CN114957460A - Antigen binding proteins that cross-neutralize staphylococcus aureus toxins and uses thereof - Google Patents

Antigen binding proteins that cross-neutralize staphylococcus aureus toxins and uses thereof Download PDF

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CN114957460A
CN114957460A CN202210152322.2A CN202210152322A CN114957460A CN 114957460 A CN114957460 A CN 114957460A CN 202210152322 A CN202210152322 A CN 202210152322A CN 114957460 A CN114957460 A CN 114957460A
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ser
amino acid
acid sequence
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安毛毛
李博华
侯炜彤
郭诗雨
邱熙然
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Xingji Biology Suzhou Co ltd
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Xingji Biology Suzhou Co ltd
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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
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • 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
    • 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
    • C07K2317/20—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

Abstract

The present application relates to an isolated antigen binding protein targeting staphylococcus aureus and derived proteins thereof, which are capable of cross-neutralizing activity against a variety of toxins of staphylococcus aureus. The application also discloses the application of the separated antigen binding protein and the derived protein thereof in the field of treating and/or preventing staphylococcus aureus infection.

Description

Antigen binding proteins that cross-neutralize staphylococcus aureus toxins and uses thereof
Technical Field
The application relates to the field of biomedicine, in particular to an antigen binding protein specifically binding to staphylococcus aureus toxin.
Background
Staphylococcus aureus belongs to the genus Staphylococcus, is an important gram-positive pathogenic bacterium, and can cause local infections such as pyogenic infection, pneumonia, pseudomembranous enteritis, pericarditis, etc., and systemic infections such as septicemia, sepsis, etc. Staphylococcus aureus is primarily pathogenic by producing toxins and invasive enzymes including hemolysins, leukocidins, enterotoxins, coagulases, and the like.
Hemolysin can be divided into four types of alpha-hemolysin, beta-hemolysin, gamma-hemolysin and delta-hemolysin according to different antigens. Gamma-hemolysin is ubiquitously expressed in all staphylococcus aureus and its gene product can produce two types of toxins: HlgAB and HlgCB. Gamma-hemolysin is highly effective in killing human immune cells, including: neutrophils, lymphocytes and macrophages, also destroy human erythrocytes. Leukocidin (PVL) is an extracellular toxin produced by staphylococcus aureus, belonging to the family of membrane-boring toxins, and is composed of two subunits, the "S" and "F" types. The F and S components of staphylococcus aureus toxin can form not only homologous but also non-homologous pairings. Inactivation of individual components is unlikely to be effective against infection by staphylococcus aureus due to the abundance and heterogeneity of the toxin family. Therefore, there is a need to develop drugs that are effective against different two-component staphylococcus aureus.
Disclosure of Invention
The present application provides an isolated antigen binding protein having one or more of the following properties: 1) can specifically bind to staphylococcus aureus toxin; 2) is capable of specifically binding to one or more of the following staphylococcus aureus toxins: HlgA, HlgB, LukD and LukF; 3) capable of neutralizing the activity of one or more staphylococcus aureus toxins; 4) can be used for treating diseases, disorders and/or complications caused by Staphylococcus aureus. The isolated antigen binding protein disclosed by the application can have in vitro affinity, neutralizing activity and pharmacodynamic activity in animal bodies on double-group toxins, and has a protective effect on mouse infection caused by staphylococcus aureus.
In one aspect, the present application provides an isolated antigen binding protein comprising at least one CDR in the VH of a heavy chain variable region of an antibody, said VH comprising the amino acid sequence set forth in SEQ ID NO: 53.
In certain embodiments, the isolated antigen binding protein comprises HCDR3, wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 1.
In certain embodiments, the isolated antigen binding protein comprises HCDR2, wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID No. 2.
In certain embodiments, the isolated antigen binding protein comprises HCDR1, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID No. 3.
In certain embodiments, the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, the HCDR1 comprises the amino acid sequence set forth in SEQ ID No. 3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID No. 2, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 1.
In certain embodiments, the isolated antigen binding protein comprises H-FR1, the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO. 45.
In certain embodiments, H-FR1 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO 10, SEQ ID NO 14, and SEQ ID NO 18.
In certain embodiments, the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 46.
In certain embodiments, H-FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 11, SEQ ID NO. 15, and SEQ ID NO. 19.
In certain embodiments, the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 47.
In certain embodiments, H-FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO 12, SEQ ID NO 16, and SEQ ID NO 20.
