Antigen-binding proteins targeting pneumolysin and their applications.

Abstract

The present application relates to an isolated antigen binding protein, comprising at least one CDR within an antibody heavy chain variable region VH, said VH comprising the amino acid sequence shown in SEQ ID NO: 39. The isolated antigen binding protein is capable of specifically binding to pneumolysin protein and variants thereof, and the present application also provides an application of the isolated antigen binding protein in the prevention and / or treatment of diseases and / or symptoms caused by Streptococcus pneumoniae infection.

Description

Detailed Description of the Invention

[0001] [Technical field] The present application relates to the biomedical field, specifically to antigen-binding proteins capable of targeting the pneumolysin protein (PLY), and their application in the field of treatment and prevention of Streptococcus pneumoniae infections.

[0002] [Background technology] Streptococcus pneumoniae, also known as the pneumococcus, is widely colonized in the human nasopharynx and can cause infections of the lungs and central nervous system when the body's resistance is weakened, and in severe cases can cause bacteremia or sepsis.

[0003] Hemolysin (PLY) is an important virulence factor of Streptococcus pneumoniae and plays a key role in the S. pneumoniae infection process. PLY can destroy lung tissue epithelial, endothelial, and immune cells, increase vascular permeability and the colonization, spread, and invasion of S. pneumoniae, affect the lung function of patients, and cause acute lung injury and even the formation of acute respiratory distress syndrome. Therefore, there is an urgent need to develop effective drugs against S. pneumoniae infection.

[0004] [Summary of the Invention] The present application provides isolated antigen-binding proteins having one or more properties selected from: 1) being capable of specifically binding to pneumolysin protein and its variants; 2) having high affinity and neutralizing activity for pneumolysin protein; 3) being capable of effectively blocking infections caused by Streptococcus pneumoniae; and 4) being capable of effectively preventing and / or treating diseases and / or symptoms caused by Streptococcus pneumoniae.

[0005] Meanwhile, the present application provides an isolated antigen binding protein, comprising at least one CDR within an antibody heavy chain variable region VH, said VH comprising the amino acid sequence shown in SEQ ID NO:39.

[0006] In some embodiments, the isolated antigen binding protein comprises an HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.

[0007] In some embodiments, the isolated antigen binding protein comprises an HCDR2, wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2.

[0008] In some embodiments, the isolated antigen binding protein comprises an HCDR1, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

[0009] In some embodiments, the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, said HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:1.

[0010] In some embodiments, the isolated antigen binding protein comprises H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:32.

[0011] In some embodiments, the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:15.

[0012] In some embodiments, the isolated antigen binding protein comprises H-FR2, wherein the H-FR2 is positioned between the HCDR1 and the HCDR2, and wherein the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:33.

[0013] In some embodiments, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:16.

[0014] In some embodiments, the isolated antigen binding protein comprises H-FR3, wherein said H-FR3 is positioned between said HCDR2 and said HCDR3, and said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:34.

[0015] In some embodiments, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:17.

[0016] In some embodiments, the isolated antigen binding protein comprises an H-FR4, wherein the N-terminus of the H-FR4 is linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:35.

[0017] In some embodiments, the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:18.

[0018] In some embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:32, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:33, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:34, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:35.

[0019] In some embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:15, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:16, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:17, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:18.

[0020] In some embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4 selected from any one of the following groups: 1) said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 8, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 9, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 10; and 2) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 15, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 16, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 17, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 18.

[0021] In some embodiments, the isolated antigen binding protein comprises a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:39.

[0022] In some embodiments, the VH of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:24.

[0023] In some embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.

[0024] In some embodiments, the heavy chain constant region of the isolated antigen binding protein is derived from a human IgG constant region.

[0025] In some embodiments, the heavy chain constant region of the isolated antigen binding protein can be derived from a human IgG1 heavy chain constant region.

[0026] In some embodiments, the heavy chain constant region of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:26.

[0027] In some embodiments, the isolated antigen binding protein comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:28 or SEQ ID NO:30.

[0028] In some embodiments, the isolated antigen binding protein comprises at least one CDR within an antibody light chain variable region VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO:40.

[0029] In some embodiments, the isolated antigen binding protein comprises an LCDR3, wherein the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:4.

[0030] In some embodiments, the isolated antigen binding protein comprises an LCDR2, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5.

[0031] In some embodiments, the isolated antigen binding protein comprises an LCDR1, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:6.

[0032] In some embodiments, the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:4.

[0033] In some embodiments, the isolated antigen binding protein comprises L-FR1, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:36.

[0034] In some embodiments, the L-FR1 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:19.

[0035] In some embodiments, the isolated antigen binding protein comprises an L-FR2, wherein the L-FR2 is located between the LCDR1 and LCDR2, and wherein the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:37.

[0036] In some embodiments, the L-FR2 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:20.

[0037] In some embodiments, the isolated antigen binding protein comprises an L-FR3, wherein the L-FR3 is positioned between the LCDR2 and the LCDR3, and wherein the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:38.

[0038] In some embodiments, the L-FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:13 or SEQ ID NO:21.

[0039] In some embodiments, the isolated antigen binding protein comprises an L-FR4, wherein the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:14.

[0040] In some embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein said L-FR1 comprises the amino acid sequence set forth in SEQ ID NO:36, said L-FR2 comprises the amino acid sequence set forth in SEQ ID NO:37, said L-FR3 comprises the amino acid sequence set forth in SEQ ID NO:38, and said L-FR4 comprises the amino acid sequence set forth in SEQ ID NO:14.

[0041] In some embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein said L-FR1 comprises the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:19, said L-FR2 comprises the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:20, said L-FR3 comprises the amino acid sequence set forth in SEQ ID NO:13 or SEQ ID NO:21, and said L-FR4 comprises the amino acid sequence set forth in SEQ ID NO:14.

[0042] In some embodiments, the isolated antigen binding protein comprises an L-FR1, an L-FR2, an L-FR3, and an L-FR4 selected from the group consisting of any one of the following: 1) said L-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, said L-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, said L-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, and said L-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 14; and 2) the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 20, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 21, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 14.

[0043] In some embodiments, the isolated antigen binding protein comprises a VL, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO:40.

[0044] In some embodiments, the VL of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:25.

[0045] In some embodiments, the isolated antigen binding protein comprises an antibody light chain constant region.

[0046] In some embodiments, the light chain constant region of the isolated antigen binding protein is derived from a human Igκ constant region.

[0047] In some embodiments, the light chain constant region of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:27.

[0048] In some embodiments, the isolated antigen binding protein comprises a light chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:31.

[0049] In some embodiments, the isolated antigen binding protein comprises a VH and a VL selected from any one of the following groups: 1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 22, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 23; and 2) the VH comprises the amino acid sequence shown in SEQ ID NO:24, and the VL comprises the amino acid sequence shown in SEQ ID NO:25.

[0050] In some embodiments, the isolated antigen binding protein comprises an antibody or an antigen-binding fragment thereof.

[0051] In some embodiments, the antigen-binding fragment comprises a Fab, a Fab', an Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv, and / or a dAb.

[0052] In some embodiments, the antibody is one or more selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0053] In some embodiments, the isolated antigen binding protein is capable of specifically binding to a pneumolysin protein.

