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WO2022199527A1 - 靶向肺炎链球菌溶血素的抗原结合蛋白及其用途 - Google Patents

靶向肺炎链球菌溶血素的抗原结合蛋白及其用途 Download PDF

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Publication number
WO2022199527A1
WO2022199527A1 PCT/CN2022/082011 CN2022082011W WO2022199527A1 WO 2022199527 A1 WO2022199527 A1 WO 2022199527A1 CN 2022082011 W CN2022082011 W CN 2022082011W WO 2022199527 A1 WO2022199527 A1 WO 2022199527A1
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seq
amino acid
acid sequence
binding protein
isolated antigen
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PCT/CN2022/082011
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English (en)
French (fr)
Inventor
安毛毛
郭诗雨
陈思敏
邱熙然
李博华
Original Assignee
星济生物(苏州)有限公司
昕曜生物(上海)有限公司
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Priority to CN202280024001.2A priority Critical patent/CN117062833A/zh
Priority to KR1020237036175A priority patent/KR20230162797A/ko
Priority to JP2023558259A priority patent/JP2024513746A/ja
Priority to US18/283,429 priority patent/US20240174736A1/en
Priority to EP22774186.5A priority patent/EP4317182A1/en
Priority to BR112023019358A priority patent/BR112023019358A2/pt
Publication of WO2022199527A1 publication Critical patent/WO2022199527A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
    • G01N2333/3156—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae [Pneumococcus]

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen binding protein capable of targeting pneumolysin protein (PLY), and its application in the field of treating and preventing Streptococcus pneumoniae infection.
  • PLY pneumolysin protein
  • Streptococcus pneumoniae also known as pneumococcus
  • pneumococcus is widely colonized in the nasopharynx of humans. When the body's resistance is low, it can cause lung or central nervous system infections, and in severe cases, bacteremia or sepsis.
  • Hemolysin is an important virulence factor of Streptococcus pneumoniae and plays an important role in the infection process of Streptococcus pneumoniae.
  • PLY can destroy lung tissue epithelium, endothelium and immune cells, increase vascular permeability and Streptococcus pneumoniae colonization, spread and invasion, and affect the lung function of patients, resulting in acute lung injury and even acute respiratory distress syndrome. Therefore, there is an urgent need to develop effective drugs against Streptococcus pneumoniae infection.
  • the present application provides an isolated antigen-binding protein having one or more of the following properties: 1) capable of specifically binding to pneumolysin protein and its variants; 2) to pneumolysin The protein has high affinity and neutralizing activity; 3) can effectively block infection caused by Streptococcus pneumoniae; 4) can effectively prevent and/or treat diseases and/or conditions caused by Streptococcus pneumoniae.
  • the application provides an isolated antigen-binding protein comprising at least one CDR in the variable VH of an antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO:39.
  • the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:1.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:2.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:3.
  • the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2 , and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the isolated antigen binding protein comprises H-FR1
  • the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1
  • the H-FR1 comprises SEQ ID NO: The amino acid sequence shown in 32.
  • the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:15.
  • the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO:33 amino acid sequence.
  • the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:16.
  • the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO:34 amino acid sequence.
  • the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:17.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: The amino acid sequence shown in 35.
  • the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:18.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 32, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:33, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:34, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:35.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprising SEQ ID NO:7 or SEQ ID NO:15
  • the amino acid sequence the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 16
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 18.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4 selected from any of the following groups:
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 8
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 9 amino acid sequence
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 10;
  • 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 amino acid sequence
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the isolated antigen binding protein comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:39.
  • 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.
  • the isolated antigen binding protein comprises an antibody heavy chain constant region.
  • the heavy chain constant region of the isolated antigen binding protein is derived from a human IgG constant region.
  • the heavy chain constant region of the isolated antigen binding protein is derived from a human IgGl heavy chain constant region.
  • the heavy chain constant region of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:26.
  • the isolated antigen binding protein comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:28 or SEQ ID NO:30.
  • the isolated antigen binding protein comprises at least one CDR in the variable region VL of an antibody light chain, the VL comprising the amino acid sequence set forth in SEQ ID NO:40.
  • the isolated antigen binding protein comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:4.
  • the isolated antigen binding protein comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5.
  • the isolated antigen binding protein comprises LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:6.
  • the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5 , and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:4.
  • the isolated antigen binding protein comprises L-FR1
  • the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1
  • the L-FR1 comprises SEQ ID NO: The amino acid sequence shown in 36.
  • the L-FR1 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:19.
  • the isolated antigen-binding protein comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO:37 amino acid sequence.
  • the L-FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:20.
  • 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.
  • the isolated antigen binding protein comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises SEQ ID NO: The amino acid sequence shown in 14.
  • the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:37, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:38, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:14.
  • the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, the L-FR1 comprising SEQ ID NO: 11 or SEQ ID NO: 19
  • the amino acid sequence of the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 20
  • the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 21
  • the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 14.
  • the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4 selected from any of the following groups:
  • the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 11
  • the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 12
  • the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 13 amino acid sequence
  • the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 14;
  • 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 amino acid sequence
  • the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 14.
  • the isolated antigen binding protein comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:40.
  • 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.
  • the isolated antigen binding protein comprises an antibody light chain constant region.
  • the light chain constant region of the isolated antigen binding protein is derived from a human IgK constant region.
  • the light chain constant region of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:27.
  • the isolated antigen binding protein comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:31.
  • the isolated antigen binding protein comprises a VH and VL selected from any of the following groups:
  • VH comprises the amino acid sequence shown in SEQ ID NO: 22
  • VL comprises the amino acid sequence shown in SEQ ID NO: 23
  • VH comprises the amino acid sequence shown in SEQ ID NO: 24
  • VL comprises the amino acid sequence shown in SEQ ID NO: 25.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises a Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, di-scFv and/or dAb.
  • the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the isolated antigen binding protein is capable of specifically binding pneumolysin protein.
  • the pneumolysin comprises wild-type pneumolysin protein and variants thereof.
  • the pneumolysin protein comprises a pneumolysin protein variant with a deletion of amino acid 146 compared to wild-type pneumolysin protein.
  • the isolated antigen binding protein is capable of preventing and/or treating diseases and/or disorders caused by Streptococcus pneumoniae.
  • the disease and/or disorder comprises a complication of a disease and/or disorder caused by Streptococcus pneumoniae.
  • the application also provides a polypeptide molecule comprising the isolated antigen binding protein.
  • the polypeptide molecule comprises a fusion protein.
  • the present application also provides immunoconjugates comprising the isolated antigen binding protein.
  • the present application also provides a nucleic acid molecule encoding the isolated antigen binding protein or the polypeptide molecule.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the present application also provides a cell comprising the nucleic acid molecule or the vector.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a method for preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the antigen binding protein is expressed.
  • the isolated antigen-binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition described herein are used alone or in combination with Use in combination with other drugs.
  • the present application also provides that the isolated antigen-binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition are in Use in the preparation of medicaments for the prevention and/or treatment of diseases and/or disorders.
  • the disease and/or disorder and its complications are caused or mediated by Streptococcus pneumoniae.
  • the disease and/or disorder comprises a complication of a disease and/or disorder caused or mediated by Streptococcus pneumoniae.
  • the present application also provides a method for detecting pneumolysin protein in a sample, the method comprising administering the isolated antigen binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule , the carrier, the cell and/or the pharmaceutical composition.
  • the present application also provides a kit for detecting pneumolysin protein in a sample, the kit comprising the isolated antigen-binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid The molecule, the carrier, the cell and/or the pharmaceutical composition.
  • the present application also provides that the isolated antigen-binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition are in Use in the preparation of a kit for detecting the presence and/or content of pneumolysin protein in a sample.
  • Figure 1 shows the assay of the binding activity of the antigen-binding protein described in the present application to PLY protein.
  • Figure 2 shows the assay of the binding activity of the antigen-binding protein described in the present application to PLYmut protein.
  • Figure 4 shows the protective effect of the antigen-binding protein described in the present application on PLY toxin model mice.
  • Figure 5 shows the protective effect of the antigen binding proteins described in the present application on mice infected with Streptococcus pneumoniae.
  • the term “pneumolysin” is used interchangeably with “Pneumolysin”, “PLY” and “pneumolysin protein” and generally refers to a multifunctional virulence factor contained in Streptococcus pneumoniae.
  • the term may encompass wild-type pneumolysin proteins and variants, analogs, homologues and functionally active fragments thereof.
  • the variant of the pneumolysin protein may comprise a mutation that deletes amino acid 146 as compared to wild-type hemolysin.
  • the functionally active fragment may be a moiety capable of eliciting a humoral and/or cellular immune response in a host.
  • isolated generally refers to artificial means obtained from the natural state. If an "isolated" substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of.
  • isolated does not exclude the admixture of artificial or synthetic materials, nor does it exclude the presence of other impurities that do not affect the activity of the material.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding capacity that has been removed from its naturally occurring state.
  • isolated antigen-binding protein may comprise an antigen-binding moiety and, optionally, a framework or framework portion that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding moiety to the antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives.
  • Such frameworks include, but are not limited to, antibody-derived framework regions comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic framework regions comprising, eg, biocompatible polymers. See, eg, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004).
  • antigen binding proteins include but are not limited to: human antibodies; humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies; trifunctional antibodies; tetrabodies; Fab, Fab', Fv fragments, Bs - Fv, F(ab') 2 , F(ab) 2 , scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.
  • CDRs also referred to as “complementarity determining regions” generally refers to regions within the variable domains of antibodies, the sequences of which are highly variable and/or form structurally defined loops.
  • an antibody typically includes six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • naturally occurring camelid antibodies consisting only of heavy chains are also functional and stable in the absence of light chains. See, eg, 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 a variety of coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, Kabat/Chothia, etc. in combination. These coding systems are known in the art, see eg http://www.bioinf.org.uk/abs/index.html#kabatnum.
  • the amino acid sequence numbering of the antigen binding protein can be 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.
  • the CDRs of the antigen binding proteins can be determined according to the Kabat numbering system (see eg Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition , U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • variable domains of native heavy and light chains each comprise four FR regions, namely 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).
  • variable domain and “variable region” are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain.
  • the variable domains of the heavy and light chains may be referred to as “ VH “ and “ VL “, respectively (or “VH” and “VL”, respectively). These domains are usually the most variable part of the antibody (relative to other antibodies of the same type) and contain the antigen binding site.
  • variable generally refers to the fact that certain segments of the variable domains may differ greatly in sequence between antibodies.
  • the variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved portions of variable domains are referred to as framework regions (FRs).
  • FRs framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, most adopting a ⁇ -sheet configuration, connected by three CDRs that form loops connecting, and in some cases forming part of, the ⁇ -sheet structure .
  • the CDRs in each chain are held in close proximity by the FR regions, and the CDRs from the other chain together contribute to 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)).
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide that includes an antigen-binding site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutant and transplanted antibodies.
  • the term “antibody” also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specific binding to pneumolysin protein). Typically, such fragments should include an antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibody is composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contains 10 antigen-binding sites, while IgA antibody includes 2-5 that can be combined with J chain to form multivalent Combined basic 4-chain unit.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is connected to the H chain by one covalent disulfide bond, while the two H chains are connected 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 intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other end.
  • VL corresponds to VH and CL corresponds to the first constant domain (CH1) of the heavy chain.
  • Particular amino acid residues are thought to form the interface between the light and heavy chain variable domains.
  • the VH and VL pair together to form a single antigen binding site.
  • immunoglobulins can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Based on the amino acid sequence of the heavy chain (CH) constant domains, immunoglobulins can be divided into different classes or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • the term "antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, pneumolysin).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • Fab generally refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested with papain as described above. Papain digestion of the antibody yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region (CH1) of the H chain (VH).
  • Fab' fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment.
