaaca aaagccaggc 120
cagtcccctg tgctggtcat ctatcaagat aacaagcggc cctcagggat ccctgagcga 180
ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240
gatgaggctg actattactg tcaggcgtgg gacagcagta cttatgtctt cggatctggg 300
accaagctga ccgtcctagg tacggtggct gcaccatctg tcttcatctt cccgccatct 360
gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctacccc 420
agagaagcca aagtgcagtg gaaggtggac aacgccctgc agagcggaaa cagccaggaa 480
agcgtgacag agcaggattc caaggattcc acatacagcc tgagcagcac actgacactg 540
tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgacaca ccagggactg 600
tcctcccctg tgacaaagag cttcaacaga ggagaatgc 639
<210> 84
<211> 213
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-012 light chain
<400> 84
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Tyr Val
85 90 95
Phe Gly Ser Gly Thr Lys Leu Thr Val Leu Gly Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 85
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 VH (nucleotides)
<400> 85
caggtcacct tgaaggagtc tgggggaggc ttgatccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctgggtt caccgtcagt agcaaccaca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaatt atttatagcg atggtaccac atactacgca 180
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240
caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agggcgatac 300
agtggctggt actttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354
<210> 86
<211> 327
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 VL (nucleotides)
<400> 86
tcctatgagc tgactcagcc accctctgtg tctgtgtccc caggacagac agcctcaatc 60
acctgctctg gagatgattt gggtagcaaa tatgcttcct ggtatcaaca gaagccaggc 120
cagtcccctg tcttggtcat ttaccaggat gaccagcggc cctcagggat ccctgagcgg 180
ttctctggct ccaactctgg gaacactgcc accctgacca tcagcgggac tcaggctatg 240
gatgaggctg actattactg tcagacgtgg gaccacggca cctccaagta tgtcttcgga 300
cctgggacca agctcaccgt cctaggt 327
<210> 87
<211> 1344
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 heavy chain (nucleotide)
<400> 87
caggtcacct tgaaggagtc tgggggaggc ttgatccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctgggtt caccgtcagt agcaaccaca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaatt atttatagcg atggtaccac atactacgca 180
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240
caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agggcgatac 300
agtggctggt actttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcgtcg 360
accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420
gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480
tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540
tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600
tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660
tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780
acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840
gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900
taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960
aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga agagatgacc 1080
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200
tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320
agcctctccc tgtctccggg taaa 1344
<210> 88
<211> 448
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 heavy chain
<400> 88
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Arg Tyr Ser Gly Trp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 89
<211> 645
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 light chain (nucleotide)
<400> 89
tcctatgagc tgactcagcc accctctgtg tctgtgtccc caggacagac agcctcaatc 60
acctgctctg gagatgattt gggtagcaaa tatgcttcct ggtatcaaca gaagccaggc 120
cagtcccctg tcttggtcat ttaccaggat gaccagcggc cctcagggat ccctgagcgg 180
ttctctggct ccaactctgg gaacactgcc accctgacca tcagcgggac tcaggctatg 240
gatgaggctg actattactg tcagacgtgg gaccacggca cctccaagta tgtcttcgga 300
cctgggacca agctcaccgt cctaggtacg gtggctgcac catctgtctt catcttcccg 360
ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420
taccccagag aagccaaagt gcagtggaag gtggacaacg ccctgcagag cggaaacagc 480
caggaaagcg tgacagagca ggattccaag gattccacat acagcctgag cagcacactg 540
acactgtcca aggccgacta cgagaagcac aaggtgtacg cctgcgaagt gacacaccag 600
ggactgtcct cccctgtgac aaagagcttc aacagaggag aatgc 645
<210> 90
<211> 215
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013 light chain
<400> 90
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asp Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp His Gly Thr Ser Lys
85 90 95
Tyr Val Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 91
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067/070 HCDR1
<400> 91
Asp Tyr Ala Met Asn
1 5
<210> 92
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-069 HCDR1
<400> 92
Asp Tyr Ala Ile Asn
1 5
<210> 93
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-070 HCDR2
<400> 93
Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Thr Ser
1 5 10 15
Val Lys Gly
<210> 94
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067/069/070 HCDR3
<400> 94
Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu Phe Asn Tyr Trp
1 5 10 15
Tyr Phe Asp Leu
20
<210> 95
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-068 HCDR3
<400> 95
Asp Gly Ser Asp Glu Ile Arg Tyr Leu Asp Trp Leu Phe Asn Tyr Trp
1 5 10 15
Tyr Phe Asp Leu
20
<210> 96
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067/068/069/070 LCDR1
<400> 96
Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Glu
1 5 10 15
<210> 97
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067/068/069/070 LFR1
<400> 97
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu His Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys
20
<210> 98
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067/068/069/070 LFR2
<400> 98
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Pro
1 5 10
<210> 99
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-070 HFR3
<400> 99