[0054] In some embodiments, the pneumolysin comprises wild-type pneumolysin protein and variants thereof.

[0055] In some embodiments, the pneumolysin protein comprises a pneumolysin protein variant having an amino acid deletion at position 146 relative to wild-type pneumolysin protein.

[0056] In some embodiments, the isolated antigen binding protein is capable of preventing and / or treating disease and / or symptoms caused by Streptococcus pneumoniae.

[0057] In some embodiments, the disease and / or condition comprises a complication of a disease and / or condition caused by Streptococcus pneumoniae.

[0058] However, the present application further provides a polypeptide molecule comprising said isolated antigen binding protein.

[0059] In some embodiments, the polypeptide molecule comprises a fusion protein.

[0060] However, the present application further provides an immunoconjugate comprising said isolated antigen-binding protein.

[0061] Meanwhile, the present application further provides a nucleic acid molecule encoding said isolated antigen binding protein or said polypeptide molecule.

[0062] On the other hand, the present application further provides a vector comprising said nucleic acid molecule.

[0063] Meanwhile, the present application further provides a cell comprising said nucleic acid molecule or said vector.

[0064] Meanwhile, the present application further provides a pharmaceutical composition comprising said isolated antigen-binding protein, said polypeptide molecule, said immunoconjugate, said nucleic acid molecule, said vector and / or said cell, and optionally a pharma- ceutically acceptable carrier.

[0065] Meanwhile, the present application further provides a method of preparing said isolated antigen binding protein, comprising culturing said cell under conditions to express said antigen binding protein.

[0066] Alternatively, the antigen-binding proteins, the polypeptide molecules, the immunoconjugates, the nucleic acid molecules, the vectors, the cells and / or the pharmaceutical compositions described in the present application may be used alone or in combination with other drugs.

[0067] Meanwhile, the present application further provides the use of said isolated antigen-binding protein, said polypeptide molecule, said immunoconjugate, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a disease and / or a condition.

[0068] In some embodiments, the disease and / or condition and its complications are caused or mediated by Streptococcus pneumoniae.

[0069] In some embodiments, the disease and / or condition comprises a complication of a disease and / or condition caused or mediated by Streptococcus pneumoniae.

[0070] Meanwhile, the present application further provides a method for detecting pneumolysin protein in a sample, the method comprising administering the isolated antigen-binding protein, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0071] On the other hand, the present application further provides a reagent kit for detecting pneumolysin protein in a sample, comprising the isolated antigen-binding protein, the polypeptide molecule, the immune complex, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0072] Meanwhile, the present application further provides the use of said isolated antigen-binding protein, said polypeptide molecule, said immune complex, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the manufacture of a reagent kit for detecting the presence and / or content of pneumolysin protein in a sample.

[0073] Those skilled in the art can readily recognize other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be understood by those skilled in the art, the contents of this application may enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the drawings and description of the present application are merely illustrative and not restrictive.

[0074] Specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention related to this application can be better understood with reference to the exemplary embodiments described in detail below and the accompanying drawings, the brief description of which is as follows: [Brief description of the drawings]

[0075] [Figure 1] FIG. 1 shows the detection of the binding activity of the antigen-binding protein described in the present application to the PLY protein. [Diagram 2] FIG. 2 shows the detection of the binding activity of the antigen-binding protein described in the present application against the PLYmut protein. [Diagram 3] FIG. 3 shows the neutralizing activity of the antigen-binding protein of the present application against the rabbit erythrocyte hemolytic activity of pneumolysin in vitro. [Figure 4] FIG. 4 shows the protective effect of the antigen-binding protein described in this application against PLY toxin model mice. [Diagram 5] FIG. 5 shows the protective effect of the antigen binding proteins described in this application on mice infected with pneumococcus.

[0076] [Mode for carrying out the invention] Hereinafter, the present application will be described in detail with reference to specific examples, so that those skilled in the art can easily understand the advantages and effects of the present invention based on the contents disclosed in the present specification.

[0077] [Term definition] In the present application, the term "pneumolysin" can be used interchangeably with "Pneumolysin", "PLY" and "pneumolysin protein" and generally refers to a multifunctional virulence factor contained in Streptococcus pneumoniae. In the present application, the term covers wild-type pneumolysin protein and its variants, analogs, homologs and functionally active fragments. For example, the variant of pneumolysin protein can include a mutation in which an amino acid at position 146 is deleted compared to the wild-type hemolysin. For example, the functionally active fragment can be a portion capable of eliciting a humoral and / or cellular immune response in a host.

[0078] In this application, the term "isolated" generally means obtained in a natural state by artificial means. When a particular "isolated" substance or component occurs in nature, the natural environment in which it exists may have been altered, or the substance may have been isolated from the natural environment, or both. For example, a non-isolated polynucleotide or polypeptide may naturally occur in an animal organism, and the same polynucleotide or polypeptide of high purity isolated in this naturally occurring state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic mixtures, or other impurities that do not affect the activity of the substance.

[0079] In this application, the term "isolated antigen-binding protein" refers to a protein having antigen-binding ability, generally free from its naturally occurring state. The "isolated antigen-binding protein" may include an antigen-binding portion and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding to an antigen. The antigen-binding protein may include, for example, a protein framework region (FR) derived from an antibody, or an alternative protein framework region or an artificial framework region with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, framework regions derived from antibodies in which mutations have been introduced to stabilize the three-dimensional structure of the antigen-binding protein, and completely synthetic framework regions, including, for example, biocompatible polymers. See, e.g., Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Illustrative antigen binding proteins include, but are not limited to, human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, single chain antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, Fab, Fab', Fv fragments, Bs-Fv, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies and fragments thereof.

[0080] In this application, the term "CDR", also referred to as "complementarity determining region", generally refers to a region in the variable domain of an antibody, which is highly variable in sequence and / or forms a structurally defined loop. Typically, an antibody contains six CDRs, three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, a naturally occurring camelid antibody consisting of only heavy chains may function normally and be stable in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996). Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, and the combined consideration of Kabat / Chothia. These coding systems are well known in the art, see in particular http: / / www.bioinf.org.uk / abs / index.html#kabatnum. For example, the numbering of the amino acid sequence of the antigen-binding protein can be determined according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; http: / / imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000 Nucleic Acids Res. 28: 219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31: 307-310; Lefranc et al., 2005, DevComp Immunol 29: 185-203).For example, the CDRs of the antigen-binding protein can be determined by the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY AcadSci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0081] In this application, the term "FR" generally refers to the more highly conserved parts of antibody variable domains, known as framework regions. Typically, naturally occurring heavy and light chain variable domains each contain four FR regions, four in VH (H-FR1, H-FR2, H-FR3 and H-FR4) and four in VL (L-FR1, L-FR2, L-FR3 and L-FR4).

[0082] In this application, the terms "variable domain" and "variable region" can be used interchangeably and typically refer to a portion of an antibody heavy and / or light chain. The variable domains of the heavy and light chains are each referred to as "V H " and "V L " (or 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 the antigen-binding sites.