  • a Fab' fragment can include all of the light chain, all of the variable regions of the heavy chain, and all or part of the first and second constant regions of the heavy chain.
  • Fab' fragments may also include part or all of the 220-330 amino acid residues of the heavy chain.
  • F(ab')2 generally refers to antibody fragments produced by pepsin digestion of whole antibodies.
  • the F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds.
  • F(ab')2 fragments have bivalent antigen-binding activity and are capable of cross-linking antigens.
  • Fv fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions.
  • Heavy chain variable regions and light chain variable regions include, for example, CDRs.
  • Fv fragments include all or part of the amino-terminal variable regions of the heavy and light chains of about 110 amino acids.
  • 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, wherein the light and heavy chains are variable
  • the regions are contiguous (eg, via synthetic linkers such as short flexible polypeptide linkers) and are capable of being expressed as single-chain polypeptides, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a scFv may have the VL and VH variable regions described in any order (eg, with respect to the N- and C-termini of the polypeptide), and the scFv may include a VL-linker-VH or VH-linker-VL can be included.
  • the term “dAb” generally refers to an antigen-binding fragment having a VH domain, a VL domain, or a VH domain or a VL domain, see eg Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.
  • the term “dAb” generally includes sdAbs.
  • the term “sdAb” generally refers to a single domain antibody. Single-domain antibodies generally refer to antibody fragments consisting only of the variable region (VH domain) of an antibody heavy chain or the variable region (VL) of an antibody light chain.
  • the term "monoclonal antibody” generally refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are usually highly specific for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the modifier "monoclonal” denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • the monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from antibodies from experimental animals such as rodents ("parent antibodies”), and the constant regions are derived from human antibodies, so that the resulting chimeric antibody has the potential to elicit an adverse immune response in human individuals compared to the parent antibody Decreased sex.
  • 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 by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a “humanized antibody” retains antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human antibodies may comprise minimally chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be used with a non-human species (donor antibody) (such as alpaca, mouse, etc.) having the desired properties, affinity and/or ability , rat, rabbit or non-human primate) CDR region residue substitutions.
  • donor antibody such as alpaca, mouse, etc.
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.
  • Fully human antibody generally refers to an antibody comprising only human immunoglobulin protein sequences.
  • a fully human antibody may contain murine sugar chains if it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells.
  • mouse antibody or “rat antibody” refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.
  • Fully human antibodies can be generated 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 make antibodies are described in US Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as the use of libraries, are known in the art.
  • polypeptide molecule and “polypeptide”, “peptide” are used interchangeably and generally refer to a polymer of amino acid residues.
  • fusion protein generally refers to a polypeptide having at least two moieties covalently linked together. Each of these moieties can be polypeptides with different properties.
  • the property may be a biological property, such as in vitro or in vivo activity.
  • the properties can also be simple chemical or physical properties, such as binding to target molecules, catalysis of reactions, etc.
  • the two moieties can be linked by a single peptide bond or by a peptide linker.
  • nucleic acid molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or analogs isolated from their natural environment or synthetically synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • a vector can transform, transduce or transfect a host cell so that the elements of genetic material it carries are expressed in the host cell.
  • vectors can include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes of P1 origin (PAC); bacteriophages such as lambda phage or M13 Bacteriophages and animal viruses, etc.
  • Animal virus species used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses vesicle virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses vesicle virus
  • a vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the carrier may also include components to assist its entry into
  • the term "cell” generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject plasmid or vector, comprising a nucleic acid molecule described herein or a nucleic acid molecule described herein Carrier.
  • a cell can include the progeny of a single cell. Progeny may not necessarily be identical (in morphology or in genome) to the original parent cell due to natural, accidental or intentional mutation.
  • Cells can include cells transfected in vitro with the vectors described herein.
  • the cells can be bacterial cells (eg, E.
  • yeast cells or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells , NS0 cells.
  • COS cells Chinese Hamster Ovary (CHO) cells
  • CHO-K1 cells Chinese Hamster Ovary (CHO) cells
  • LNCAP cells HeLa cells
  • HEK293 cells COS-1 cells
  • NS0 cells eukaryotic cells
  • the term “immunoconjugate” generally refers to the conjugation of the other agents (eg, chemotherapeutic agents, radioactive elements, cytostatic and cytotoxic agents) to the antibody or antigen-binding fragment thereof (eg, A conjugate formed by covalently linking a linking molecule), the conjugate can specifically bind to the antigen on the target cell through the antibody or antigen-binding fragment thereof, and deliver the other agent to the target cell.
  • the other agents eg, chemotherapeutic agents, radioactive elements, cytostatic and cytotoxic agents
  • the term "pharmaceutical composition” generally refers to a composition for the prevention/treatment of a disease or disorder.
  • the pharmaceutical composition may comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a carrier described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or suitable preservatives preparation.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.
  • the term "pharmaceutically acceptable carrier” generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that, at the doses and concentrations employed, are effective for the cells or mammals to which they are exposed. non-toxic.
  • Physiologically acceptable carriers can 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 nonionic surfactants.
  • the term "specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can be found in a heterogeneous population of molecules, including biomolecules
  • the presence of the target determines the presence of the target.
  • an antibody that specifically binds a target (which can be an epitope) can be an antibody that binds to that target with greater affinity, avidity, easier, and/or for a longer duration than it binds to other targets .
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include, but does not require, exclusive binding.
  • an antigen binding protein is specific for a particular epitope that cross-reacts with multiple antigens, wherein the specific antibody is capable of binding multiple antigens that carry the cross-reactive epitopes.
  • antigen binding protein binding sites and/or antigen binding proteins with specific binding cross-reactive epitopes are also referred to as multispecific or cross specific binding site antigen binding proteins, respectively.
  • an antigen binding protein can have a multispecific binding site that specifically binds to epitopes that cross-react with multiple different antigens.
  • the term "subject” generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues with the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, a substitution, deletion or A protein or polypeptide with the addition of one or more amino acids.
  • the functional variant may comprise at least 1, such as 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions that have been made , a protein or polypeptide with amino acid changes, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the protein or polypeptide prior to alteration.
  • the substitutions can be conservative substitutions.
  • the homolog may be at least about 85% (eg, an antibody or fragment thereof) of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to the pneumolysin protein). , having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more ) proteins or polypeptides with sequence homology.
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences. "Percent sequence homology" can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences.
  • FASTA and BLAST A description of the FASTA algorithm can be found in 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, “Rapid and Sensitive Protein Similarity Search", Science, 227: 1435-1441, 1989.
  • a description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", J. Molecular Biology, 215: 403-410 , 1990.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 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%.
  • the CDRs of antibodies are part of the variable region. Amino acid residues in this region can make contact with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, Kabat/Chothia, etc. in combination. These coding systems are known in the art, see eg http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR regions according to the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR regions.
  • the CDRs encompass CDR sequences that are divided according to any CDR division; variants thereof are also encompassed, the variants comprising substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR .
  • variants thereof are also encompassed, the variants comprising substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR .
  • homologues thereof are also encompassed, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 92%, amino acid sequences of about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the isolated antigen binding protein can be defined by IMGT.
  • the application provides an isolated antigen-binding protein, which can comprise at least one CDR in the VH of the variable region of an antibody heavy chain, and the VH can comprise the amino acid sequence shown in SEQ ID NO:39.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:24.
  • the HCDR of the isolated antigen-binding protein can be divided in any form, as long as the VH is the same as the amino acid sequence shown in any one of SEQ ID NO: 39, the HCDR obtained by dividing in any form can fall into the within the scope of protection of this application.
  • the antigen-binding protein isolated in this application may comprise the CDRs shown in the following table.
  • the isolated antigen binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 1.
  • the isolated antigen binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, and the HCDR2 may comprise the amino acid shown in SEQ ID NO:2 sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 1.
  • the isolated antigen binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO: The amino acid sequence shown in 32.
  • 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.
  • 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.
  • the isolated antigen binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO: 34 amino acid sequence.
  • the H-FR3 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:17.
  • the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: 35 amino acid sequence.
  • 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.
  • the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 32, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:33, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:34, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:35 amino acid sequence.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:7
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 10.
  • 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
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO, respectively, in sequence : 1, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and the amino acid sequence shown in SEQ ID NO: 18.
  • the isolated antigen binding protein may comprise a VH comprising the amino acid sequence shown in SEQ ID NO:39.
  • the VH may comprise the amino acid sequence set forth in any one of SEQ ID NO:22.
  • the VH may comprise the amino acid sequence set forth in any one of SEQ ID NO:24.
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region.
  • the antibody heavy chain constant region can be derived from the heavy chain constant region of any immunoglobulin, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody heavy chain constant region can be derived from a human IgG heavy chain constant region.
  • the heavy chain constant region of the immunoglobulin may comprise mutants thereof.
  • the antibody heavy chain constant region may be derived from the heavy chain constant region of any one of human IgG1-4.
  • the isolated antigen binding protein can be derived from a human IgGl heavy chain constant region.
  • the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:26.
  • the isolated antigen binding protein may comprise a heavy chain.
  • the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:28.
  • the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:30.
  • the isolated antigen binding protein may comprise at least one CDR in the variable region VL of the antibody light chain, and the VL may comprise the amino acid sequence shown in SEQ ID NO:40.
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:25.
  • the LCDR of the isolated antigen-binding protein can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NO: 40, the LCDR obtained by dividing in any form can fall into the within the scope of protection of this application.
  • the antigen-binding protein isolated in this application can comprise the CDRs shown in the following table.
  • the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6, and the LCDR2 may comprise the amino acid shown in SEQ ID NO:5 sequence, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the isolated antigen binding protein may comprise L-FR1, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 may comprise SEQ ID NO: The amino acid sequence shown in 36.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:19.
  • the isolated antigen-binding protein can comprise L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 can comprise SEQ ID NO: 37 amino acid sequence.
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:12.
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen-binding protein may comprise L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise SEQ ID NO: 38 amino acid sequence.
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:13.
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the isolated antigen-binding protein may comprise L-FR4, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO: 14 amino acid sequence.
  • the isolated antigen-binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4, and 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 amino acid sequence.
  • the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 19
  • the L-FR2 may comprise the amino acid sequence set forth 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
  • the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 12
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13
  • the amino acid sequence shown, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 19
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 20
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 21
  • the amino acid sequence shown, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14.
  • LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO, respectively, in sequence :4, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and the amino acid sequence shown in SEQ ID NO: 14.
  • the isolated antigen binding protein may comprise an antibody light chain constant region.
  • the light chain constant region can be derived from a human antibody light chain constant region.
  • the light chain constant region can be derived from a human IgK constant region.
  • the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:27.
  • the isolated antigen binding protein may comprise a light chain.
  • the light chain of the isolated antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:29.
  • the light chain of the isolated antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:31.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.
  • the HCDR1 of the isolated antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:2, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:1
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:4. amino acid sequence shown.
  • the isolated antigen binding protein may comprise VH and VL.
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:22
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:23.
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:24
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:25.
  • the isolated antigen-binding protein may comprise an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragments may include Fab, Fab', Fv fragments, F(ab') 2 , F(ab) 2 , scFv, di-scFv and/or dAbs.
  • the antibodies may include monoclonal antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies.
  • the VH of the humanized antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the VL of the humanized antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:25.
  • amino acid residue groups include those with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains ( For example, tyrosine, phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eglycine, as
  • one or more amino acid residues in the CDR regions of the isolated antigen binding proteins described herein can be replaced with other amino acid residues of the same side chain group.
  • Those skilled in the art know that some conservative sequence modifications will not abolish antigen binding, see, 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.
  • antigen binding proteins with pneumolysin protein antigen affinity can be screened by immunizing mice with pneumolysin mutants, obtaining mouse splenocytes and fusing myeloma cells.
  • the post-translational modification site of the antigen-binding protein obtained by screening can also be optimized to obtain a humanized sequence.