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile Ala Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Thr Gln Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 100
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-065 HCDR3
<400> 100
Gly Gly Asn Ser Asp Trp Asn Phe Asp Tyr
1 5 10
<210> 101
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-066 HCDR3
<400> 101
Gly Arg Asn Ser Gly Phe Ser Phe Asp Tyr
1 5 10
<210> 102
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-065/YN-066 LCDR3
<400> 102
Gln Thr Trp Asp Tyr Trp Thr Ser Lys Tyr Val
1 5 10
<210> 103
<211> 109
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-065/YN-066 VL
<400> 103
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asp Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp Tyr Trp Thr Ser Lys
85 90 95
Tyr Val Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 104
<211> 215
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-065/YN-066 light chain
<400> 104
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asp Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp Tyr Trp Thr Ser Lys
85 90 95
Tyr Val Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 105
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-065 VH
<400> 105
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Gly Asn Ser Asp Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 106
<211> 448
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-065 heavy chain
<400> 106
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Gly Asn Ser Asp Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 107
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-066 VH
<400> 107
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Arg Asn Ser Gly Phe Ser Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 108
<211> 448
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-066 heavy chain
<400> 108
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Arg Asn Ser Gly Phe Ser Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 109
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067/YN-068/YN-069/YN-070 VL
<400> 109
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu His Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Pro Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 110
<211> 218
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067/YN-067 Fab/YN-068/YN-068
Fab/YN-069/YN-069Fab/YN-070/YN-070 Fab light chain
<400> 110
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu His Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Pro Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 111
<211> 131
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067 VH
<400> 111
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser
130
<210> 112
<211> 461
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067 heavy chain
<400> 112
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 113
<211> 236
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-067Fab heavy chain
<400> 113
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
225 230 235
<210> 114
<211> 131
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-068 VH
<400> 114
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Leu Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser
130
<210> 115
<211> 461
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-068 heavy chain
<400> 115
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Leu Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 116
<211> 236
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-068Fab heavy chain
<400> 116
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Leu Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
225 230 235
<210> 117
<211> 131
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-069 VH
<400> 117
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Ile Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser
130
<210> 118
<211> 461
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-069 heavy chain
<400> 118
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Ile Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 119
<211> 236
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-069Fab heavy chain
<400> 119
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Ile Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
225 230 235
<210> 120
<211> 131
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-070 VH
<400> 120
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Thr
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Gln Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser
130
<210> 121
<211> 461
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-070 heavy chain
<400> 121
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Thr
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Gln Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 122
<211> 236
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN070 Fab heavy chain
<400> 122
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Thr
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Gln Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Glu Ile Arg Tyr Phe Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
225 230 235
<210> 123
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 HCDR1 general formula
<220>
<221> X4
<222> (4)..(4)
<223> X4 = M or I
<220>
<221> X5
<222> (5)..(5)
<223> X5 = S or N
<220>
<221> X5
<222> (5)..(5)
<223> X5 = S or N
<400> 123
Asp Tyr Ala Xaa Xaa
1 5
<210> 124
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 HCDR2 general formula
<220>
<221> X15
<222> (15)..(15)
<223> X15 = T or A
<400> 124
Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Xaa Ser
1 5 10 15
Val Lys Gly
<210> 125
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 HCDR3 general formula
<220>
<221> X5
<222> (5)..(5)
<223> X5 = G or E
<220>