[0083] In this application, the term "variable" generally refers to the fact that certain segments of the variable domains may differ substantially in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) of the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions, which usually adopt a β-pleated sheet structure, forming a cyclic link and sometimes forming part of the β-pleated sheet structure. The CDRs in each chain are held in close proximity by the FR regions and, together with the CDRs in the other chain, facilitate the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0084] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. For purposes of the present invention, unless modified by the term "intact", such as "intact antibody", the term "antibody" also includes antibody fragments, such as, for example, Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function (e.g., specifically bind to pneumolysin protein). Typically, such fragments include an antigen-binding domain. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetramer units and another polypeptide called J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2-5 basic 4-chain units that can combine with J chains and polymerize to form multivalent aggregates. In the case of IgG, the 4-chain unit is usually about 150,000 daltons. Each L chain is linked to a H chain by a covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the α and γ chains, respectively, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains. The pairing of VH and VL forms an antigen-binding site.For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and chapter 6. L chains from any vertebrate species can be classified into one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domain. Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of the heavy chain (CH) constant domain. There are currently five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains called α, δ, ε, γ and μ, respectively.

[0085] In the present application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind to an antigen (e.g., a pneumolysin protein). In the present application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0086] In this application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. As mentioned above, intact antibodies can be digested with papain. After an antibody is digested with papain, two identical antigen-binding fragments are produced: a "Fab" fragment and a remaining "Fc" fragment (i.e., as above for the Fc region). The Fab fragment can consist of the complete light and heavy chain variable regions, as well as the first constant region (CH1) of the heavy chain (VH).

[0087] In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is slightly larger than a Fab fragment. For example, a Fab' fragment may contain all of the light chain, all of the variable domain of the heavy chain, and the first and second constant domains of all or part of the heavy chain. For example, a Fab' fragment may contain some or all of amino acid residues 220-330 of the heavy chain.

[0088] In this application, the term "F(ab')2" generally refers to an antibody fragment generated by pepsin digestion of an intact antibody. The F(ab')2 fragment contains two Fab fragments linked by disulfide bonds and a portion of the hinge region. The F(ab')2 fragment has bivalent antigen-binding activity and can cross-link antigens.

[0089] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which contains all or a portion of the heavy and light chain variable regions and lacks the heavy and light chain constant regions. The heavy and light chain variable regions include, for example, the CDRs. For example, the Fv fragment contains all or a portion of the amino-terminal variable regions of the heavy and light chains, about 110 amino acids.

[0090] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, where the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker, such as a short flexible polypeptide linker), capable of being expressed as a single polypeptide chain, and where the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and may comprise a VL-linker-VH or a VH-linker-VL.

[0091] In this application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, see for example Ward et al. (Nature, 1989 Oct 12;341(6242):544-6), Holt et al., Trends Biotechnol., 2003,21(11):484-490; and for example WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd. The term "dAb" generally includes sdAb. The term "sdAb" generally refers to a single domain antibody. A single domain antibody generally refers to an antibody fragment consisting of only an antibody heavy chain variable region (VH domain) or an antibody light chain variable region (VL).

[0092] In this application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules of single molecular composition. Generally, monoclonal antibodies are highly specific for a single antigenic site. And, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by the culture of a hybridoma, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies as used in this application can be produced in hybridoma cells or can be produced by recombinant DNA methods.

[0093] In this application, the term "chimeric antibody" generally refers to an antibody whose variable regions are derived from one species and whose constant regions are derived from another species. Typically, the variable regions are derived from an antibody of a laboratory animal such as a rodent (the "parent antibody"), and the constant regions are derived from a human antibody, thus reducing the likelihood that the resulting chimeric antibody will provoke an adverse immune response in a human individual compared to the parent antibody.

[0094] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody have been replaced with the corresponding amino acids from a human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids are also permitted, so long as the antibody retains its ability to bind to a particular antigen. A humanized antibody optionally includes at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains similar antigen specificity as the original antibody. A "humanized" form of a non-human antibody can be a chimeric antibody that contains minimal sequences derived from a non-human immunoglobulin. In some cases, residues from the CDR regions of a human immunoglobulin (acceptor antibody) can be replaced with residues from the CDR regions of a non-human species (donor antibody) (e.g., alpaca, mouse, rat, rabbit, or non-human primate) having the desired properties, affinities, and / or capacities. In some cases, residues from the FR regions of a human immunoglobulin can be replaced with the corresponding non-human residues. In addition, a humanized antibody can include amino acid modifications that are not present in the acceptor antibody or the donor antibody. These modifications can be made to further refine antibody performance, such as binding affinity.

[0095] The term "fully humanized antibody" generally refers to an antibody that contains only human immunoglobulin sequences. If produced in a mouse, mouse cell, or a hybridoma derived from a mouse cell, a fully humanized antibody may contain mouse glycosylation. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to produce antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140. Other techniques, such as using libraries, are well known in the art.

[0096] In this application, the terms "polypeptide molecule", "polypeptide" and "peptide" can be used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked portions, each of which can be a polypeptide with different properties. The properties can be biological properties, such as in vitro or in vivo activity. The properties can also be simple chemical or physical properties, such as binding to a target molecule, catalysis of a reaction, etc. The two portions can be linked by a single peptide bond or a peptide linker.

[0097] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length, either deoxyribonucleotides or ribonucleotides, in an isolated form or analogues either isolated from their natural environment or artificially synthesized.

[0098] In this application, the term "vector" generally refers to a nucleic acid delivery tool into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. A vector can transform, transduce, or transfect a host cell to express the genetic material element that it carries in the host cell. By way of example, vectors include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain an origin of replication. Vectors may further comprise components that aid in entry into cells, such as, but not limited to, a viral particle, a liposome, or a protein membrane.

[0099] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that can be or has been a recipient of a subject's plasmid or vector, including a nucleic acid molecule described in the present invention or a vector described in the present invention. A cell may include the progeny of a single cell. Due to natural, accidental, or deliberate mutations, the progeny is not necessarily completely identical (either in the form of the total DNA complement or in the genome) to the original parent cell. A cell may include a cell that has been transfected in vitro with a vector of the present application. A cell may be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell, etc.

[0100] In this application, the term "immunoconjugate" generally refers to a complex formed by conjugating (e.g., covalently binding by a binding molecule) said other reagent (e.g., chemotherapeutic agents, radioactive elements, cytostatic agents and cytotoxic agents) to said antibody or antigen-binding fragment thereof, which is capable of delivering said other reagent to a target cell by specifically binding said antibody or antigen-binding fragment thereof to an antigen on the target cell.

[0101] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise an isolated antigen-binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and an optional pharma- ceutically acceptable adjuvant. The pharmaceutical composition may further comprise an appropriate formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Preferably, the acceptable components of the composition are non-toxic to the recipient at the dosages and concentrations used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0102] In this application, the term "pharmaceutically acceptable carrier" generally includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed at the dosages and concentrations used. Physiologically acceptable carriers include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium and / or non-ionic surfactants.