  • pneumolysin antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art.
  • the antigen binding proteins of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (eg, Western blot), flow cytometry (eg, FACS), immunohistochemistry, immunofluorescence, and the like antigen-binding activity.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting eg, Western blot
  • flow cytometry eg, FACS
  • immunohistochemistry eg, immunofluorescence, and the like antigen-binding activity.
  • the isolated antigen binding protein is capable of specifically binding pneumolysin.
  • the pneumolysin may comprise wild-type pneumolysin and variants thereof.
  • the variant of pneumolysin may comprise a deletion of amino acid 146 compared to wild-type pneumolysin.
  • the amino acid sequence of wild-type pneumolysin can be determined from the Uniprot database (Entry ID: Q04IN8).
  • the binding of the isolated antigen-binding protein to pneumolysin can be detected by ELISA method.
  • the antigen-binding proteins described herein may be present at less than or equal to about 0.03 ⁇ g/ml, less than or equal to about 0.028 ⁇ g/ml, less than or equal to about 0.026 ⁇ g/ml, less than or equal to about 0.024 ⁇ g/ml, less than or equal to about 0.024 ⁇ g/ml equal to about 0.022 ⁇ g/ml, less than or equal to about 0.02034 ⁇ g/ml, less than or equal to about 0.020 ⁇ g/ml, less than or equal to about 0.018 ⁇ g/ml, less than or equal to about 0.01706 ⁇ g/ml, less than or equal to about 0.016 ⁇ g/ml ml, less than or equal to about 0.014 ⁇ g/ml, less than or equal to about 0.012 ⁇ g/ml
  • the isolated antigen binding protein is capable of neutralizing the biological activity of pneumolysin protein.
  • recombinant pneumolysin can be mixed with the isolated antigen binding protein described herein, added to rabbit red blood cells, and the antihemolytic activity of the antigen binding protein can be tested.
  • the isolated antigen binding protein is capable of preventing and/or treating diseases and/or disorders.
  • the disease and/or disorder may be caused or mediated by Streptococcus pneumoniae.
  • the disease and/or disorder may comprise a complication of a disease and/or disorder caused or mediated by Streptococcus pneumoniae.
  • the disease and/or disorder may comprise sepsis and/or bacteremia.
  • Polypeptide molecules Polypeptide molecules, nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions
  • the application provides polypeptide molecules that may comprise the isolated antigen binding proteins described herein.
  • the polypeptide molecule may comprise a fusion protein.
  • the polypeptide molecule may be a fusion protein.
  • the fusion protein may comprise a multispecific antibody.
  • the application provides isolated nucleic acid molecules that can encode the isolated antigen binding proteins described herein.
  • it may be produced or synthesized by: (i) amplified in vitro, eg, by polymerase chain reaction (PCR) amplification; (ii) recombinantly produced by cloning; (iii) purified (iv) synthetic, such as by chemical synthesis.
  • PCR polymerase chain reaction
  • the present application provides a vector, which can comprise the nucleic acid molecule described herein.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell.
  • the vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used, eg, in genetic engineering.
  • the vector is an expression vector.
  • the carrier may also include components to assist its entry into cells, such as viral particles, liposomes or protein coats, but not only these materials.
  • the present application provides a cell, which may comprise the nucleic acid molecule described herein or the vector described herein.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into such host cells, eg, eukaryotic cells, such as cells from plants, fungi or yeast cells, and the like.
  • the cells can be bacterial cells (eg, E.
  • yeast cells or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2/0 cells, NSO cells or myeloma cells.
  • CHO Chinese Hamster Ovary
  • LNCAP low-density lipoprotein
  • HeLa cells HeLa cells
  • 293T cells HeLa cells
  • COS-1 cells COS-1 cells
  • SP2/0 cells nuclear-derived cells
  • NSO cells myeloma cells.
  • the vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the present application also provides immunoconjugates, which may comprise the isolated antigen binding proteins described herein.
  • an isolated antigen binding protein or fragment thereof described herein can be linked to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, and the like.
  • the attachment can be through one or more covalent bonds, or non-covalent interactions, and can include chelation.
  • linkers which may be known in the art, can be used to form immunoconjugates.
  • immunoconjugates can be provided in the form of fusion proteins, which can be expressed from polynucleotides encoding the immunoconjugates.
  • the immunoconjugate may also comprise, for example, an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the antibody and therapeutic agent can be cross-linked by a linker that is cleavable, such as a peptidic, disulfide, or hydrazone-like linker.
  • the present application also provides a pharmaceutical composition, which can comprise the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the immunoconjugate described in the present application, the The nucleic acid molecule, the vector described herein and/or the cell described herein, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.
  • the pharmaceutical compositions may also contain more than one active compound, typically those active compounds with 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, as well as on the clinical parameters of the subject.
  • the pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents compatible with pharmaceutical administration, and is generally safe and nontoxic .
  • the pharmaceutical composition may comprise parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.
  • the present application provides methods of making the isolated antigen binding proteins.
  • the method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed.
  • these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application.
  • animals can be immunized with an attenuated strain of pneumolysin or a fragment thereof.
  • the attenuated S. pneumoniae strain may comprise a deletion mutation at position 146 compared to the wild type.
  • Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • pneumolysin can be used as an immunogen (antigen) for the production of non-human antibodies specific for pneumolysin, which are screened for biological activity.
  • the priming immunogen can be full-length pneumolysin, including native homodimers, or peptides containing single/multiple epitopes.
  • the immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art.
  • the present application provides that the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition are prepared Use in a medicament for the prevention and/or treatment of a disease and/or disorder.
  • the present application also provides a method for preventing and/or treating a disease and/or disorder, the method may comprise administering to a subject in need thereof the isolated antigen-binding protein, the polypeptide molecule, The nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition.
  • the administration can be carried out by different means, eg intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition described in the present application can be used with for the prevention and/or treatment of diseases and/or disorders.
  • the disease and/or disorder may be caused or mediated by Streptococcus pneumoniae.
  • the disease and/or disorder may be a complication of a disease and/or disorder caused or mediated by Streptococcus pneumoniae.
  • the disease and/or disorder may include bacterial infection.
  • the disease and/or disorder may include bacteremia and/or sepsis.
  • the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition can be used alone, or It may be used in combination with other drugs to prevent and/or treat diseases and/or conditions.
  • the other drug can be any drug that is currently known to have antibacterial effects.
  • the disease and/or condition may be due to Streptococcus pneumoniae infection, or may be a related disease caused by other infections.
  • the present application also provides a method for detecting pneumolysin protein in a sample, the method comprising administering the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the The cell, the immunoconjugate and/or the pharmaceutical composition.
  • the method for detecting pneumolysin protein in a sample may be an in vitro method.
  • an isolated antigen binding protein described herein is contacted with an ex vivo sample, and the presence and/or amount of pneumolysin in the sample is detected.
  • the method of detecting pneumolysin protein in a sample is non-therapeutic.
  • the method of detecting pneumolysin protein in a sample is not a diagnostic method.
  • the present application also provides a reagent or kit for detecting pneumolysin protein in a sample, which comprises the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the The cell, the immunoconjugate and/or the pharmaceutical composition.
  • the present application also provides that the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition are in Use in the preparation of a kit for detecting the presence and/or content of pneumolysin in a sample.
  • the amino acid sequence of pneumolysin (PLY, Entry ID: Q04IN8) was determined using the Uniprot database, and the alanine at position 146 was deleted according to the full-length amino acid sequence of hemolysin as the amino acid of the attenuated PLY mut.
  • the amino acid sequence is The template was limited to NdeI/XhoI, and a His tag was introduced at the C-terminus. It was handed over to Beijing Qingke Biotechnology Co., Ltd. for codon optimization and gene synthesis to obtain the plasmids of PLY and PLY mut proteins.
  • the recombinant plasmid was transformed into E.coli BL21(DE3)plysS, and the E.coli expression system was used to recombinantly express PLY and PLYmut recombinant proteins.
  • mice were immunized by subcutaneous multi-point immunization method.
  • the subcutaneous multi-point immunization process is shown below.
  • Myeloma cells P3X63Ag8.653 were fused with mouse spleen cells immunized with PLY mut antigen by PEG method.
  • the fused hybridoma cells were seeded in 96-well plates for culture. Hybridoma cells were then cultured using HAT medium and HT medium. When the cell supernatant in the 96-well plate was slightly yellow, 100 ⁇ l of the cell culture supernatant was taken, and ELISA method was used to screen the hybridoma clones that bind to the PLYmut antigen. After culturing the hybridoma parent clones with stronger affinity, subcloning was carried out by limiting dilution method, and monoclonal cells were selected. The monoclonal supernatant was screened by ELISA to screen 2E5 monoclonal cells that had strong affinity with PLYmut protein and secreted antibody, and the secreted monoclonal antibody was named m2E5.
  • RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.).
  • the total RNA was reversed into cDNA using a reverse transcription kit (Takara).
  • Use primers to amplify cDNA fragments (Anke Krebber. 1997), use DNA product purification kit (Beijing Tiangen Biochemical Technology Co., Ltd.) to purify PCR products, and use ligation kit (Beijing Qingke Biotechnology Co., Ltd.) to connect to T vector
  • ligation kit Beijing Qingke Biotechnology Co., Ltd.
  • m2E5 heavy chain variable region (m2E5VH) SEQ ID NO: 22
  • the underlined part represents the CDR sequence obtained by dividing using the IMGT numbering scheme.
  • the germline sequence closest to the mouse antibody 2E5 was selected as the template, and m2E5 was humanized, and the The post-translational modification sites of the antigen-binding protein were optimized to obtain the sequences of the heavy chain variable region (hu2E5VH3) and light chain variable region (hu2E5VL0) of the humanized antibody 2E5H3L0 as follows.
  • Humanized antigen binding protein 2E5 heavy chain variable region (hu2E5VH3) SEQ ID NO:24
  • Humanized antigen binding protein 2E5 light chain variable region (hu2E5VL0) SEQ ID NO: 25
  • the underlined part represents the CDR sequence obtained by dividing using the IMGT numbering scheme.
  • heavy chain amino acid sequence and light chain (c2E5VLCL) amino acid sequence of the chimeric antibody 2E5HL are:
  • the heavy chain (hu2E5VH3CH) amino acid sequence and light chain (hu2E5VLOCL) amino acid sequence of the 2E5H3L0 humanized antibody are:
  • the heavy chain expression vector pcDNA3.4-m2E5VHCH of the 2E5HL chimeric antibody and the light chain expression vector pcDNA3.4-m2E5VLCL were co-transfected into Expi293F cells, and the Expi293F expression system was used to transiently express 2E5HL. Finally, the chimeric antibody protein was purified with Protein A.
  • the heavy chain expression vector pcDNA3.4-hu2E5VH3CH of the 2E5H3L0 humanized antibody and the light chain expression vector pcDNA3.4-hu2E5VL0CL were co-transfected into Expi293F cells, and the Expi293F expression system was used to transiently express 2E5H3L0.
  • the humanized antibody protein was purified with Protein A. .
  • the purified 2E5HL chimeric antibody and 2E5H3L0 humanized antibody were identified by ELISA for antigen-binding activity: 0.1 ⁇ g/well of PLY and PLYmut proteins were coated on a 96-well microtiter plate (Thermo), incubated at 4°C overnight, and buffered with PBST. After washing the plate, add 200 ⁇ l of 5% BSA to each well and incubate at 37°C for 1 hour; after blocking, wash the plate with PBST buffer, and add 0.1 ⁇ g of 2E5HL human diluted in the first hole to the microtiter plate.
  • the EC 50 value of 2E5HL is 0.01706 ⁇ g/ml, and the EC 50 value of 2E5H3L0 is 0.02034 ⁇ g/ml; the binding results of antibody and PLYmut protein are shown in Figure 2.