In certain embodiments, the isolated antigen binding protein comprises H-FR4, the N-terminus of H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO. 48.
In certain embodiments, H-FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 13 or SEQ ID NO. 17.
In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, said H-FR1 comprising the amino acid sequence set forth in SEQ ID NO:45, said H-FR2 comprising the amino acid sequence set forth in SEQ ID NO:46, said H-FR3 comprising the amino acid sequence set forth in SEQ ID NO:47, and said H-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 48.
In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, said H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO 10, SEQ ID NO 14, and SEQ ID NO 18, said H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO 11, SEQ ID NO 15, and SEQ ID NO 19, said H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO 12, SEQ ID NO 16, and SEQ ID NO 20, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO 13 or SEQ ID NO 17.
In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4 selected from any one of the group consisting of:
1) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 10, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 11, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 12, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 13;
2) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO. 14, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO. 15, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO. 16, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO. 17;
3) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 18, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 15, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 16, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 17;
4) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 18, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 19, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 16, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 17;
5) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 18, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 15, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 20, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 17; and
6) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 18, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 19, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 20, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 17.
In certain embodiments, the isolated antigen binding protein comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 53.
In certain embodiments, the VH of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO 30, 31, 32, 33, 34, and 35.
In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.
In certain embodiments, the heavy chain constant region is derived from a human IgG constant region.
In certain embodiments, the heavy chain constant region is derived from a human IgG1 heavy chain constant region.
In certain embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 42.
In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain comprising an amino acid sequence set forth in any one of SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, and SEQ ID NO 61.
In certain embodiments, the isolated antigen binding protein comprises at least one CDR in the variable region VL of an antibody, said VL comprising the amino acid sequence set forth in SEQ ID NO: 54.
In certain embodiments, the isolated antigen binding protein comprises LCDR3, wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID No. 4.
In certain embodiments, the isolated antigen binding protein comprises LCDR2, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID No. 5.
In certain embodiments, the isolated antigen binding protein comprises LCDR1, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 44.
In certain embodiments, the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9.
In certain embodiments, the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, said LCDR1 comprising the amino acid sequence set forth in SEQ ID No. 44, said LCDR2 comprising the amino acid sequence set forth in SEQ ID No. 5, and said LCDR3 comprising the amino acid sequence set forth in SEQ ID No. 4.
In certain embodiments, the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, said LCDR1 comprising the amino acid sequence set forth in any one of SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No.9, said LCDR2 comprising the amino acid sequence set forth in SEQ ID No. 5, and said LCDR3 comprising the amino acid sequence set forth in SEQ ID No. 4.
In certain embodiments, the isolated antigen binding protein comprises L-FR1, the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1, and the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO. 49.
In certain embodiments, L-FR1 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:25, and SEQ ID NO: 29.
In certain embodiments, the isolated antigen binding protein comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 50.
In certain embodiments, L-FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 22 or SEQ ID NO. 26.
In certain embodiments, the isolated antigen binding protein comprises L-FR3, the L-FR3 is located between the LCDR2 and LCDR3, and the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO. 51.
In certain embodiments, L-FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 23 or SEQ ID NO. 27.
In certain embodiments, the isolated antigen binding protein comprises L-FR4, the N-terminus of L-FR4 is linked directly or indirectly to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO. 52.
In certain embodiments, the L-FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 24 or SEQ ID NO. 28.
In certain embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4, said L-FR1 comprising the amino acid sequence set forth in SEQ ID NO. 49, said L-FR2 comprising the amino acid sequence set forth in SEQ ID NO. 50, said L-FR3 comprising the amino acid sequence set forth in SEQ ID NO. 51, and said L-FR4 comprising the amino acid sequence set forth in SEQ ID NO. 52.
In certain embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4, said L-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:25, and SEQ ID NO:29, said L-FR2 comprising the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:26, said L-FR3 comprising the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:27, and said L-FR4 comprising the amino acid sequence set forth in SEQ ID NO:24 or SEQ ID NO: 28.
In certain embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4 selected from any one of the group consisting of:
1) the L-FR1 comprises an amino acid sequence shown as SEQ ID NO. 21, the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 22, the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 23, and the L-FR4 comprises an amino acid sequence shown as SEQ ID NO. 24;
2) the L-FR1 comprises an amino acid sequence shown as SEQ ID NO. 25, the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 26, the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 27, and the L-FR4 comprises an amino acid sequence shown as SEQ ID NO. 28; and
3) the L-FR1 comprises an amino acid sequence shown as SEQ ID NO. 29, the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 26, the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 27, and the L-FR4 comprises an amino acid sequence shown as SEQ ID NO. 28.