<221> X9
<222> (9)..(9)
<223> X9 = F or L
<400> 125
Asp Gly Ser Asp Xaa Ile Arg Tyr Xaa Asp Trp Leu Phe Asn Tyr Trp
1 5 10 15
Tyr Phe Asp Leu
20
<210> 126
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 LCDR1 general formula
<220>
<221> X16
<222> (16)..(16)
<223> X16 = D or E
<400> 126
Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Xaa
1 5 10 15
<210> 127
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 LFR1 general formula
<220>
<221> X12
<222> (12)..(12)
<223> X12= P or H
<400> 127
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Xaa Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys
20
<210> 128
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 LFR2 general formula
<220>
<221> X12
<222> (12)..(12)
<223> X12= L or P
<400> 128
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Xaa Leu Ile Tyr
1 5 10 15
<210> 129
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 HFR3 general formula
<220>
<221> X23
<222> (23)..(23)
<223> X23 = E or Q
<400> 129
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile Ala Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Lys Thr Xaa Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 130
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013/065/YN-066 HCDR3 general formula
<220>
<221> X2
<222> (2)..(2)
<223> X2 = R or G
<220>
<221> X3
<222> (3)..(3)
<223> X3 = Y or N
<220>
<221> X5
<222> (5)..(5)
<223> X5 = G or D
<220>
<221> X6
<222> (6)..(6)
<223> X6 = W or F
<220>
<221> X7
<222> (7)..(7)
<223> X7 = Y or N or S
<400> 130
Gly Xaa Xaa Ser Xaa Xaa Xaa Phe Asp Tyr
1 5 10
<210> 131
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013/065/YN-066 LCDR3 general formula
<220>
<221> X5
<222> (5)..(5)
<223> X5 = H or Y
<220>
<221> X6
<222> (6)..(6)
<223> X6 = G or W
<400> 131
Gln Thr Trp Asp Xaa Xaa Thr Ser Lys Tyr Val
1 5 10
<210> 132
<211> 109
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013/065/YN-066 VL formula
<220>
<221> X92
<222> (92)..(92)
<223> X92= H or Y
<220>
<221> X93
<222> (93)..(93)
<223> X93= G or W
<400> 132
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asp Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp Xaa Xaa Thr Ser Lys
85 90 95
Tyr Val Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 133
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013/065/YN-066 VH general formula
<220>
<221> X99
<222> (99)..(99)
<223> X99= R or G
<220>
<221> X100
<222> (100)..(100)
<223> X100= Y or N
<220>
<221> X102
<222> (102)..(102)
<223> X102= G or D
<220>
<221> X103
<222> (103)..(103)
<223> X103= W or F
<220>
<221> X104
<222> (104)..(104)
<223> X104= Y or N or S
<400> 133
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Xaa Xaa Ser Xaa Xaa Xaa Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 134
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 VL general formula
<220>
<221> X12
<222> (12)..(12)
<223> X12= P or H
<220>
<221> X39
<222> (39)..(39)
<223> X39= D or E
<220>
<221> X51
<222> (51)..(51)
<223> X51= L or P
<400> 134
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Xaa Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Xaa Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Xaa Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 135
<211> 131
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 VH general formula
<220>
<221> X34
<222> (34)..(34)
<223> X34= M or I
<220>
<221> X35
<222> (35)..(35)
<223> X35= S or N
<220>
<221> X54
<222> (54)..(54)
<223> X54= A or T
<220>
<221> misc_feature
<222> (64)..(64)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> X91
<222> (91)..(91)
<223> X91= E or Q
<220>
<221> X105
<222> (105)..(105)
<223> X105= G or E
<220>
<221> X109
<222> (109)..(109)
<223> X109= F or L
<400> 135
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Xaa Xaa Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Xaa
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Xaa Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Xaa Ile Arg Tyr Xaa Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser
130
<210> 136
<211> 215
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013/065/YN-066 light chain general formula
<220>
<221> 92
<222> (92)..(92)
<223> X92= H or Y
<220>
<221> 93
<222> (93)..(93)
<223> X93= G or W
<400> 136
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Asp Leu Gly Ser Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asp Gln Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Asp Xaa Xaa Thr Ser Lys
85 90 95
Tyr Val Phe Gly Pro Gly Thr Lys Leu Thr Val Leu Gly Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 137
<211> 448
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-013/065/YN-066 heavy chain general formula
<220>
<221> X99
<222> (99)..(99)
<223> X99= R or G
<220>
<221> X100
<222> (100)..(100)
<223> X100= Y or N
<220>
<221> X102
<222> (102)..(102)
<223> X102= G or D
<220>
<221> X103
<222> (103)..(103)
<223> X103= W or F
<220>
<221> X104
<222> (104)..(104)
<223> X104= Y or N or S
<400> 137
Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Xaa Xaa Ser Xaa Xaa Xaa Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 138
<211> 218
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 light chain general formula
<220>
<221> X12
<222> (12)..(12)
<223> X12= P or H
<220>
<221> X39
<222> (39)..(39)
<223> X39= D or E
<220>
<221> X51
<222> (51)..(51)
<223> X51= L or P
<400> 138
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Xaa Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asn Gly Tyr Asn Tyr Leu Xaa Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Xaa Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95
Leu Gln Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 139
<211> 461
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> YN-011/067/068/069/070 general formula of heavy chain
<220>
<221> X34
<222> (34)..(34)
<223> X34= M or I
<220>
<221> X35
<222> (35)..(35)