  • the EC 50 value was 0.01582 ⁇ g/ml, and the EC 50 value of 2E5H3L0 was 0.01617 ⁇ g/ml.
  • the results showed that both the humanized antibody 2E5H3L0 and the chimeric antibody 2E5HL had strong affinity with both pneumolysin PLY and PLYmut proteins.
  • recombinant pneumolysin PLY protein and chimeric antibody 2E5HL or humanized antibody 2E5H3L0 were mixed in a certain proportion and added to a 96-well plate (Thermo), incubated at 37°C for 10min, added with 5% rabbit erythrocytes, and incubated at 37°C for 1h .
  • the sample was centrifuged at 3000 rpm for 5 min, and the absorbance was detected at a wavelength of 405 nm using a microplate reader (Multiskcin FC, Thermo), and its anti-hemolytic activity was measured.
  • Streptococcus pneumoniae toxin has rabbit erythrocyte hemolytic activity, while both chimeric antibody 2E5HL and humanized antibody 2E5H3L0 can effectively neutralize the rabbit erythrocyte hemolytic activity of Streptococcus pneumoniae toxin in a dose-dependent manner.
  • mice were pre-injected with 1.25 mg/kg chimeric antibody 2E5HL, humanized antibody 2E5H3L0 and human IgG antibody (as a control), and 0.75 ⁇ g pneumolysin PLY protein was injected into the tail vein 1 h later. Observe the survival time of mice.
  • Example 9 The protective effect of the antigen-binding protein of the present application on mice infected with Streptococcus pneumoniae
  • mice were injected with 2 ⁇ 10 8 CFU of Streptococcus pneumoniae into the tail vein 2 hours in advance, and 2 hours later, 30 mg/kg of 2E5HL, 2E5H3L0 or human IgG (control) were injected into the tail vein respectively, and the survival of the mice in each group was observed and recorded.

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Abstract

提供一种分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:39所示的氨基酸序列。所述分离的抗原结合蛋白能够特异性结合肺炎链球菌溶血素蛋白及其变体，还提供了所述分离的抗原结合蛋白在预防和/或治疗肺炎链球菌感染的疾病和/或病症中的用途。

Description

靶向肺炎链球菌溶血素的抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种能够靶向肺炎链球菌溶血素蛋白(PLY)的抗原结合蛋白，及其在治疗和预防肺炎链球菌感染领域的应用。
背景技术
肺炎链球菌又称肺炎球菌，广泛定植在人类鼻咽部，在机体抵抗力低下时可引起肺部或者中枢神经系统感染，严重时形成菌血症或者脓毒症。
溶血素(PLY)是肺炎链球菌重要的毒力因子，在肺炎链球菌形成感染过程中发挥重要作用。PLY可以破坏肺组织上皮、内皮及免疫细胞，增加血管通透性及肺炎链球菌定植、传播和侵袭，并影响患者肺功能，形成急性肺损伤，甚至急性呼吸窘迫综合征。因此，亟需开发有效的对抗肺炎链球菌感染的药物。
发明内容
本申请提供了一种分离的抗原结合蛋白，其具有下述性质中的一种或多种：1)能够特异性结合肺炎链球菌溶血素蛋白及其变体；2)对肺炎链球菌溶血素蛋白具有高亲和力及中和活性；3)能有效阻断肺炎链球菌形成的感染；4)能够有效预防和/或治疗肺炎链球菌引起的疾病和/或病症。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:39所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述 HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:32所示的氨基酸序列。
在某些实施方式中，所述H-FR1包含SEQ ID NO:7或SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述H-FR2包含SEQ ID NO:8或SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:34所示的氨基酸序列。
在某些实施方式中，所述H-FR3包含SEQ ID NO:9或SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述H-FR4包含SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:32所示的氨基酸序列，所述H-FR2包含SEQ ID NO:33所示的氨基酸序列，所述H-FR3包含SEQ ID NO:34所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:7或SEQ ID NO:15所示的氨基酸序列，所述H-FR2包含SEQ ID NO:8或SEQ ID NO:16所示的氨基酸序列，所述H-FR3包含SEQ ID NO:9或SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含选自下述任一组的H-FR1，H-FR2，H-FR3和H-FR4：
1)所述H-FR1包含SEQ ID NO:7所示的氨基酸序列，所述H-FR2包含SEQ ID NO:8所示的氨基酸序列，所述H-FR3包含SEQ ID NO:9所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:10所示的氨基酸序列；以及
2)所述H-FR1包含SEQ ID NO:15所示的氨基酸序列，所述H-FR2包含SEQ ID NO:16 所示的氨基酸序列，所述H-FR3包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VH，所述VH包含SEQ ID NO:39所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的VH包含SEQ ID NO:22或SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链恒定区。
在某些实施方式中，所述分离的抗原结合蛋白的重链恒定区源自人IgG恒定区。
在某些实施方式中，所述分离的抗原结合蛋白的重链恒定区源自人IgG1重链恒定区。
在某些实施方式中，所述分离的抗原结合蛋白的重链恒定区包含SEQ ID NO:26所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括重链，所述重链包含SEQ ID NO:28或SEQ ID NO:30所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:40所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR3，所述LCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR2，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1，所述LCDR1包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:36所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的L-FR1包含SEQ ID NO:11或SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR2，所述L-FR2位于所述LCDR1和所述LCDR2之间，且所述L-FR2包含SEQ ID NO:37所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的L-FR2包含SEQ ID NO:12或SEQ ID NO:20所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR3，所述L-FR3位于所述LCDR2和所述LCDR3之间，且所述L-FR3包含SEQ ID NO:38所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的L-FR3包含SEQ ID NO:13或SEQ ID NO:21所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR4，所述L-FR4的N端与所述LCDR3的C端直接或间接相连，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:36所示的氨基酸序列，所述L-FR2包含SEQ ID NO:37所示的氨基酸序列，所述L-FR3包含SEQ ID NO:38所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:11或SEQ ID NO:19所示的氨基酸序列，所述L-FR2包含SEQ ID NO:12或SEQ ID NO:20所示的氨基酸序列，所述L-FR3包含SEQ ID NO:13或SEQ ID NO:21所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含选自下述任一组的L-FR1，L-FR2，L-FR3和L-FR4：
1)所述L-FR1包含SEQ ID NO:11所示的氨基酸序列，所述L-FR2包含SEQ ID NO:12所示的氨基酸序列，所述L-FR3包含SEQ ID NO:13所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列；以及
2)所述L-FR1包含SEQ ID NO:19所示的氨基酸序列，所述L-FR2包含SEQ ID NO:20所示的氨基酸序列，所述L-FR3包含SEQ ID NO:21所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VL，所述VL包含SEQ ID NO:40所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的所述VL包含SEQ ID NO:23或SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链恒定区。
在某些实施方式中，所述分离的抗原结合蛋白的所述轻链恒定区源自人Igκ恒定区。
在某些实施方式中，所述分离的抗原结合蛋白的所述轻链恒定区包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链，所述轻链包含SEQ ID NO:29或SEQ ID NO:31所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含选自下述任一组的VH和VL：
1)所述VH包含SEQ ID NO:22所示的氨基酸序列，且所述VL包含SEQ ID NO:23所示的氨基酸序列；以及
2)所述VH包含SEQ ID NO:24所示的氨基酸序列，且所述VL包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述抗体选自下组中的一种或多种：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述分离的抗原结合蛋白能够特异性结合肺炎链球菌溶血素蛋白。
在某些实施方式中，所述肺炎链球菌溶血素包含野生型肺炎链球菌溶血素蛋白及其变体。
在某些实施方式中，所述肺炎链球菌溶血素蛋白包含与野生型肺炎链球菌溶血素蛋白相比，第146位氨基酸缺失的肺炎链球菌溶血素蛋白变体。
在某些实施方式中，所述分离的抗原结合蛋白能够预防和/或治疗肺炎链球菌引起的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肺炎链球菌引起的疾病和/或病症的并发症。
另一方面，本申请还提供了多肽分子，其包含所述分离的抗原结合蛋白。
在某些实施方式中，所述多肽分子包含融合蛋白。
另一方面，本申请还提供了免疫缀合物，其包含所述分离的抗原结合蛋白。
另一方面，本申请还提供了核酸分子，其编码所述分离的抗原结合蛋白或所述多肽分子。
另一方面，本申请还提供了载体，其包含所述核酸分子。
另一方面，本申请还提供了细胞，其包含所述核酸分子或所述载体。
另一方面，本申请还提供了药物组合物，其包含所述分离的抗原结合蛋白、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的载剂。
另一方面，本申请还提供了制备所述分离的抗原结合蛋白的方法，所述方法包括在使得所述抗原结合蛋白表达的条件下，培养所述细胞。
另一方面，本申请所述分离的抗原结合蛋白、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物单独使用或与其它药物联合使用。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
在某些实施方式中，所述疾病和/或病症及其并发症由肺炎链球菌引起或介导。
在某些实施方式中，所述疾病和/或病症包括肺炎链球菌引起或介导的疾病和/或病症的并发症。
另一方面，本申请还提供了检测样品中肺炎链球菌溶血素蛋白的方法，所述方法包括施用所述分离的抗原结合蛋白、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
另一方面，本申请还提供了检测样品中肺炎链球菌溶血素蛋白的试剂盒，所述试剂盒包括所述分离的抗原结合蛋白、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物在制备试剂盒中的用途，所述试剂盒用于检测样品中肺炎链球菌溶血素蛋白的存在和/或含量。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请所述抗原结合蛋白与PLY蛋白的结合活性测定。
图2显示的是本申请所述抗原结合蛋白与PLYmut蛋白的结合活性测定。
图3显示的是本申请所述抗原结合蛋白在体外对肺炎链球菌溶血素的兔红细胞溶血活性的中和活性。
图4显示的是本申请所述抗原结合蛋白对PLY毒素模型小鼠的保护作用。
图5显示的是本申请所述抗原结合蛋白对肺炎链球菌感染小鼠的保护作用。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“肺炎链球菌溶血素”可以与“Pneumolysin”、“PLY”和“肺炎链球菌溶血素蛋白”互换使用，通常指肺炎链球菌含有的一种多功能毒力因子。在本申请中，该术语可涵盖野生型肺炎链球菌溶血素蛋白及其变体、类似物、同源物和功能活性片段。例如，所述肺炎链球菌溶血素蛋白的变体可以包含与野生型溶血素相比，发生第146位氨基酸缺失的突变。例如，所述功能活性片段可以是能够在宿主中引起体液和/或细胞免疫应答的部分。
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“分离的抗原结合蛋白”通常指脱离了其天然存在状态的具有抗原结合能力的蛋白。该“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的CDR或CDR衍生物的备选蛋白框架区或人工框架区。此类框架包括，但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突变的抗体来源的框架区以及包含例如生物相容性聚合物的完全合成的框架区。参见例如Korndorfer等,2003,Proteins:Structure,Function,andBioinformatics,53(1):121-129(2003)；Roque等,Biotechnol.Prog.20:639-654(2004)。抗原结合蛋白的实例包括但不限于：人抗体；人源化抗体；嵌合抗体；重组抗体；单链抗体；双功能抗体；三功能抗体；四功能抗体；Fab，Fab’，Fv片 段，Bs-Fv，F(ab’) 2，F(ab) 2，scFv，di-scFv，dAb，IgD抗体；IgE抗体；IgM抗体；IgG1抗体；IgG2抗体；IgG3抗体；或IgG4抗体以及其片段。