[0103] In this application, the term "specifically binds" or "specific" generally refers to a measurable and reproducible interaction, such as the binding of an antibody to a target, where the presence of the target can be determined in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to that target with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In some embodiments, specific binding may include exclusive binding, but this is not required. The term is also applicable, for example, when an antigen binding protein is specific for a particular epitope that cross-reacts with multiple antigens, and a specific antibody can bind to multiple antigens with cross-reactive epitopes. Such antigen binding protein binding sites and / or antigen binding proteins that specifically bind to cross-reactive epitopes are also referred to as multispecific binding sites or cross-specific binding sites antigen binding proteins, respectively. For example, an antigen binding protein may have a multispecific binding site that specifically binds to epitopes that cross-react with several different antigens.

[0104] In this application, the term "subject" generally refers to a human or a non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0105] In the present application, such proteins, polypeptides and / or amino acid sequences should be understood to include variants or homologues having at least the same or similar function as the protein or polypeptide in question.

[0106] In the present application, the variant may be, for example, a protein or polypeptide obtained by substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to a pneumolysin protein). For example, the functional variant may include a protein or polypeptide having an amino acid modification by substitution, deletion, and / or insertion of at least 1, such as 1-30, 1-20 or 1-10, even more such as 1, 2, 3, 4 or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide before modification (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 ability) of the protein or polypeptide before modification. For example, the substitution may be a conservative substitution.

[0107] In the present application, the homologue may be a protein or polypeptide having at least about 85% (e.g., 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 even higher) sequence homology with the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to a pneumolysin protein).

[0108] In this application, the homology generally refers to the closeness, similarity, or relatedness between two or more sequences. The method of calculating "percent sequence homology" is to compare two sequences aligned within a comparison window, determine the number of positions where 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, Gln, Cys, and Met) is present in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions within the comparison window (i.e., the window size), and multiply the result by 100 to calculate the percent sequence homology. Alignment for determining percent sequence homology can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared or within a region of the target sequence. The homology may be measured by FASTA and BLAST. For a description of the FASTA algorithm, see W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J. Lipman and W.R. Pearson, "Fast and Sensitive Protein Similarity Searching," Science, 227:1435-1441, 1989. For a description of the BLAST algorithm, see S. Altschul, W. Gish, W. Miller, E.W. Myers and D. Lipman, "Basic Local Alignment Search Tools," Journal of Molecular Biology, 215:403-410, 1990.

[0109] In this application, the term "comprise" generally means "comprise", "contain", or "include". In some cases, it also means "is" or "consists of".

[0110] In this application, the term "about" generally means a variation of 0.5%-10% above and below the specified value, for example, a variation of 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 and below the specified value.

[0111] [Detailed Description of the Invention] Isolated antigen-binding proteins The CDR of an antibody is also called the complementarity determining region, and is a part of the variable region. The amino acid residues of the region may contact the antigen or the epitope of the antigen. The antibody CDR can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and the comprehensive consideration of Kabat / Chothia. These coding systems are well known in the art, see http: / / www.bioinf.org.uk / abs / index.html#kabatnum for example. Those skilled in the art can determine the CDR region using different coding systems according to the sequence and structure of the antibody. The CDR region may differ depending on the coding system. In the present application, CDR encompasses the CDR sequence divided according to any CDR division method; it also encompasses variants of the amino acid sequence of the CDR in which one or more amino acids are substituted, deleted, and / or added. For example, 1-30, 1-20 or 1-10, further for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids are substituted, deleted and / or inserted; further including homologues thereof, said homologues may be amino acid sequences having at least about 85% (e.g., 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 higher) sequence identity with the amino acid sequence of said CDR. In the present application, said isolated antigen binding protein may be defined by IMGT.

[0112] Meanwhile, the present application provides an isolated antigen-binding protein, which may comprise at least one CDR in an antibody heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 39. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 24. In the present application, the HCDR of the isolated antigen-binding protein may be split in any manner, and as long as the VH is identical to any amino acid sequence shown in any one of SEQ ID NO: 39, the HCDR obtained by splitting in any manner also falls within the scope of protection of the present application.

[0113] For example, by dividing the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:24 in the manner set forth in Table 1, an isolated antigen binding protein according to the present application may comprise the CDRs set forth in the table below.

[0114] [Table 1]

[0115] In the present application, the isolated antigen binding protein may comprise an HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1.

[0116] In the present application, the isolated antigen binding protein may comprise an HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2.

[0117] In the present application, the isolated antigen binding protein may comprise an HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0118] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:1.

[0119] In some embodiments, the isolated antigen binding protein may comprise H-FR1, the C-terminus of the H-FR1 being directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:32.

[0120] In the present application, the H-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:15.

[0121] In the present application, the isolated antigen binding protein may comprise H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:33.

[0122] In the present application, the H-FR2 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:16.

[0123] In the present application, the isolated antigen binding protein may comprise an H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:34.

[0124] In the present application, the H-FR3 of the isolated antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:17.

[0125] In the present application, the isolated antigen binding protein may comprise H-FR4, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:35.

[0126] In the present application, the H-FR4 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:18.

[0127] In the present application, the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 32, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 33, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 34, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 35. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 15, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 16, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 17, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 18. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:7, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:8, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:9, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:10. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:15, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:16, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:17, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:18.

[0128] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and H-FR4. For example, the HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively, in order. For example, HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein can comprise the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

[0129] In the present application, the isolated antigen binding protein may comprise a VH, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 39. For example, the VH may comprise the amino acid sequence set forth in any one of SEQ ID NO: 22. For example, the VH may comprise the amino acid sequence set forth in any one of SEQ ID NO: 24.

[0130] In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region. For example, the antibody heavy chain constant region may be derived from any immunoglobulin heavy chain constant region, including IgM, IgD, IgG, IgA and IgE. For example, the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In the present application, the immunoglobulin heavy chain constant region may include variants thereof. In the present application, the antibody heavy chain constant region may be derived from any of the heavy chain constant regions of human IgG1-4. For example, the isolated antigen binding protein may be derived from a human IgG1 heavy chain constant region. For example, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0131] In the present application, the isolated antigen binding protein can comprise a heavy chain. For example, the heavy chain can comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain can comprise the amino acid sequence shown in SEQ ID NO: 30.

[0132] In the present application, the isolated antigen-binding protein may comprise at least one CDR in an antibody light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 40. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 25. In the present application, the LCDR of the isolated antigen-binding protein may be split in any manner, and as long as the VL is identical to any amino acid sequence shown in any one of SEQ ID NO: 40, the LCDR obtained by splitting in any manner also falls within the scope of protection of the present application.

[0133] For example, by dividing the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:25 in the manner set forth in Table 2, an isolated antigen binding protein according to the present application may comprise the CDRs set forth in the table below.

[0134] [Table 2]

[0135] In the present application, the isolated antigen binding protein may comprise an LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:4.

[0136] In the present application, the isolated antigen binding protein may comprise an LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5.

[0137] In the present application, the isolated antigen binding protein may comprise an LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6.

[0138] In the present application, the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:5, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:4.

[0139] In the present application, the isolated antigen binding protein may comprise L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 36. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 19.

[0140] In the present application, the isolated antigen binding protein may comprise an L-FR2, wherein the L-FR2 is located between the LCDR1 and LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 37. For example, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 20.

[0141] In the present application, the isolated antigen binding protein may comprise an L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 38. For example, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 21.