In certain embodiments, the isolated antigen binding protein comprises a VL comprising the amino acid sequence set forth in SEQ ID NO 54.
In certain embodiments, the VL comprises an amino acid sequence set forth in any one of SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, and SEQ ID NO 40.
In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region.
In certain embodiments, the light chain constant region is derived from a human Ig kappa constant region.
In certain embodiments, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 43.
In certain embodiments, the isolated antigen binding protein comprises an antibody light chain comprising an amino acid sequence set forth in any one of SEQ ID NO 56, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, and SEQ ID NO 66.
In certain embodiments, the isolated antigen binding protein comprises any one of the group VH and VL selected from:
1) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
2) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
3) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
4) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
5) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises { SEQ ID NO:40
The amino acid sequence shown;
6) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
7) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
8) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
9) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
10) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
11) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
12) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
13) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
14) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
15) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
16) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
17) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
18) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
19) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
20) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
21) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
22) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
23) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
24) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
25) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
26) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
27) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
28) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
29) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
30) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
31) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
32) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
33) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
34) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
35) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40; and
36) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41.
In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
In certain embodiments, the antigen binding fragment comprises a Fab, Fab ', Fv fragment, F (ab') 2 ，F(ab) 2 scFv, di-scFv and/or dAb.
In certain embodiments, the antibody is selected from one or more of the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
In certain embodiments, the isolated antigen binding protein is capable of specifically binding to staphylococcus aureus toxin.
In certain embodiments, the staphylococcus aureus toxin comprises a leukocidin.
In certain embodiments, the staphylococcus aureus toxin is selected from one or more of the group consisting of: HlgA, HlgB, LukD and LukF.
In certain embodiments, the isolated antigen binding protein is capable of neutralizing the activity of one or more s.aureus virulence factors selected from the group consisting of: HlgA, HlgB, LukD and LukF.
In certain embodiments, the isolated antigen binding protein is capable of treating a disease and/or disorder caused by staphylococcus aureus.
In certain embodiments, the disease and/or disorder comprises a complication of a disease and/or disorder caused by staphylococcus aureus.
In another aspect, the present application also provides a polypeptide molecule comprising the isolated antigen binding protein.
In certain embodiments, the polypeptide molecule comprises a fusion protein.
In another aspect, the present application also provides immunoconjugates comprising the isolated antigen binding proteins.
In another aspect, the present application also provides a nucleic acid molecule encoding the isolated antigen binding protein or the polypeptide molecule.
In another aspect, the present application also provides a vector comprising the nucleic acid molecule.
In another aspect, the present application also provides a cell comprising the nucleic acid molecule or the vector.
In another aspect, the present application also provides a pharmaceutical composition comprising the isolated antigen binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, and/or the cell, and optionally a pharmaceutically acceptable carrier.
In another aspect, the present application also provides a method of making the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the antigen binding protein is expressed.
In another aspect, the present application also provides the use of the isolated antigen binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.
In certain embodiments, the disease and/or disorder and its complications are caused or mediated by staphylococcus aureus.
In certain embodiments, the disease and/or condition comprises a complication of a disease and/or condition caused or mediated by staphylococcus aureus.
In certain embodiments, the disease and/or condition comprises sepsis and/or bacteremia.
In another aspect, the isolated antigen binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition described herein are used alone or in combination with other drugs.
In another aspect, the present application also provides a method of detecting staphylococcus aureus leukocidin in a sample, the method comprising administering the isolated antigen binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
In another aspect, the present application also provides a kit for detecting staphylococcus aureus leukocidin in a sample, the kit comprising administering the isolated antigen binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
In another aspect, the present application also provides the use of the isolated antigen binding protein, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a kit for detecting the presence and/or amount of staphylococcus aureus leukocidin in a sample.
Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.
Drawings
Specific features of the invention to which this application relates are set forth in the following claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:
FIGS. 1A-D show assays for the binding activity of antigen binding proteins described herein to Staphylococcus aureus leukocidin.
FIGS. 2A-C show in vitro neutralization of Staphylococcus aureus leukocidin activity assays for antigen binding proteins described herein.
FIG. 3 shows the protective effect of the antigen binding proteins described herein on S.aureus infected mice.