在本申请中，术语“CDR”也称“互补决定区”，通常指抗体可变结构域中的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在某些实施方案中，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。参见，例如，Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、AbM、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。例如，所述抗原结合蛋白的氨基酸序列编号可以按照IMGT编号方案(IMGT,the international ImMunoGeneTics information system@imgt.cines.fr；http：//imgt.cines.fr；Lefranc等,1999,Nucleic Acids Res.27:209-212；Ruiz等,2000 Nucleic Acids Res.28:219-221；Lefranc等,2001,Nucleic Acids Res.29:207-209；Lefranc等,2003,Nucleic Acids Res.31:307-310；Lefranc等,2005,DevComp Immunol 29:185-203)。例如，所述抗原结合蛋白的CDR可以根据Kabat编号系统确定(参见例如Kabat EA&Wu TT(1971)Ann NY AcadSci 190:382-391和Kabat EA et al.,(1991)Sequences of Proteins of Immunological Interest,FifthEdition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)。
在本申请中，术语“FR”通常指抗体可变结构域的更高度保守的部分，其被称为框架区。通常，天然重链和轻链的可变结构域各自包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3和L-FR4)。
在本申请中，术语“可变结构域”与“可变区”可以互换使用，通常指抗体重链和/或轻链的一部分。重链和轻链的可变结构域可以分别称为“V H”和“V L”(或者分别称为“VH”和“VL”)。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。
在本申请中，术语“可变”通常指在抗体之间可变结构域的某些区段在序列上可能存在较大差异。可变结构域介导抗原结合并决定特定抗体对其特定抗原的特异性。然而，可变性并非在整个可变结构域范围内均匀分布。它通常集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大多数采用β-折叠构型，通过三个CDR连 接，其形成环形连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成(参见Kabat et al,Sequences of Immunological Interest,Fifth Edition,National Institute of Health,Bethesda,Md.(1991))。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab') 2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合肺炎链球菌溶血素蛋白)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994，第71页和第6章。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。根据重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。
在本申请中，术语“抗原结合片段”通常指具有特异结合抗原(例如，肺炎链球菌溶血素)能力的一个或多个片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
在本申请中，术语“Fab”通常指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶 消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区，同上)。Fab片段可以由一条完整的L链与一条重链的可变区和该H链(VH)的第一恒定区(CH1)组成。
在本申请中，术语“Fab′片段”通常指人单克隆抗体的单价抗原结合片段，该片段比Fab片段稍大。例如，Fab′片段可以包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有的220-330个氨基酸残基。
在本申请中，术语“F(ab')2”通常指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段具有二价抗原结合活性并且能够交联抗原。
在本申请中，术语“Fv片段”通常指人单克隆抗体的单价抗原结合片段，包括所有或部分重链可变区和轻链可变区，并且缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区包括例如CDR。例如，Fv片段包括重链和轻链的约110个氨基酸的所有或部分氨基端可变区。
在本申请中，术语“scFv”通常指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
在本申请中，术语“dAb”通常是指具有VH域、VL域或具有VH域或VL域的抗原结合片段，参考例如Ward等人(Nature,1989Oct 12；341(6242)：544-6)，参考Holt等人,Trends Biotechnol.,2003,21(11)：484-490；以及参考例如WO 06/030220、WO 06/003388和DomantisLtd的其它公布的专利申请。术语“dAb”通常包括sdAb。术语“sdAb”通常指单域抗体。单域抗体通常指仅由抗体重链可变区(VH结构域)或抗体轻链的可变区(VL)组成的抗体片段。
在本申请中，术语“单克隆抗体”通常指单分子组成的抗体分子制备物。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重 组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体的CDR区以外的部分或全部氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如羊驼，小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
术语“全人源抗体”通常指仅包含人类免疫球蛋白蛋白质序列的抗体。如果其是在小鼠中、在小鼠细胞中或在衍生自小鼠细胞的杂交瘤中生产，那么全人源抗体可能含有鼠糖链。类似地，“小鼠抗体”或“大鼠抗体”分别指仅包含小鼠或大鼠免疫球蛋白序列的抗体。可通过噬菌体展示或其它分子生物学方法，在人体内、在具有人类免疫球蛋白种系序列的转基因动物体内生成全人源抗体。可用于制造抗体的示例性技术在美国专利：6,150,584、6,458,592、6,420,140中描述。其它技术，如使用文库，是本领域已知的。
在本申请中，术语“多肽分子”和“多肽”、“肽”可以互换使用，通常指氨基酸残基的聚合物。术语“融合蛋白”通常指具有至少两个共价连接在一起的部分的多肽。其中每个部分可以是具有不同特性的多肽。该特性可以是生物学性质，例如体外或体内活性。该性质也可以是简单的化学或物理性质，例如与靶分子的结合，反应的催化等。这两个部分可以通过单个肽键或通过肽接头连接。
在本申请中，术语“核酸分子”通常指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元 件在宿主细胞内得以表达。举例来说，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类可以包括逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本发明所述的核酸分子或本发明所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本申请所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞。
在本申请中，术语“免疫缀合物”通常指所述其他试剂(例如，化疗剂、放射性元素、细胞生长抑制剂和细胞毒性剂)与所述抗体或其抗原结合片段缀合(例如，通过连接分子共价相连)而形成的缀合物，该缀合物可以通过所述抗体或其抗原结合片段与靶细胞上的抗原特异性结合，将所述其他试剂递送至靶细胞。
在本申请中，术语“药物组合物”通常指用于预防/治疗疾病或病症的组合物。所述药物组合物可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的载剂”通常包括药剂学可接受的载剂、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。生理学可接受的载体可包括例如缓冲剂，抗氧化剂，低分子量(少于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子，例如钠，和/或非离子表面活性剂。
在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，例如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体可以是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更长的持续时间结合该靶标的抗体。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在某些实施方案中，特异性结合可以包括但不要求排他性地结合。该术语也适用于，如抗原结合蛋白对与多个抗原交叉反应的特定表位是有特异性的，其中，特异性的抗体能结合携带交叉反应表位的多种抗原。这种抗原结合蛋白的结合位点和/或具有特异性结合交叉反应表位的抗原结合蛋白，分别也被称为多特异性或交叉特异性结合位点的抗原结合蛋白。例如，抗原结合蛋白可具有与多个不同抗原交叉反应的表位特异性结合的多特异性结合位点。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，特异性结合肺炎链球菌溶血素蛋白的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，特异性结合肺炎链球菌溶血素蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met) 的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，术语“包含”通常是指包括、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下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％、或10％的范围内变动。
发明详述
分离的抗原结合蛋白
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在本申请中，所述分离的抗原结合蛋白可以通过IMGT定义。
一方面，本申请提供一种分离的抗原结合蛋白，其可以包含抗体重链可变区VH中的至少一个CDR，所述VH可以包含SEQ ID NO:39所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:22或SEQ ID NO:24所示的氨基酸序列。在本申请中，所述分离的抗原结合蛋白的HCDR可以通过任何形式划分，只要VH与SEQ ID NO:39中任一项所示的氨基酸序列相同，以任何形式划分得到的HCDR都可落入本申请的保护范围内。
例如，通过表1的方法划分SEQ ID NO:22或SEQ ID NO:24所示的氨基酸序列，本申请中分离的抗原结合蛋白可以包含如下表所示的CDR。
表1.不同方式划分SEQ ID NO:22或SEQ ID NO:24所示的氨基酸序列得到的CDR序列
Figure PCTCN2022082011-appb-000001
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR2，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1可以包含SEQ ID NO:32所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的H-FR1可以包含SEQ ID NO:7或SEQ ID NO:15所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR2，所述H-FR2位于所述HCDR1 和所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:33所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的H-FR2可以包含SEQ ID NO:8或SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:34所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的H-FR3可以包含SEQ ID NO:9或SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:35所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的H-FR4可以包含SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1可以包含SEQ ID NO:32所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:33所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:34所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:35所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:7或SEQ ID NO:15所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:8或SEQ ID NO:16所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:9或SEQ ID NO:17所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:7所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:8所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:9所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:10所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:15所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:16所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2，HCDR3，H-FR1，H-FR2，H-FR3和H-FR4。例如，所述分离的抗原结合蛋白的HCDR1，HCDR2，HCDR3，H-FR1，H-FR2，H-FR3和H-FR4可分别依次包含SEQ ID NO:3、SEQ ID NO:2、SEQ ID NO:1、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10所示的氨基酸序列。例如，所述分离的抗原结合蛋白的HCDR1，HCDR2，HCDR3，H-FR1，H-FR2，H-FR3和H-FR4可分别依次包含SEQ ID NO:3、SEQ ID NO:2、SEQ ID NO:1、SEQ ID NO:15、SEQ ID  NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含VH，所述VH包含SEQ ID NO:39所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:22中任一项所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:24中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包括抗体重链恒定区。例如，所述抗体重链恒定区可以源自任一种免疫球蛋白的重链恒定区，包括IgM、IgD、IgG、IgA和IgE。例如，所述抗体重链恒定区可以源自人IgG重链恒定区。在本申请中，所述免疫球蛋白的重链恒定区可以包含其突变体。在本申请中，所述抗体重链恒定区可以源自人IgG1-4中任一种的重链恒定区。例如，所述分离的抗原结合蛋白可以源自人IgG1重链恒定区。例如，所述重链恒定区可以包含SEQ ID NO:26所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含重链。例如，所述重链可以包含SEQ ID NO:28所示的氨基酸序列。例如，所述重链可以包含SEQ ID NO:30所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含抗体轻链可变区VL中的至少一个CDR，所述VL可以包含SEQ ID NO:40所示的氨基酸序列。例如，所述VL可以包含SEQ ID NO:23或SEQ ID NO:25所示的氨基酸序列。在本申请中，所述分离的抗原结合蛋白的LCDR可以通过任何形式划分，只要VL与SEQ ID NO:40中任一项所示的氨基酸序列相同，以任何形式划分得到的LCDR都可落入本申请的保护范围内。