[0142] In the present application, the isolated antigen binding protein may comprise an L-FR4, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:14.

[0143] In the present application, the isolated antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 36, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 37, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 38, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 19, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 20, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 21, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14. For example, the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 11, the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 12, the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 13, and the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 14. For example, the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 19, the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 20, the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 21, and the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 14.

[0144] In the present application, the isolated antigen binding protein may comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4. For example, the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, in sequence. For example, LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:14, respectively.

[0145] In the present application, the isolated antigen binding protein may comprise a VL, and the VL may comprise the amino acid sequence set forth in SEQ ID NO: 40. For example, the VL may comprise the amino acid sequence set forth in SEQ ID NO: 23. For example, the VL may comprise the amino acid sequence set forth in SEQ ID NO: 25.

[0146] In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region. For example, the light chain constant region may be derived from a human antibody light chain constant region. For example, the light chain constant region may be derived from a human Igκ constant region. For example, the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO:27.

[0147] In the present application, the isolated antigen binding protein may comprise a light chain. For example, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 29. For example, the light chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 31.

[0148] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. For example, the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:1, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:5, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:4.

[0149] In the present application, the isolated antigen binding protein may comprise a VH and a VL. For example, the VH may comprise the amino acid sequence set forth in SEQ ID NO:22, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:23. For example, the VH may comprise the amino acid sequence set forth in SEQ ID NO:24, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:25.

[0150] In the present application, the isolated antigen binding protein may comprise an antibody or an antigen binding fragment thereof. In the present application, the antigen binding fragment may comprise a Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and / or dAb. In the present application, the antibody may comprise a monoclonal antibody, a chimeric antibody, a humanized antibody and / or a fully humanized antibody. For example, the VH of the humanized antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 24. For example, the VL of the humanized antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 25.

[0151] It should be noted that the isolated antigen binding proteins described in the present application may comprise heavy and / or light chain sequences having one or more conservative sequence modifications. "Conservative sequence modifications" refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications may be introduced into the antibodies of the present invention by standard techniques known in the art, such as by site-directed mutagenesis or PCR-mediated directed mutagenesis. Conservative amino acid substitutions are those that replace an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are already known in the art. These families include amino acids containing alkaline side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR regions of the isolated antigen binding proteins described in the present application can be replaced with other amino acid residues from the same side chain family. As will be known to those of skill in the art, some conservative sequence modifications do not disrupt antigen binding.For example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol.10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0152] In this application, the pneumolysin mutant can be used to immunize mice, and the resulting mouse spleen cells can be fused with myeloma cells to screen for antigen-binding proteins that have pneumolysin protein antigen affinity. In this application, the post-translational modification site of the screened antigen-binding protein can also be optimized to obtain a humanized sequence.

[0153] The pneumolysin antigen binding proteins described herein can be identified, screened, or characterized by a variety of detection methods known in the art.

[0154] For example, the antigen-binding activity of the antigen-binding proteins of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblot (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like.

[0155] In the present application, the isolated antigen-binding protein can specifically bind to pneumolysin. In the present application, the pneumolysin can include wild-type pneumolysin and its mutants. In the present application, the pneumolysin mutant has 146 amino acids deleted compared to the wild-type pneumolysin. In the present application, the amino acid sequence of the wild-type pneumolysin can be determined from the Uniprot database (Entry ID: Q04IN8).

[0156] In the present application, the binding of the isolated antigen-binding protein to pneumolysin can be detected by ELISA. For example, the antigen-binding protein described in the present application has an EC of about 0.03 μg / ml or less, about 0.028 μg / ml or less, about 0.026 μg / ml or less, about 0.024 μg / ml or less, about 0.022 μg / ml or less, about 0.02034 μg / ml or less, about 0.020 μg / ml or less, about 0.018 μg / ml or less, about 0.01706 μg / ml or less, about 0.016 μg / ml or less, about 0.014 μg / ml or less, about 0.012 μg / ml or less, or about 0.010 μg / ml or less. 50 It can bind to pneumolysin protein at this value.

[0157] In the present application, the isolated antigen-binding protein can neutralize the biological activity of pneumolysin protein. For example, recombinant pneumolysin was mixed with the isolated antigen-binding protein described in the present application, and rabbit red blood cells were added to detect the anti-hemolytic activity of the antigen-binding protein.

[0158] In the present application, the isolated antigen binding protein can prevent and / or treat a disease and / or condition. In the present application, the disease and / or condition can be caused or mediated by Streptococcus pneumoniae. In the present application, the disease and / or condition can include complications of a disease and / or condition caused or mediated by Streptococcus pneumoniae. For example, the disease and / or condition can include sepsis and / or bacteremia.

[0159] Polypeptide molecules, nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions On the other hand, the present application provides polypeptide molecules that can comprise the isolated antigen binding proteins described in the present application.

[0160] In the present application, the polypeptide molecule may comprise a fusion protein. In the present application, the polypeptide molecule may be a fusion protein. In the present application, the fusion protein may comprise a multispecific antibody.

[0161] In contrast, the present application provides an isolated nucleic acid molecule capable of encoding an isolated antigen binding protein described herein, e.g., that has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) recombinantly produced, e.g., by cloning, (iii) purified, e.g., by enzymatic cleavage and gel electrophoretic fractionation, or (iv) synthetically produced or synthesized, e.g., chemically synthesized.

[0162] On the other hand, the present application provides a vector that may contain the nucleic acid molecule described in the present application. The vector may also contain other genes, such as, for example, marker genes that allow the selection of the vector under suitable conditions in a suitable host cell. In addition, the vector may also contain expression control elements that allow the appropriate expression of the coding region in a suitable 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, etc. The vector may transform, transduce, or transfect a host cell to express the genetic material elements that it carries in the host cell. The vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used, for example, in genetic engineering. For example, the vector is an expression vector. The vector may also include, but is not limited to, components that aid in cell entry, such as, for example, viral particles, liposomes, or protein membranes.

[0163] Alternatively, the present application provides cells that may include the nucleic acid molecules described herein or the vectors described herein. In some embodiments, each or each host cell may include one or more of the nucleic acid molecules or vectors described herein. In some embodiments, each or each host cell may include a plurality (e.g., two or more) or a plurality (e.g., two or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein may be introduced into the host cell, e.g., a eukaryotic cell, e.g., a cell derived from a plant, a fungus or a yeast cell. In some embodiments, the cell may be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a 293T cell, a COS-1 cell, a SP2 / 0 cell, a NS0 cell, or a myeloma cell. The vectors described herein may be introduced into the host cell by methods known in the art, e.g., electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0164] On the other hand, the present application provides immunoconjugates that can include the isolated antigen-binding proteins described in the present application.

[0165] In some embodiments, the isolated antigen-binding protein or fragment thereof described herein can be conjugated to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, etc. The conjugation can be via one or more covalent or non-covalent interactions, and can include chelation. Various linkers known in the art can be used to form the immunoconjugate. Furthermore, the immunoconjugate can be provided in the form of a fusion protein that can be expressed by a polynucleotide encoding the immunoconjugate. The immunoconjugate can further include, for example, an antibody-drug conjugate (ADC). In an ADC, the antibody and the therapeutic agent can be crosslinked by a cleavable linker, such as a peptide linker, a disulfide linker, or a hydrazone linker.