FIG. 4 shows the results of affinity assays for antigen binding proteins of the present application and Staphylococcus aureus leukocidin HlgB.
FIG. 5 shows the results of affinity assays for antigen binding proteins of the present application and Staphylococcus aureus leukocidin HlgB.
FIG. 6 shows the results of the affinity assays for antigen binding proteins of the present application and Staphylococcus aureus leukocidin HlgB
FIGS. 7A-D show affinity assays for antigen binding proteins described herein and Staphylococcus aureus leukocidin.
FIG. 8 shows the results of testing the therapeutic effect of the antigen binding proteins described herein using a model of Staphylococcus aureus alpha-toxin induced mouse sepsis.
FIG. 9 shows the results of evaluating the anti-infective pharmacodynamic effects of the antigen binding proteins described herein using a mouse bacteremia model induced by Staphylococcus aureus.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.
Definition of terms
In the present application, the term "Staphylococcus aureus" is used interchangeably with "Staphylococcus aureus", "s. In the present application, the term may encompass any species and form of s.aureus and variants, homologues and functionally active fragments thereof, and the term "s.aureus" may also include any toxin secreted by s.aureus and variants, homologues and functionally active fragments of the secreted toxin. The term "staphylococcus aureus toxin" may be used interchangeably with "staphylococcus aureus virulence factor". The class of the term "leukocidin" includes, but is not limited to, S components such as LukS-PV, HlgA (γ -hemolysin), HlgC (γ -hemolysin), LukE from Staphylococcus aureus, LukS-I (from Staphylococcus intermedia); and F components, such as LukF-PV, LukF' -PV, HlgB (gamma-hemolysin), LukD from Staphylococcus aureus, and LukF-I (from Staphylococcus intermedia). The present application may encompass one or more of a variety of leukocidins, as well as variant, homologues, analogues and functionally active fragments of leukocidins.
In the present application, the term "isolated" generally refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" does not exclude the presence of other impurities which do not affect the activity of the substance, mixed with artificial or synthetic substances.
In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding capacity that is free from its naturally occurring state. The "isolated antigen binding protein" may comprise a portion that binds an antigen and, optionally, a framework or framework portion that allows the antigen binding portion to adopt a conformation that facilitates binding of the antigen binding portion to an antigen. The antigen binding protein may comprise, for example, antibody-derived protein Framework Regions (FRs) 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, for example, to stabilize the three-dimensional structure of the antigen binding protein, as well as fully synthetic framework regions comprising, for example, biocompatible polymers. See, e.g., Korndorfer et al, 2003, Proteins: Structure, Function, andBioinformatics,53(1): 121-; roque et al, Biotechnol.prog.20:639-654 (2004). Examples of antigen binding proteins include, but are not limited to: a human antibody; a humanized antibody; a chimeric antibody; a recombinant antibody; a single chain antibody; a bifunctional antibody; a trifunctional antibody; a tetra-functional antibody; fab, Fab ', Fv fragments, Bs-Fv, F (ab') 2 ，F(ab) 2 scFv, di-scFv, dAb, IgD antibodies; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or IgG4 antibodies and fragments thereof.
In the present application, the term "CDR", also known as "complementarity determining region", generally refers to the region in an antibody variable domain whose sequence is highly variable and/or forms structurally defined loops. Typically, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR 3). In certain embodiments, naturally occurring camelid antibodies consisting of only heavy chains are also capable of normal and stable function in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature 363: 446-; 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, collectively Kabat/Chothia, and the like. These coding systems are known in the art and can be found in particular, for example, http:// www.bioinf.org.uk/abs/index. For example, the amino acid sequence numbering of the antigen binding protein may be according to the IMGT numbering scheme (IMGT, the International ImmunoGeneTiCs information system @ IMGT. circuits. fr; http:// IMGT. circuits. fr; Lefranc et al, 1999, Nucleic Acids Res.27: 209. 212; Ruiz et al, 2000Nucleic Acids Res.28: 219. 221; Lefranc et al, 2001, Nucleic Acids Res.29: 207. 209; Lefranc et al, 2003, Nucleic Acids Res.31: 307. 310; Lefranc et al, 2005, Comppon. 29: 185. Im. 203). For example, the CDRs of the antigen binding protein may be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acadsi 190:382-391 and Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, FifthEdion, U.S. department of Health and Human Services, NIH Publication No. 91-3242).