例如，通过表2的方法划分SEQ ID NO:23或SEQ ID NO:25所示的氨基酸序列，本申请中分离的抗原结合蛋白可以包含如下表所示的CDR。
表2.不同方式划分SEQ ID NO:23或SEQ ID NO:25所示的氨基酸序列得到的CDR序列
Figure PCTCN2022082011-appb-000002
在本申请中，所述分离的抗原结合蛋白可以包含LCDR3，所述LCDR3可以包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR2，所述LCDR2可以包含SEQ ID  NO:5所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR1，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR1，LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:5所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1可以包含SEQ ID NO:36所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:11所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR2，所述L-FR2位于所述LCDR1和所述LCDR2之间，且所述L-FR2可以包含SEQ ID NO:37所示的氨基酸序列。例如，所述L-FR2可以包含SEQ ID NO:12所示的氨基酸序列。例如，所述L-FR2可以包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3可以包含SEQ ID NO:38所示的氨基酸序列。例如，所述L-FR3可以包含SEQ ID NO:13所示的氨基酸序列。例如，所述L-FR3可以包含SEQ ID NO:21所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1可以包含SEQ ID NO:36所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:37所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:38所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:14所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:11或SEQ ID NO:19所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:12或SEQ ID NO:20所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:13或SEQ ID NO:21所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:14所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:11所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:12所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:13所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:14所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:19所示的氨基酸序列，所述L-FR2 可以包含SEQ ID NO:20所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:21所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR1，LCDR2，LCDR3，L-FR1，L-FR2，L-FR3和L-FR4。例如，所述分离的抗原结合蛋白的LCDR1，LCDR2，LCDR3，L-FR1，L-FR2，L-FR3和L-FR4可分别依次包含SEQ ID NO:6、SEQ ID NO:5、SEQ ID NO:4、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列。例如，所述分离的抗原结合蛋白的LCDR1，LCDR2，LCDR3，L-FR1，L-FR2，L-FR3和L-FR4可分别依次包含SEQ ID NO:6、SEQ ID NO:5、SEQ ID NO:4、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含VL，所述VL包含SEQ ID NO:40所示的氨基酸序列。例如，所述VL可以包含SEQ ID NO:23所示的氨基酸序列。例如，所述VL可以包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含抗体轻链恒定区。例如，所述轻链恒定区可以源自人抗体轻链恒定区。例如，所述轻链恒定区可以源自人Igκ恒定区。例如，所述轻链恒定区可以包含SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含轻链。例如，所述分离的抗原结合蛋白的轻链可以包含SEQ ID NO:29所示的氨基酸序列。例如，所述分离的抗原结合蛋白的轻链可以包含SEQ ID NO:31所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3。例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所示HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:5所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含VH和VL。例如，所述VH可以包含SEQ ID NO:22所示的氨基酸序列，且所述VL可以包含SEQ ID NO:23所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:24所示的氨基酸序列，且所述VL可以包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含抗体或其抗原结合片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。 在本申请中，所述抗体可以包括单克隆抗体、嵌合抗体、人源化抗体和/或全人源抗体。例如，所述人源化抗原结合蛋白的VH可以包含SEQ ID NO:24所示的氨基酸序列。例如，所述人源化抗原结合蛋白的VL可以包含SEQ ID NO:25所示的氨基酸序列。
此外，需要说明的是，本申请所述分离的抗原结合蛋白可以包含与其存在一个或多个保守序列修饰的重链和/或轻链序列。所谓“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请所述分离的抗原结合蛋白中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。在某些实施方式中，本申请所述分离的抗原结合蛋白的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换。本领域内的技术人员知道，一些保守序列修改不会使抗原结合性消失，具体可以参见，例如，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。
在本申请中，可以通过使用肺炎链球菌溶血素突变体对小鼠进行免疫，获取小鼠脾细胞与骨髓瘤细胞融合筛选具有肺炎链球菌溶血素蛋白抗原亲和力的抗原结合蛋白。在本申请中，还可以对筛选得到的抗原结合蛋白翻译后修饰位点进行优化，获得人源化序列。
可以通过本领域已知的各种测定鉴别、筛选或表征本申请所述的肺炎链球菌溶血素抗原结合蛋白。
例如，可通过已知方法诸如酶联免疫吸附测定(ELISA)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FACS)、免疫组织化学、免疫荧光等来测试本申请抗原结合蛋白的抗原结合活性。
在本申请中，所述分离的抗原结合蛋白能够特异性结合肺炎链球菌溶血素。在本申请中，所述肺炎链球菌溶血素可以包含野生型肺炎链球菌溶血素以及其变体。在本申请中，所述肺 炎链球菌溶血素的变体可以包含与野生型肺炎链球菌溶血素相比，第146位氨基酸缺失。在本申请中，野生型肺炎链球菌溶血素的氨基酸序列可以通过Uniprot数据库(Entry ID：Q04IN8)确定。
在本申请中，所述分离的抗原结合蛋白与肺炎链球菌溶血素的结合可以通过ELISA方法进行检测。例如，本申请所述的抗原结合蛋白可以以小于或等于约0.03μg/ml，小于或等于约0.028μg/ml，小于或等于约0.026μg/ml，小于或等于约0.024μg/ml，小于或等于约0.022μg/ml，小于或等于约0.02034μg/ml，小于或等于约0.020μg/ml，小于或等于约0.018μg/ml，小于或等于约0.01706μg/ml，小于或等于约0.016μg/ml，小于或等于约0.014μg/ml，小于或等于约0.012μg/ml，小于或等于约0.010μg/ml的EC 50值与肺炎链球菌溶血素蛋白结合。
在本申请中，所述分离的抗原结合蛋白能够中和肺炎链球菌溶血素蛋白的生物学活性。例如，可以将重组肺炎链球菌溶血素与本申请所述分离的抗原结合蛋白混合，加入兔红细胞，检测所述抗原结合蛋白的抗溶血活性。
在本申请中，所述分离的抗原结合蛋白能够预防和/或治疗疾病和或/病症。在本申请中，所述疾病和/或病症可以由肺炎链球菌引起或介导。在本申请中，所述疾病和/或病症可以包含由肺炎链球菌引起或介导的疾病和/或病症的并发症。例如，所述疾病和/或病症可以包含脓毒症和/或菌血症。
多肽分子、核酸分子、载体、细胞、免疫缀合物和药物组合物
另一方面，本申请提供了多肽分子，其可以包含本申请所述的分离的抗原结合蛋白。
在本申请中，所述多肽分子可以包含融合蛋白。在本申请中，所述多肽分子可以为融合蛋白。在本申请中，所述融合蛋白可以包括多特异性抗体。
另一方面，本申请提供了分离的核酸分子，其可以编码本申请所述的分离的抗原结合蛋白。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的；(ii)通过克隆重组产生的；(iii)纯化的，例如通过酶切和凝胶电泳分级分离；或者(iv)合成的，例如通过化学合成。
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。所述载体可以包括，例如质粒、粘粒、 病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
另一方面，本申请提供了一种细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。在某些实施方式中，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、293T细胞、COS-1细胞、SP2/0细胞、NS0细胞或骨髓瘤细胞。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
另一方面，本申请还提供了免疫缀合物，其可以包含本申请所述的分离的抗原结合蛋白。
在某些实施方式中，可以将本申请所述的分离的抗原结合蛋白或其片段与另一试剂，如化学治疗剂、毒素、免疫治疗剂、成像探针、分光镜探针等的连接。该连接可以通过一个或多个共价键，或非共价相互作用，并且可以包括螯合作用。可以使用多种接头(所述接头可以为本领域所知)以形成免疫缀合物。此外，可以以融合蛋白质的形式提供免疫缀合物，其可以由编码免疫缀合物的多核苷酸表达。所述免疫缀合物还可以包含例如抗体-药物缀合物(ADC)。在ADC中，抗体和治疗剂可以通过接头交联，该接头可切割，例如肽类接头、二硫类接头或腙类接头。
另一方面，本申请还提供了药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的多肽分子、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)佐剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在某些实施方案中，所述药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量可以取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。
在某些实施方案中，所述药学上可接受的载剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在某些实施方案中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。
制备方法
另一方面，本申请提供了制备所述分离的抗原结合蛋白的方法。所述方法可包括，在使得所述的抗原结合蛋白表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
任何适于产生单克隆抗体的方法都可用于产生本申请的抗原结合蛋白。例如，可以用肺炎链球菌溶血素减毒株或其片段免疫动物。例如，所述肺炎链球菌减毒株可以包含与野生型相比的第146位缺失突变。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的肺炎链球菌溶血素都可以作为免疫原(抗原)，用于产生对肺炎链球菌溶血素特异的非人抗体，筛选所述抗体的生物学活性。例如，激发免疫原可以是全长的肺炎链球菌溶血素，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。
方法和用途
另一方面，本申请提供了所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防和/或治疗疾病和/或病症的方法，所述方法可以包括向有需要的受试者施用本申请所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物。
在本申请中，所述施用可以通过不同方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
另一方面，本申请所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、 所述细胞、所述免疫缀合物和/或所述药物组合物，其可以用于预防和/或治疗疾病和/或病症。
在本申请中，所述疾病和/或病症可以由肺炎链球菌引起或介导。
在本申请中，所述疾病和/或病症可以是肺炎链球菌引起或介导的疾病和/或病症的并发症。
在本申请中，所述疾病和/或病症可以包括细菌感染。
在本申请中，所述疾病和/或病症可以包括菌血症和/或脓毒症。
在本申请中，所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物可以单独使用，也可以与其他药物联合使用预防和/或治疗疾病和/或病症。在某些实施方式中，所述其他药物可以是目前已知的任何具有抗菌作用的药物。所述疾病和/或病症可以是由于肺炎链球菌感染引起的，也可以是由其他感染引起的相关疾病。
另一方面，本申请还提供了检测样品中肺炎链球菌溶血素蛋白的方法，所述方法包括施用所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物。
在本申请中，所述检测样品中肺炎链球菌溶血素蛋白的方法可以是体外方法。例如，使本申请所述的分离的抗原结合蛋白和离体的样品接触，检测样品中肺炎链球菌溶血素的存在和/或含量。在某些情形中，所述检测样品中肺炎链球菌溶血素蛋白的方法为非治疗目的。在某些情形中，所述检测样品中肺炎链球菌溶血素蛋白的方法不是诊断方法。