[0166] Meanwhile, the present application further provides pharmaceutical compositions, which may comprise an isolated antigen binding protein described in the present application, a polypeptide molecule described in the present application, an immunoconjugate described in the present application, a nucleic acid molecule described in the present application, a vector described in the present application, and / or a cell described in the present application, and an optional pharma- ceutically acceptable carrier.

[0167] In some embodiments, the pharmaceutical composition may further comprise a suitable formulation of one or more (pharmaceutical effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. Preferably, the acceptable components of the composition are non-toxic to recipients at the dosages and concentrations used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0168] In some embodiments, the pharmaceutical composition may contain two or more active compounds, which are generally active compounds that have complementary activities that do not adversely affect each other.The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0169] In some embodiments, the pharma- ceutically acceptable carrier includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents compatible with drug administration, and are generally safe and non-toxic.

[0170] In some embodiments, the pharmaceutical composition may include parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration, or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, administration of the pharmaceutical composition may be accomplished in a variety of ways, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the pharmaceutical composition may be administered without interruption. Uninterrupted (or continuous) administration may be accomplished by a miniature pump system worn by the patient, which may meter the flow of therapeutic agent into the patient's body, for example as described in WO2015 / 036583.

[0171] Preparation method Meanwhile, the present application provides a method for preparing said isolated antigen-binding protein, said method comprising culturing a host cell described in the present application under conditions for expressing said antigen-binding protein. For example, methods such as a suitable medium, a suitable temperature and a suitable incubation time well known to those skilled in the art can be used.

[0172] Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application. For example, an animal can be immunized with an attenuated strain of pneumolysin or a fragment thereof. For example, the attenuated strain of S. pneumoniae can contain a mutation that deletes position 146 compared to the wild type. Any suitable immunization method can be used, including adjuvants, immunostimulants, and repeated inoculations for boosting immunity, and one or more methods can be used.

[0173] Any suitable form of pneumolysin can be used as an immunogen (antigen) to generate non-human antibodies specific to pneumolysin and screen the biological activity of the antibodies. For example, the eliciting immunogen can be full-length pneumolysin, including the natural homodimer, or single / multiple epitope-containing peptides. The immunogen can be used alone or in combination with one or more immunogenicity enhancing agents known in the art.

[0174] Methods and Applications Meanwhile, the present application provides the use of said isolated antigen-binding protein, said polypeptide molecule, said nucleic acid molecule, said vector, said cell, said immunoconjugate and / or said pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a disease and / or a symptom.

[0175] Meanwhile, the present application further provides a method for preventing and / or treating a disease and / or condition comprising administering to a subject in need thereof an isolated antigen binding protein, said polypeptide molecule, said nucleic acid molecule, said vector, said cell, said immunoconjugate and / or said pharmaceutical composition described in the present application.

[0176] In the present application, said administration can be performed in various ways, for example, intravenously, intratumorally, intraperitoneally, subcutaneously, intramuscularly, topically or intradermally.

[0177] On the other hand, the isolated antigen-binding proteins, said polypeptide molecules, said nucleic acid molecules, said vectors, said cells, said immunoconjugates and / or said pharmaceutical compositions described in the present application can be used for the prevention and / or treatment of diseases and / or conditions.

[0178] In the present application, said diseases and / or conditions may be caused or mediated by Streptococcus pneumoniae.

[0179] In the present application, said disease and / or condition may be a complication of a disease and / or condition caused or mediated by Streptococcus pneumoniae.

[0180] In the present application, said diseases and / or conditions include bacterial infections.

[0181] In the present application, the disease and / or condition may include bacteremia and / or sepsis.

[0182] In the present application, the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition may be used alone or in combination with other drugs to prevent and / or treat a disease and / or condition. In some embodiments, the other drug may be any currently known drug with antibacterial activity. The disease and / or condition may be a Streptococcus pneumoniae infection or a related disease caused by other infections.

[0183] Meanwhile, the present application further provides a method for detecting pneumolysin in a sample, the method comprising administering the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex and / or the pharmaceutical composition.

[0184] In the present application, the method for detecting pneumolysin in a sample can be an in vitro method. For example, an isolated antigen binding protein described in the present application is contacted with an ex vivo sample and the presence and / or content of pneumolysin in the sample is detected. In some embodiments, the method for detecting pneumolysin in a sample is not for therapeutic purposes. In some embodiments, the method for detecting pneumolysin in a sample is not a diagnostic method.

[0185] On the other hand, the present application further provides a reagent or reagent kit for detecting pneumolysin in a sample, comprising the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex and / or the pharmaceutical composition.

[0186] Meanwhile, the present application further provides the use of said isolated antigen-binding protein, said polypeptide molecule, said nucleic acid molecule, said vector, said cell, said immune complex and / or said pharmaceutical composition in the manufacture of a reagent kit for detecting the presence and / or content of pneumolysin in a sample.

[0187] The following examples are not limited by theory, but are merely intended to illustrate the antigen-binding proteins, preparation methods and applications of the present application, and are not intended to limit the scope of the present invention. EXAMPLES

[0188] Example 1 Expression of pneumolysin (PLY) recombinant protein The amino acid sequence of pneumolysin (PLY, Entry ID: Q04IN8) was determined using the Uniprot database, and the 146th alanine was deleted from the full-length amino acid sequence of pneumolysin to obtain the attenuated PLY mut amino acid sequence. The amino acid sequence was used as a template to define the NdeI / XhoI cleavage site, and a His tag was introduced at the C-terminus. Codon optimization and gene synthesis were performed by Tsingke Biotechnology Co., Ltd. (● is the character for "hand" under "respect") to obtain the plasmids for PLY and PLY mut proteins. The recombinant plasmids were transformed into E. coli BL21(DE3)plysS, and the PLY and PLY mut recombinant proteins were expressed in the E. coli expression system.

[0189] Example 2 Immunization of Balb / C mice with PLY mut antigen The prepared PLY mut protein was used to immunize five Balb / C mice by subcutaneous multipoint immunization, and the process of subcutaneous multipoint immunization was as follows.

[0190] [Table 3]

[0191] Example 3 Fusion of spleen cells from immunized mice with myeloma cells and screening for antigen-binding proteins with affinity for the PLYmut antigen Myeloma cells P3X63Ag8.653 were fused with mouse spleen cells immune to the PLY mut antigen by the PEG method. The fused hybridoma cells were seeded and cultured in a 96-well plate. Hybridoma cell culture was then performed using HAT and HT media. When the cell supernatant in the 96-well plate turned slightly yellow, 100 μl of the cell culture supernatant was taken and hybridoma clone cells with the ability to bind to the PLY mut antigen were screened using the ELISA method. Hybridoma parent clone cells with relatively strong affinity were further cultured, and then subcloned by limiting dilution to select monoclonal cells. The monoclonal supernatant was used to select 2E5 monoclonal cells with a secreted antibody that had a strong affinity for the PLYmut protein by ELISA, and the secreted monoclonal antibody was named m2E5.