<223> X35= S or N
<220>
<221> X54
<222> (54)..(54)
<223> X54= A or T
<220>
<221> misc_feature
<222> (64)..(64)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> X91
<222> (91)..(91)
<223> X91= E or Q
<220>
<221> X105
<222> (105)..(105)
<223> X105= G or E
<220>
<221> X109
<222> (109)..(109)
<223> X109= F or L
<400> 139
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30
Ala Xaa Xaa Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Xaa
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Xaa Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Gly Ser Asp Xaa Ile Arg Tyr Xaa Asp Trp Leu
100 105 110
Phe Asn Tyr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
115 120 125
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
130 135 140
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
145 150 155 160
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
165 170 175
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
180 185 190
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
195 200 205
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
210 215 220
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460

Claims (21)

1. An antibody or antigen-binding fragment thereof capable of binding to an IL17 protein, comprising an antibody light chain or fragment thereof comprising LCDR1, LCDR2 and LCDR3 and an antibody heavy chain or fragment thereof comprising HCDR1, HCDR2 and HCDR3, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence selected from any one of:
(1) The amino acid sequence of the LCDR1 is shown as SEQ ID NO.40, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.41, the amino acid sequence of the LCDR3 is shown as SEQ ID NO.42, and the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 37; the amino acid sequence of the HCDR2 is shown as SEQ ID NO.38, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 39;
(2) The amino acid sequence of the LCDR1 is shown as SEQ ID NO.40, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.41, the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 102, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.37, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.38, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 100; and
(3) The amino acid sequence of the LCDR1 is shown as SEQ ID NO.40, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.41, the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 102, the amino acid sequence of the HCDR1 is shown as SEQ ID NO.37, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.38, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 101.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a monoclonal antibody.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises: single chain antibodies and/or humanized antibodies.
4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises: chimeric antibodies and/or fully human antibodies.
5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody light chain or fragment thereof comprises a light chain variable region (VL) and the amino acid sequence of the light chain variable region VL is as set forth in any one of SEQ ID NO 52, SEQ ID NO 54 and SEQ ID NO 103.
6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody light chain or fragment thereof further comprises a human constant region or a murine constant region.
7. The antibody or antigen-binding fragment thereof of claim 1, the antibody light chain or fragment thereof comprises a human constant region, and the human constant region comprises a human Ig kappa or Ig lambda constant region.
8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the antibody light chain or fragment thereof is set forth in any one of SEQ ID NO 72, SEQ ID NO 90, and SEQ ID NO 104.
9. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody heavy chain or fragment thereof comprises a heavy chain variable region VH having an amino acid sequence as set forth in any one of SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 105, and SEQ ID NO 107.
10. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody heavy chain or fragment thereof further comprises a human constant region or a murine constant region.
11. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody heavy chain or fragment thereof comprises a human constant region, and the human constant region comprises a human IgG constant region.
12. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of said antibody heavy chain or fragment thereof is set forth in any one of SEQ ID NO 70, SEQ ID NO 88, SEQ ID NO 106, and SEQ ID NO 108.
13. The antibody or antigen-binding fragment thereof of claim 1, wherein the IL17 protein is a human IL17 protein.
14. The antibody or antigen-binding fragment thereof of claim 1, wherein the IL17 protein comprises an IL17A protein and/or an IL17F protein.
15. An isolated one or more nucleic acid molecules encoding the antibody or antigen-binding fragment thereof of any one of claims 1-14.
16. A vector comprising the nucleic acid molecule of claim 15.
17. A host cell comprising the nucleic acid molecule of claim 15 or the vector of claim 16.
18. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-14, the method comprising culturing the host cell of claim 17 under conditions such that the antibody or antigen-binding fragment thereof of any one of claims 1-14 is expressed.
19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, and/or the host cell of claim 17, and optionally a pharmaceutically acceptable adjuvant.
20. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-14 in the preparation of an agent for inhibiting the binding of an IL17 protein to an IL17 ligand.
21. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-14 in the preparation of an agent for inhibiting the expression of chemokine CXCL1.
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