另一方面，本申请还提供了检测样品中肺炎链球菌溶血素蛋白的试剂或试剂盒，其包含所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述多肽分子、所述核酸分子、所述载体、所述细胞、所述免疫缀合物和/或所述药物组合物在制备试剂盒中的用途，所述试剂盒用于检测样品中肺炎链球菌溶血素的存在和/或含量。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的抗原结合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1 肺炎链球菌溶血素(PLY)重组蛋白的表达
利用Uniprot数据库确定肺炎链球菌溶血素(PLY，Entry ID：Q04IN8)的氨基酸序列，并根据溶血素全长氨基酸序列缺失第146位的丙氨酸作为减毒的PLY mut的氨基酸，以氨基 酸序列为模板，限定酶切位点为NdeI/XhoI，C末端引入His标签，交由北京擎科生物科技有限公司进行密码子优化和基因合成，获得PLY、PLY mut蛋白的质粒。将重组质粒转化至大肠杆菌E.coli BL21(DE3)plysS，并利用大肠杆菌表达系统，重组表达PLY和PLY mut重组蛋白。
实施例2 PLY mut抗原免疫Balb/C小鼠
采用制备的PLY mut蛋白，皮下多点免疫方法对5只Balb/C小鼠进行免疫，皮下多点免疫流程如下所示。
Figure PCTCN2022082011-appb-000003
实施例3 免疫小鼠脾细胞与骨髓瘤细胞融合及筛选具有PLYmut抗原亲和力的抗原结合蛋白
采用PEG法将骨髓瘤细胞P3X63Ag8.653与免疫PLY mut抗原的小鼠脾脏细胞进行融合。将融合后的杂交瘤细胞接种于96孔板中培养。后续使用HAT培养基和HT的培养基进行杂交瘤细胞培养。培养至96孔板细胞上清液微黄时，取100μl细胞培养上清液，采用ELISA方法进行筛选具有与PLY mut抗原结合的杂交瘤克隆细胞。将亲和力较强的杂交瘤母克隆细胞继续培养后，通过有限稀释法进行亚克隆，并选取单克隆细胞。将单克隆上清液通过ELISA方法筛选出与PLYmut蛋白有强亲和力的分泌抗体的2E5单克隆细胞，分泌出的单克隆抗体命名为m2E5。
实施例4 特异性鼠源抗原结合蛋白序列的获得
利用RNAfast200试剂盒(上海飞捷生物技术有限公司)提取1×10 6分泌抗PLY mut抗体的2E5单克隆细胞总RNA。并利用反转录试剂盒(Takara)，将总RNA反转成cDNA。利用引物扩增cDNA片段(Anke Krebber.1997)，利用DNA产物纯化试剂盒(北京天根生化科 技有限公司)将PCR产物纯化，利用连接试剂盒(北京擎科生物科技有限公司)连接到T载体上，并转化到感受态细胞中，委托上海擎科生物进行序列测定，鼠源单克隆抗体m2E5的重链可变区(m2E5VH)和轻链可变区(m2E5VL)测序结果如下。
m2E5重链可变区(m2E5VH)SEQ ID NO:22
Figure PCTCN2022082011-appb-000004
m2E5轻链可变区(m2E5VL)SEQ ID NO:23
Figure PCTCN2022082011-appb-000005
其中下划线部分表示使用IMGT编号方案划分得到的CDR序列。
实施例5 抗PLY鼠源抗原结合蛋白的人源化
通过NCBI数据库比对(https://www.ncbi.nlm.nih.gov/igblast/)，选取与鼠源抗体2E5(m2E5)最接近的germline序列为模板，对m2E5进行人源化，并对抗原结合蛋白翻译后修饰位点进行优化，获得人源化抗体2E5H3L0的重链可变区(hu2E5VH3)和轻链可变区(hu2E5VL0)序列如下。
人源化抗原结合蛋白2E5重链可变区(hu2E5VH3)SEQ ID NO:24
Figure PCTCN2022082011-appb-000006
人源化抗原结合蛋白2E5轻链可变区(hu2E5VL0)SEQ ID NO:25
Figure PCTCN2022082011-appb-000007
其中下划线部分表示使用IMGT编号方案划分得到的CDR序列。
实施例6 抗PLY抗原结合蛋白的表达与抗原结合活性检测
将m2E5重链可变区的C端与人免疫球蛋白γ1(IgG1)的恒定区的N端相连并构建至pcDNA3.4载体，获得2E5鼠/人嵌合抗体(2E5HL)的重链表达载体pcDNA3.4-c2E5VHCH。将2E5轻链可变区的C端与轻链kappa恒定区的N端相连并构建至pcDNA3.4载体，获得2E5嵌合抗体轻链表达载体pcDNA3.4-c2E5VLCL。
其中嵌合抗体2E5HL的重链(c2E5VHCH)氨基酸序列及轻链(c2E5VLCL)氨基酸序列为：
c2E5VHCH SEQ ID NO:28：
Figure PCTCN2022082011-appb-000008
c2E5VLCL SEQ ID NO:29：
Figure PCTCN2022082011-appb-000009
将2E5人源化重链可变区的C端与人免疫球蛋白γ1(IgG1)的恒定区的N端相连并构建至pcDNA3.4载体，获得人源化重链表达载体pcDNA3.4-hu2E5VH3CH。将2E5人源化轻链可变区的C端与轻链kappa恒定区的N端相连并构建至pcDNA3.4载体，获得人源化轻链表达载体pcDNA3.4-hu2E5VL0CL。
2E5H3L0人源化抗体的重链(hu2E5VH3CH)氨基酸序列及轻链(hu2E5VL0CL)氨基酸序列为：
hu2E5VH3CH SEQ ID NO:30：
Figure PCTCN2022082011-appb-000010
hu2E5VL0CL SEQ ID NO:31：
Figure PCTCN2022082011-appb-000011
将2E5HL嵌合抗体的重链表达载体pcDNA3.4-m2E5VHCH与轻链表达载体pcDNA3.4- m2E5VLCL共转染Expi293F细胞，利用Expi293F表达系统瞬时表达2E5HL，最后用Protein A纯化嵌合抗体蛋白。将2E5H3L0人源化抗体的重链表达载体pcDNA3.4-hu2E5VH3CH与轻链表达载体pcDNA3.4-hu2E5VL0CL共转染Expi293F细胞，利用Expi293F表达系统瞬时表达2E5H3L0，最后用Protein A纯化人源化抗体蛋白。纯化获得的2E5HL嵌合抗体和2E5H3L0人源化抗体用ELISA方法进行抗原结合活性鉴定：96孔酶标板(Thermo)中包被PLY和PLYmut蛋白0.1μg/孔，4℃孵育过夜，使用PBST缓冲液洗板后，每孔加入200μl的5％BSA放置于37℃孵育1小时；封闭结束使用PBST缓冲液进行洗板，并在酶标板中加入首孔为0.1μg倍比稀释后的2E5HL人鼠嵌合抗体或2E5H3L0人源化抗体，4℃孵育过夜；一抗过夜后进行洗板，在酶标板中加入1：10000稀释的HRP标记山羊抗人IgG二抗，每孔100μl，37℃孵育1小时后进行洗板；每孔加入100μl TMB 37℃孵育进行显色，10min后采用终止液终止显色，酶标仪(Multiskcin FC，Thermo)读取结果数值并使用Graphpad软件作图。抗体与PLY蛋白结合情况结果如图1所示，2E5HL的EC 50值为0.01706μg/ml，2E5H3L0的EC 50值为0.02034μg/ml；抗体与PLYmut蛋白结合情况结果如图2所示，2E5HL的EC 50值为0.01582μg/ml，2E5H3L0的EC 50值为0.01617μg/ml.结果证明，人源化抗体2E5H3L0和嵌合抗体2E5HL均与肺炎链球菌溶血素PLY和PLYmut蛋白均具有强亲和力。
实施例7 体外中和肺炎链球菌毒素活性测定
将6.25ng重组肺炎链球菌溶血素PLY蛋白与嵌合抗体2E5HL或人源化抗体2E5H3L0以一定比例混合加入到96孔板(Thermo)，37℃孵育10min，加入5％兔红细胞，37℃孵育1h。将样品3000rpm离心5min，利用酶标仪(Multiskcin FC，Thermo)405nm波长处检测吸光度，测得其抗溶血活性。
结果如图3所示，肺炎链球菌毒素具有兔红细胞溶血活性，而嵌合抗体2E5HL和人源化抗体2E5H3L0均能有效中和肺炎链球菌毒素的兔红细胞溶血活性，并具有剂量依赖性。
实施例8 本申请的抗原结合蛋白对PLY毒素模型小鼠的保护作用
预先分别给C57BL/6小鼠注射剂量为1.25mg/kg的嵌合抗体2E5HL、人源化抗体2E5H3L0和人IgG抗体(作为对照)，1h后尾静脉注射0.75μg肺炎链球菌溶血素PLY蛋白，观察小鼠生存时间。
结果如图4所示，对照组小鼠全部死亡，而注射了嵌合抗体2E5HL和人源化抗体2E5H3L0的小鼠则达到了完全保护效应。
实施例9 本申请的抗原结合蛋白对肺炎链球菌感染小鼠的保护作用
取种子库肺炎链球菌，解冻后，取200μl菌液用涂布器均匀涂布在哥伦比亚血平板，培养10～12h，将哥伦比亚血平板上取出一块血琼脂(3cm×3cm)，至于15ml灭菌后的THY培养基，37℃恒温培养箱中静置培养至OD600为0.4～0.6，约需2～3h。离心收集菌体，并用PBS重悬备用。根据体重对C57BL/6J小鼠进行随机分组，分为模型组、单克隆抗体药物治疗组。提前2h给小鼠尾静脉注射2×10 8CFU的肺炎链球菌，2h后，尾静脉分别注射30mg/kg的2E5HL、2E5H3L0或人IgG(对照)，观察记录各组小鼠的生存情况。
结果如图5所示，与对照人IgG相比，嵌合抗体2E5HL和人源化抗体2E5H3L0对肺炎链球菌(SP)感染小鼠具有良好保护效应。

Claims (68)

  1. 分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:39所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。
  6. 根据权利要求4-5中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:32所示的氨基酸序列。
  7. 根据权利要求6所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:7或SEQ ID NO:15所示的氨基酸序列。
  8. 根据权利要求4-7中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:33所示的氨基酸序列。
  9. 根据权利要求8所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:8或SEQ ID NO:16所示的氨基酸序列。
  10. 根据权利要求3-9中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:34所示的氨基酸序列。
  11. 根据权利要求10所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:9或SEQ ID NO:17所示的氨基酸序列。
  12. 根据权利要求2-11中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:35所示的氨基酸序列。
  13. 根据权利要求12所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:32所示的氨基酸序列，所述H-FR2包含SEQ ID NO:33所示的氨基酸序列，所述H-FR3包含SEQ ID NO:34所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:35所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:7或SEQ ID NO:15所示的氨基酸序列，所述H-FR2包含SEQ ID NO:8或SEQ ID NO:16所示的氨基酸序列，所述H-FR3包含SEQ ID NO:9或SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:10或SEQ ID NO:18所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含选自下述任一组的H-FR1，H-FR2，H-FR3和H-FR4：
    1)所述H-FR1包含SEQ ID NO:7所示的氨基酸序列，所述H-FR2包含SEQ ID NO:8所示的氨基酸序列，所述H-FR3包含SEQ ID NO:9所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:10所示的氨基酸序列；以及
    2)所述H-FR1包含SEQ ID NO:15所示的氨基酸序列，所述H-FR2包含SEQ ID NO:16所示的氨基酸序列，所述H-FR3包含SEQ ID NO:17所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:18所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含VH，所述VH包含SEQ ID NO:39所示的氨基酸序列。
  18. 根据权利要求17所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:22或SEQ ID NO:24所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区。
  20. 根据权利要求19所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG恒定区。
  21. 根据权利要求19-20中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG1重链恒定区。
  22. 根据权利要求19-21中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区包含SEQ ID NO:26所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的分离的抗原结合蛋白，其包含重链，所述重链包含SEQ ID NO:28或SEQ ID NO:30所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包含抗体轻链可变区VL中 的至少一个CDR，所述VL包含SEQ ID NO:40所示的氨基酸序列。
  25. 根据权利要求1-24中任一项所述的分离的抗原结合蛋白，其包含LCDR3，所述LCDR3包含SEQ ID NO:4所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的分离的抗原结合蛋白，其包含LCDR2，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的分离的抗原结合蛋白，其包含LCDR1，所述LCDR1包含SEQ ID NO:6所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:4所示的氨基酸序列。
  29. 根据权利要求27-28中任一项所述的分离的抗原结合蛋白，其包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:36所示的氨基酸序列。
  30. 根据权利要求29所述的分离的抗原结合蛋白，其中所述L-FR1包含SEQ ID NO:11或SEQ ID NO:19所示的氨基酸序列。
  31. 根据权利要求27-30中任一项所述的分离的抗原结合蛋白，其包含L-FR2，所述L-FR2位于所述LCDR1和所述LCDR2之间，且所述L-FR2包含SEQ ID NO:37所示的氨基酸序列。
  32. 根据权利要求31所述的分离的抗原结合蛋白，其中所述L-FR2包含SEQ ID NO:12或SEQ ID NO:20所示的氨基酸序列。
  33. 根据权利要求26-32中任一项所述的分离的抗原结合蛋白，其包含L-FR3，所述L-FR3位于所述LCDR2和所述LCDR3之间，且所述L-FR3包含SEQ ID NO:38所示的氨基酸序列。
  34. 根据权利要求33所述的分离的抗原结合蛋白，其中所述L-FR3包含SEQ ID NO:13或SEQ ID NO:21所示的氨基酸序列。
  35. 根据权利要求25-34中任一项所述的分离的抗原结合蛋白，其包含L-FR4，所述L-FR4的N端与所述LCDR3的C端直接或间接相连，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
  36. 根据权利要求1-35中任一项所述的分离的抗原结合蛋白，其包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:36所示的氨基酸序列，所述L-FR2包含SEQ ID NO:37所示的氨基酸序列，所述L-FR3包含SEQ ID NO:38所示的氨基酸序 列，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
  37. 根据权利要求1-36中任一项所述的分离的抗原结合蛋白，其包含L-FR1，L-FR2，L-FR3和L-FR4，所述L-FR1包含SEQ ID NO:11或SEQ ID NO:19所示的氨基酸序列，所述L-FR2包含SEQ ID NO:12或SEQ ID NO:20所示的氨基酸序列，所述L-FR3包含SEQ ID NO:13或SEQ ID NO:21所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的分离的抗原结合蛋白，其包含选自下述任一组的L-FR1，L-FR2，L-FR3和L-FR4：
    1)所述L-FR1包含SEQ ID NO:11所示的氨基酸序列，所述L-FR2包含SEQ ID NO:12所示的氨基酸序列，所述L-FR3包含SEQ ID NO:13所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列；以及