[0192] Example 4 Obtaining sequences of specific mouse antigen-binding proteins Using the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.), 1 × 10 6 Total RNA was extracted from 2E5 monoclonal cells secreting anti-PLY mut antibody. Total RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara). Primers were used to amplify cDNA fragments (Anke Krebber.1997), and PCR products were purified using a DNA product purification kit (Beijing Tiangen Biochemical Technology Co., Ltd.), ligated into T vectors using a ligation kit (Beijing ●ke Biological Technology Co., Ltd.) (● indicates "hand" under "Kei"). competent cells were transformed, and sequence determination was performed by Beijing ●ke Biological Technology Co., Ltd. (● indicates "hand" under "Kei"). The sequence determination results of the heavy chain variable region (m2E5VH) and light chain variable region (m2E5VL) of mouse monoclonal antibody m2E5 were as follows:

[0193] m2E5 heavy chain variable region (m2E5VH) SEQ ID NO: 22 EVKVEESGGGSVQPGGSMKLSCVAS GYTFGDNW MNWVRQSPEKGLEWVAE IRLKSNNYAT RYAESVKGRFTISRDDSKSSVYLQMNNLRTEDTGIYYC TRTQFITSVVAPFDF WGQGTTLTVSS m2E5 light chain variable region (m2E5VL) SEQ ID NO: 23 DIVMSQSPSSLGVSVGEKITMSCKSS QSLLYSRNQKNY LAWYQQKPGQSPKLLIY W.A. STRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC QQYYRYPLT FGGGTKVEIK However, the underlined portions indicate the CDR sequences divided using the IMGT numbering scheme.

[0194] Example 5 Humanization of anti-PLY mouse antigen binding protein Based on the NCBI database alignment (https: / / www.ncbi.nlm.nih.gov / igblast / ), the germline sequence closest to mouse antibody 2E5 (m2E5) was selected as the template, m2E5 was humanized, and the post-translational modification sites of the antigen-binding protein were optimized. The sequences of the heavy chain variable region (hu2E5VH3) and light chain variable region (hu2E5VL0) of the obtained humanized antibody 2E5H3L0 are as follows:

[0195] Humanized antigen binding protein 2E5 heavy chain variable region (hu2E5VH3) SEQ ID NO: 24 EVQLVESGGGLVQPGGSLKLSCAAS GYTFGDNW MNWVRQASGKGLEWVGE IRLKSNNYAT RYAESVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC TRTQFITSVVAPFDF WGQGTTVTVSS Humanized antigen binding protein 2E5 light chain variable region (hu2E5VL0) SEQ ID NO: 25 DIVMTQSPDSLAVSLGERATINCKSS QSLLYSRNQKNY LAWYQQKPGQPPKLLIY W.A. STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYRYPLT FGGGTKVEIK However, the underlined portions indicate the CDR sequences divided using the IMGT numbering scheme.

[0196] Example 6 Expression of anti-PLY antigen-binding protein and detection of antigen-binding activity The C-terminus of the m2E5 heavy chain variable region was linked to the N-terminus of the constant region of human immunoglobulin gamma 1 (IgG1) and constructed in a pcDNA3.4 vector to obtain the heavy chain expression vector pcDNA3.4-c2E5VHCH of the 2E5 mouse / human chimeric antibody (2E5HL). The C-terminus of the 2E5 light chain variable region was linked to the N-terminus of the light chain kappa constant region and constructed in a pcDNA3.4 vector to obtain the 2E5 chimeric antibody light chain expression vector pcDNA3.4-c2E5VLCL. Among them, the amino acid sequences of the heavy chain (c2E5VHCH) and the light chain (c2E5VLCL) of the chimeric antibody 2E5HL are as follows:

[0197] c2E5VHCH SEQ ID NO: 28: EVKVEESGGGSVQPGGSMKLSCVAS GYTFGDNW MNWVRQSPEKGLEWVAE IRLKSNNYAT RYAESVKGRFTISRDDSKSSVYLQMNNLRTEDTGIYYC TRTQFITSVVAPFDFWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK c2E5VLCL SEQ ID NO: 29: DIVMSQSPSSLGVSVGEKITMSCKSS QSLLYSRNQKNY LAWYQQKPGQSPKLLIY W.A. STRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC QQYYRYPLT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0198] The C-terminus of the 2E5 humanized heavy chain variable region was linked to the N-terminus of the constant region of human immunoglobulin gamma 1 (IgG1) and constructed in a pcDNA3.4 vector to obtain a humanized heavy chain expression vector pcDNA3.4-hu2E5VH3CH. The C-terminus of the 2E5 humanized light chain variable region was linked to the N-terminus of the light chain kappa constant region and constructed in a pcDNA3.4 vector to obtain a humanized light chain expression vector pcDNA3.4-hu2E5VL0CL.

[0199] The heavy chain (hu2E5VH3CH) and light chain (hu2E5VL0CL) amino acid sequences of the 2E5H3L0 humanized antibody are as follows:

[0200] hu2E5VH3CH SEQ ID NO: 30: EVQLVESGGGLVQPGGSLKLSCAAS GYTFGDNW MNWVRQASGKGLEWVGE IRLKSNNYAT RYAESVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC TRTQFITSVVAPFDF WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hu2E5VL0CL SEQ ID NO: 31: DIVMTQSPDSLAVSLGERATINCKSS QSLLYSRNQKNY LAWYQQKPGQPPKLLIY W.A. STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYRYPLT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0201] The heavy chain expression vector pcDNA3.4-m2E5VHCH of the 2E5HL chimeric antibody was co-transfected with the light chain expression vector pcDNA3.4-m2E5VLCL into Expi293F cells, 2E5HL was instantly expressed using the Expi293F expression system, and finally the chimeric antibody protein was purified using Protein A. The heavy chain expression vector pcDNA3.4-hu2E5VH3CH of the 2E5H3L0 humanized antibody was co-transfected with the light chain expression vector pcDNA3.4-hu2E5VL0CL into Expi293F cells, 2E5H3L0 was instantly expressed using the Expi293F expression system, and finally the humanized antibody protein was purified using Protein A. The obtained 2E5HL chimeric antibody and 2E5H3L0 humanized antibody were purified, and the antigen binding activity was identified using the ELISA method. In other words, PLY and PLYmut proteins were coated at 0.1 μg / well on a 96-well ELISA plate (Thermo), incubated overnight at 4°C, washed with PBST buffer, then 200 μL of 5% BSA was added per well and incubated at 37°C for 1 hour; after blocking, the plate was washed with PBST buffer, and 2E5HL human mouse chimeric antibody or 2E5H3L0 humanized antibody was added to the ELISA plate at 0.1 μg in the first well and incubated at 4°C overnight; after the primary antibody was placed overnight, the plate was washed, and 100 μL of HRP-labeled goat anti-human IgG secondary antibody diluted 1:10000 was added to the ELISA plate at 1:10000 in ... The results were calculated using the Graphpad software and plotted using the Graphpad software. The binding status of the antibody to the PLY protein is shown in Figure 1. 50 The EC value was 0.01706 μg / ml, and the EC 50 The binding status of the antibody to the PLYmut protein was shown in FIG. 2, and the EC50 The EC value of 2E5H3L0 was 0.01582μg / ml. 50 The value was 0.01617 μg / ml. The results show that both the humanized antibody 2E5H3L0 and the chimeric antibody 2E5HL have strong affinity for pneumolysin PLY and PLYmut proteins.