    2)所述L-FR1包含SEQ ID NO:19所示的氨基酸序列，所述L-FR2包含SEQ ID NO:20所示的氨基酸序列，所述L-FR3包含SEQ ID NO:21所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:14所示的氨基酸序列。
  39. 根据权利要求1-38中任一项所述的分离的抗原结合蛋白，其包含VL，所述VL包含SEQ ID NO:40所示的氨基酸序列。
  40. 根据权利要求39所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:23或SEQ ID NO:25所示的氨基酸序列。
  41. 根据权利要求1-40中任一项所述的分离的抗原结合蛋白，其包含抗体轻链恒定区。
  42. 根据权利要求41所述的分离的抗原结合蛋白，其中所述轻链恒定区源自人Igκ恒定区。
  43. 根据权利要求41-42中任一项所述的分离的抗原结合蛋白，其中所述轻链恒定区包含SEQ ID NO:27所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的分离的抗原结合蛋白，其包括轻链，所述轻链包含SEQ ID NO:29或SEQ ID NO:31所示的氨基酸序列。
  45. 根据权利要求1-44中任一项所述的分离的抗原结合蛋白，其包含选自下述任一组的VH和VL：
    1)所述VH包含SEQ ID NO:22所示的氨基酸序列，且所述VL包含SEQ ID NO:23所示的氨基酸序列；以及
    2)所述VH包含SEQ ID NO:24所示的氨基酸序列，且所述VL包含SEQ ID NO:25所示的氨基酸序列。
  46. 根据权利要求1-45中任一项所述的分离的抗原结合蛋白，其包含抗体或其抗原结合片 段。
  47. 根据权利要求1-46中任一项所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
  48. 根据权利要求1-47中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组中的一种或多种：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  49. 根据权利要求1-48中任一项所述的分离的抗原结合蛋白，其能够特异性结合肺炎链球菌溶血素蛋白。
  50. 根据权利要求49所述的分离的抗原结合蛋白，其中所述肺炎链球菌溶血素包含野生型肺炎链球菌溶血素蛋白及其变体。
  51. 根据权利要求49-50中任一项所述的分离的抗原结合蛋白，其中所述肺炎链球菌溶血素蛋白包含与野生型肺炎链球菌溶血素蛋白相比，其146位氨基酸缺失的肺炎链球菌溶血素蛋白变体。
  52. 根据权利要求1-51中任一项所述的分离的抗原结合蛋白，其能够预防和/或治疗肺炎链球菌引起的疾病和/或病症。
  53. 根据权利要求52所述的分离的抗原结合蛋白，其中所述疾病和/或病症包括肺炎链球菌引起的疾病和/或病症的并发症。
  54. 多肽分子，其包含权利要求1-53中任一项所述的分离的抗原结合蛋白。
  55. 根据权利要求54所述的多肽分子，其包含融合蛋白。
  56. 免疫缀合物，其包含权利要求1-53中任一项所述的分离的抗原结合蛋白。
  57. 核酸分子，其编码权利要求1-53中任一项所述的分离的抗原结合蛋白或权利要求54-55中任一项所述的多肽分子。
  58. 载体，其包含权利要求57所述的核酸分子。
  59. 细胞，其包含权利要求57所述的核酸分子或权利要求58所述的载体。
  60. 药物组合物，其包含权利要求1-53中任一项所述的分离的抗原结合蛋白、权利要求54-55中任一项所述的多肽分子、权利要求56所述的免疫缀合物、权利要求57所述的核酸分子、权利要求58所述的载体和/或权利要求59所述的细胞，以及任选地药学上可接受的载剂。
  61. 制备权利要求1-53中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得所述抗原结合蛋白表达的条件下，培养权利要求59所述的细胞。
  62. 权利要求1-53中任一项所述的分离的抗原结合蛋白、权利要求54-55中任一项所述的多肽分子、权利要求56所述的免疫缀合物、权利要求57所述的核酸分子、权利要求58 所述的载体、权利要求59所述的细胞和/或权利要求60所述的药物组合物单独使用或与其它药物联合使用。
  63. 权利要求1-53中任一项所述的分离的抗原结合蛋白、权利要求54-55中任一项所述的多肽分子、权利要求56所述的免疫缀合物、权利要求57所述的核酸分子、权利要求58所述的载体、权利要求59所述的细胞和/或权利要求60所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
  64. 根据权利要求63所述的用途，其中所述疾病和/或病症及其并发症由肺炎链球菌引起或介导。
  65. 根据权利要求63-64中任一项所述的用途，其中所述疾病和/或病症包括肺炎链球菌引起或介导的疾病和/或病症的并发症。
  66. 检测样品中肺炎链球菌溶血素蛋白的方法，所述方法包括施用权利要求1-53中任一项所述的分离的抗原结合蛋白、权利要求54-55中任一项所述的多肽分子、权利要求56所述的免疫缀合物、权利要求57所述的核酸分子、权利要求58所述的载体、权利要求59所述的细胞和/或权利要求60所述的药物组合物。
  67. 检测样品中肺炎链球菌溶血素蛋白的试剂盒，所述试剂盒包括权利要求1-53中任一项所述的分离的抗原结合蛋白、权利要求54-55中任一项所述的多肽分子、权利要求56所述的免疫缀合物、权利要求57所述的核酸分子、权利要求58所述的载体、权利要求59所述的细胞和/或权利要求60所述的药物组合物。
  68. 权利要求1-53中任一项所述的分离的抗原结合蛋白、权利要求54-55中任一项所述的多肽分子、权利要求56所述的免疫缀合物、权利要求57所述的核酸分子、权利要求58所述的载体、权利要求59所述的细胞和/或权利要求60所述的药物组合物在制备试剂盒中的用途，所述试剂盒用于检测样品中肺炎链球菌溶血素蛋白的存在和/或含量。
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2075803A1 (es) * 1993-12-17 1995-10-01 Univ Oviedo Procedimiento de la deteccion de neumolisina
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6420140B1 (en) 1996-10-11 2002-07-16 Abgenix, Inc. Production of multimeric protein by cell fusion method
US6458592B1 (en) 1995-03-29 2002-10-01 Abgenix, Inc. Production of antibodies using cre-mediated site-specific recombination
WO2006003388A2 (en) 2004-06-30 2006-01-12 Domantis Limited Compositions and methods for treating inflammatory disorders
WO2006030220A1 (en) 2004-09-17 2006-03-23 Domantis Limited Compositions monovalent for cd40l binding and methods of use
CN101018863A (zh) * 2004-05-07 2007-08-15 利-安·柯卡姆 突变体肺炎链球菌溶血素蛋白质
CN101061139A (zh) * 2004-08-23 2007-10-24 吉奈斯拓公司 肺炎球菌溶血素的结合部分
WO2012064865A1 (en) * 2010-11-09 2012-05-18 The University Of Chicago Role of adam10 and its relevance to disease and therapeutics
WO2015036583A2 (en) 2013-09-13 2015-03-19 Amgen Inc. Combination of epigenetic factors and bispecific compounds targeting cd33 and cd3 in the treatment of myeloid leukemia
CN110167960A (zh) * 2017-01-03 2019-08-23 瑞泽恩制药公司 针对金黄色葡萄球菌溶血素a毒素的人类抗体

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
ES2075803A1 (es) * 1993-12-17 1995-10-01 Univ Oviedo Procedimiento de la deteccion de neumolisina
US6458592B1 (en) 1995-03-29 2002-10-01 Abgenix, Inc. Production of antibodies using cre-mediated site-specific recombination
US6420140B1 (en) 1996-10-11 2002-07-16 Abgenix, Inc. Production of multimeric protein by cell fusion method
CN101018863A (zh) * 2004-05-07 2007-08-15 利-安·柯卡姆 突变体肺炎链球菌溶血素蛋白质
WO2006003388A2 (en) 2004-06-30 2006-01-12 Domantis Limited Compositions and methods for treating inflammatory disorders
CN101061139A (zh) * 2004-08-23 2007-10-24 吉奈斯拓公司 肺炎球菌溶血素的结合部分
WO2006030220A1 (en) 2004-09-17 2006-03-23 Domantis Limited Compositions monovalent for cd40l binding and methods of use
WO2012064865A1 (en) * 2010-11-09 2012-05-18 The University Of Chicago Role of adam10 and its relevance to disease and therapeutics
WO2015036583A2 (en) 2013-09-13 2015-03-19 Amgen Inc. Combination of epigenetic factors and bispecific compounds targeting cd33 and cd3 in the treatment of myeloid leukemia
CN110167960A (zh) * 2017-01-03 2019-08-23 瑞泽恩制药公司 针对金黄色葡萄球菌溶血素a毒素的人类抗体

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
"Basic and Clinical Immunology", 1994, APPLETON & LANGE, pages: 71
ADIB CONQUY, INT. IMMUNOL., vol. 10, 1998, pages 341 - 6
BEERS, CLIN. CAN. RES., vol. 6, 2000, pages 2835 - 43
BRUMMELL, BIOCHEM, vol. 32, 1993, pages 1180 - 8
D.J. LIPMANW.R. PEARSON: "Rapid and Sensitive Protein Similarity Search", SCIENCE, vol. 227, 1989, pages 1435 - 1441
DE LOS TOYOS, J. R. ET AL.: "Functional Analysis of Pneumolysin by Use of Monoclonal Antibodies", INFECTION AND IMMUNITY, vol. 64, no. 2, 29 February 1996 (1996-02-29), XP002366575 *
HALL, J. IMMUNOL., vol. 149, 1992, pages 1605 - 12
HAMERS-CASTERMAN, NATURE, vol. 363, 1993, pages 446 - 448
HOLT, TRENDS BIOTECHNOL, vol. 21, no. 11, 2003, pages 484 - 490
KABAT EA: "Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services", 1991, NATIONAL INSTITUTE OF HEALTH, pages: 91 - 3242
KABAT EAWU TT, ANN NY ACADSCI, vol. 190, 1971, pages 382 - 391
KELLEYO'CONNELL, BIOCHEM., vol. 32, 1993, pages 6862 - 35
KOMISAROV, J. BIOL. CHEM., vol. 272, 1997, pages 26864 - 26870
KORNDORFER, PROTEINS: STRUCTURE, FUNCTION, AND BIOINFORMATICS, vol. 53, no. 1, 2003, pages 121 - 129
KUCINSKAITE-KODZE INDRE, SIMANAVICIUS MARTYNAS, DAPKUNAS JUSTAS, PLECKAITYTE MILDA, ZVIRBLIENE AURELIJA: "Mapping of Recognition Sites of Monoclonal Antibodies Responsible for the Inhibition of Pneumolysin Functional Activity", BIOMOLECULES, vol. 10, no. 7, 8 July 2020 (2020-07-08), pages 1009, XP055970747, DOI: 10.3390/biom10071009 *
LEFRANC, DEVCOMP IMMUNOL, vol. 29, 2005, pages 185 - 203
LEFRANC, NUCLEIC ACIDS RES., vol. 27, 1999, pages 209 - 212
LEFRANC, NUCLEIC ACIDS RES., vol. 29, 2001, pages 207 - 209
LEFRANC, NUCLEIC ACIDS RES., vol. 31, 2003, pages 307 - 310
ROQUE, BIOTECHNOL. PROG., vol. 20, 2004, pages 639 - 654
RUIZ, NUCLEIC ACIDS RES., vol. 28, 2000, pages 219 - 221
S. ALTSCHULW. GISHW. MILLERE.W. MYERSD. LIPMAN: "A Basic Local Alignment Search Tool", JOURNAL OF MOLECULAR BIOLOGY, vol. 215, 1990, pages 403 - 410
SHERIFF, NATURE STRUCT. BIOL., vol. 3, 1996, pages 733 - 736
W.R. PEARSOND.J. LIPMAN: "Improved Tool for Biological Sequence Comparison", PROC. SATL. ACAD. SCI., vol. 85, 1988, pages 2444 - 2448
WARD, NATURE, vol. 341, no. 6242, 12 October 1989 (1989-10-12), pages 544 - 6
WILDT, PROT. ENG., vol. 10, 1997, pages 835 - 41
YAO RUN, CUI YA-LI, YUAN JUN, WANG HONG, GONG YI, YIN YI-BING, ZHANG XUE-MEI: "Protective Effect of Mucosal Immunization with ΔA146Ply against Streptococcus pneumoniae", CHINESE JOURNAL OF BIOLOGICALS, CHINESE PREVENTIVE MEDICINE ASSOCIATION, CHINA, vol. 24, no. 5, 31 May 2011 (2011-05-31), China , pages 579 - 582, XP055970739, ISSN: 1004-5503, DOI: 10.13200/j.cjb.2011.05.88.yaor.007 *

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