[0202] Example 7 In vitro detection of neutralizing activity of Streptococcus pneumoniae toxins 6.25ng of recombinant pneumolysin PLY protein was mixed with chimeric antibody 2E5HL or humanized antibody 2E5H3L0 at a certain ratio, added to a 96-well plate (Thermo), and incubated at 37°C for 10min, then 5% rabbit red blood cells were added and incubated at 37°C for 1h. The samples were centrifuged at 3000rpm for 5min, and the antihemolytic activity was measured by detecting the absorbance at 405nm using a microplate reader (Multiscin FC, Thermo). The results, as shown in FIG. 3, show that Streptococcus pneumoniae toxins have rabbit erythrocyte hemolytic activity, and both the chimeric antibody 2E5HL and the humanized antibody 2E5H3L0 can effectively neutralize the rabbit erythrocyte hemolytic activity of Streptococcus pneumoniae toxins in a dose-dependent manner.

[0203] Example 8 Protective effect of the antigen-binding protein described in the present application on PLY toxin model mice C57BL / 6 mice were first injected with chimeric antibody 2E5HL, humanized antibody 2E5H3L0, or human IgG antibody (as a control) at a dose of 1.25 mg / kg, and then 1 h later, 0.75 μg of pneumolysin PLY protein was injected into the tail vein, and the survival time of the mice was observed.

[0204] The results are shown in FIG. 4, in which all mice in the control group died, while mice injected with chimeric antibody 2E5HL and humanized antibody 2E5H3L0 achieved complete protection.

[0205] Example 9 Protective effect of antigen-binding proteins described in this application against Streptococcus pneumoniae-infected mice S. pneumoniae was collected from the seed bank and thawed. 200 μl of the bacterial solution was evenly applied to a Columbia blood plate with an applicator and cultured for 10-12 h. Blood agar (3 cm × 3 cm) was removed from the Columbia blood plate and added to 15 ml of sterilized THY medium. The blood agar was cultured in a 37°C incubator until the OD600 reached 0.4-0.6, which took about 2-3 h. Cells were collected by centrifugation and resuspended in PBS. C57BL / 6J mice were randomly divided into a model group and a monoclonal antibody drug treatment group according to body weight. 2 × 10 8 CFU of Streptococcus pneumoniae was injected into the mice, and 2 h later, 30 mg / kg of 2E5HL, 2E5H3L0 or human IgG (control) was injected into the tail vein, respectively, and the survival status of the mice in each group was observed and recorded.

[0206] The results are shown in FIG. 5. Compared with the control human IgG, the chimeric antibody 2E5HL and the humanized antibody 2E5H3L0 have better protective effects against Streptococcus pneumoniae (SP)-infected mice.

Claims

1. 1. An isolated antigen binding protein comprising: and / or comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 1; and / or An isolated antigen binding protein comprising LCDR1, LCDR2 and LCDR3, wherein said LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, said LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5, and said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

4.

2. A peptide comprising H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:15, wherein H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:16, wherein H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:17, and wherein H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:18, and / or 2. The isolated antigen binding protein of claim 1, comprising L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, L-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 37, L-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 38, and L-FR4 comprises the amino acid sequence set forth in SEQ ID NO:

14.

3. The isolated antigen binding protein, comprising H-FR1, H-FR2, H-FR3, and H-FR4 selected from the group consisting of: 1) said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:7, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:8, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:9, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:10; and 2) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 15, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 16, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 17, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 18; and / or The isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4 selected from any one of the following groups: 1) said L-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, said L-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, said L-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, and said L-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 14; and 2) The isolated antigen-binding protein of claim 1, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 19, L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 20, L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 21, and L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

14.

4. The isolated antigen binding protein, comprising a VH, the VH comprising the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:24; and / or The isolated antigen binding protein of claim 1, wherein the isolated antigen binding protein comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO:

25.

5. The isolated antigen-binding protein of claim 1, comprising an antibody heavy chain constant region and / or an antibody light chain constant region.

6. The isolated antigen binding protein described in claim 1, comprising a heavy chain, wherein the heavy chain comprises an amino acid sequence shown in SEQ ID NO:28 or SEQ ID NO:

30.

7. The isolated antigen-binding protein of claim 1, comprising a light chain, the light chain comprising an amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:

31.

8. The antibody comprises a VH and a VL selected from any one of the following groups: 1) the VH comprises the amino acid sequence set forth in SEQ ID NO:22, and the VL comprises the amino acid sequence set forth in SEQ ID NO:23; and 2) The isolated antigen-binding protein of claim 4, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO:

25.

9. The isolated antigen-binding protein of claim 1, comprising an antibody or an antigen-binding fragment thereof.

10. The isolated antigen binding protein of claim 9, wherein the antigen binding fragment comprises a Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv and / or a dAb.

11. The isolated antigen-binding protein of claim 9, wherein the antibody is one or more selected from a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully humanized antibody.

12. A polypeptide molecule comprising the isolated antigen-binding protein of claim 1.

13. An immune complex comprising the isolated antigen-binding protein of claim 1.

14. A nucleic acid molecule encoding the isolated antigen-binding protein of claim 1 or a polypeptide molecule comprising said isolated antigen-binding protein.

15. A vector comprising the nucleic acid molecule described in claim 14.

16. A cell comprising the nucleic acid molecule described in claim 14 or a vector containing the nucleic acid molecule.

17. A pharmaceutical composition comprising the isolated antigen-binding protein of claim 1, a polypeptide molecule comprising said isolated antigen-binding protein, an immune complex comprising said isolated antigen-binding protein, a nucleic acid molecule encoding said isolated antigen-binding protein, a vector comprising said nucleic acid molecule and / or a cell comprising said nucleic acid molecule or a vector comprising said nucleic acid molecule, and optionally a pharma- ceutically acceptable carrier.

18. A method for preparing an isolated antigen-binding protein, comprising culturing a cell according to claim 16 under conditions to express the antigen-binding protein.

19. A method for detecting pneumolysin protein in a sample, the method comprising administering an isolated antigen-binding protein according to claim 1, a polypeptide molecule comprising said isolated antigen-binding protein, an immune complex comprising said isolated antigen-binding protein, a nucleic acid molecule encoding said isolated antigen-binding protein, a vector comprising said nucleic acid molecule, a cell comprising said nucleic acid molecule or a vector comprising said nucleic acid molecule, and / or a pharmaceutical composition comprising said isolated antigen-binding protein.

20. A reagent kit for detecting pneumolysin protein in a sample, comprising: an isolated antigen-binding protein according to claim 1, a polypeptide molecule comprising said isolated antigen-binding protein, an immune complex comprising said isolated antigen-binding protein, a nucleic acid molecule encoding said isolated antigen-binding protein, a vector comprising said nucleic acid molecule, a cell comprising said nucleic acid molecule or a vector comprising said nucleic acid molecule, and / or a pharmaceutical composition comprising said isolated antigen-binding protein.

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