In this application, the term "FR" generally refers to a more highly conserved portion of an antibody variable domain, which is referred to as the framework region. Typically, the variable domains of native heavy and light chains each comprise four FR regions, namely four in the VH (H-FR1, H-FR2, H-FR3 and H-FR4), and four in the VL (L-FR1, L-FR2, L-FR3 and L-FR 4).
In the present application, the terms "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 "V" respectively H "and" V L "(alternatively referred to as" VH "and" VL ", respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and comprise antigen junctionsA synthetic site.
In the present application, the term "variable" generally refers to the possibility of large differences in sequence of certain segments of the variable domains between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a circular connection, and in some cases form part of a β -sheet structure. The CDRs in each chain are held together 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)).
In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising 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 chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F (ab') 2 Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function (e.g., specifically bind Staphylococcus aureus leukocidin). 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 antibodies consist of 5 elementary heterotetrameric units with another polypeptide called J chain and contain 10 antigen-binding sites, while IgA antibodies comprise 2-5 molecules that can interact with the J chainThe binding polymerization forms a polyvalent combination of essentially 4-chain units. For IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. 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 the alpha and gamma chains, respectively, and four CH domains for the mu and epsilon isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH and CL to the first constant domain of the heavy chain (CH 1). Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The VH and VL pair together to form a single antigen-binding site. For the structure and properties of antibodies of different classes see, e.g., Basic and Clinical Immunology,8th Edition, Daniel P.Sties, Abba I.Terr and Tristram G.Parsolw (eds), Appleton&Lange, Norwalk, conn.,1994, page 71 and chapter 6. L chains from any vertebrate species can be classified into one of two distinctly different classes, termed κ and λ, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy Chain (CH), immunoglobulins can be assigned to different classes or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively.
In the present application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind an antigen (e.g., staphylococcus aureus leukocidin). In the present application, the antigen binding fragments may include Fab, Fab', F (ab) 2 Fv fragment, F (ab') 2 scFv, di-scFv and/or dAb.
In the present application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. As described above, whole antibodies can be digested with papain. Papain digestion of antibodies produces two identical antigen binding fragments, a "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region, supra). Fab fragments may consist of one complete L chain with the variable region of one heavy chain and the first constant region (CH1) of the H chain (VH).
In the present application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than the Fab fragment. For example, a Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, the Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.
In the present application, the term "F (ab') 2" generally refers to antibody fragments produced by pepsin digestion of intact antibodies. The F (ab')2 fragment contains two Fab fragments and a partial hinge region held together by disulfide bonds. F (ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigens.
In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or a portion of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, Fv fragments comprise all or a portion of the amino-terminal variable region of about 110 amino acids of the heavy and light chains.
In the present application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, a scFv can have the VL and VH variable regions described in any order (e.g., relative to the N-terminus and C-terminus of a polypeptide), and the scFv can comprise a VL-linker-VH or can comprise a VH-linker-VL.
In the present application, the term "dAb" generally refers to antigen-binding fragments having a VH domain, a VL domain or having a VH domain or a VL domain, see, for example, Ward et al (Nature,1989Oct 12; 341 (6242): 544-6), reference Holt et al, Trends Biotechnol.,2003,21 (11): 484-490; and to other published patent applications such as WO 06/030220, WO 06/003388 and domentis ltd. The term "dAb" generally includes sdabs. The term "sdAb" generally refers to single domain antibodies. Single domain antibodies generally refer to antibody fragments consisting of only the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL).
In the present application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are typically highly specific for a single antigenic site. Moreover, 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, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use herein can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.
In the present application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable regions are derived from an antibody of an experimental animal such as a rodent ("parent antibody") and the constant regions are derived from a human antibody, such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent (e.g., alpaca-derived) antibody.
In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., an alpaca antibody) are replaced with corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible as long as they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "humanized antibodies" retain antigen specificity similar to the original antibody. "humanized" forms of non-human (e.g., alpaca) antibodies may be chimeric antibodies that minimally comprise sequences derived from non-human immunoglobulins. In certain instances, CDR region residues in a human immunoglobulin (acceptor antibody) can be replaced with CDR region residues of a non-human species (donor antibody) such as alpaca, mouse, rat, rabbit, or non-human primate having the desired properties, affinities, and/or capabilities. In some cases, residues from the FR region of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may comprise amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made in order to further improve the properties of the antibody, such as binding affinity.
The term "fully human antibody" generally refers to an antibody comprising only human immunoglobulin protein sequences. A fully human antibody may contain a murine sugar chain if it is produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, "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 biological methods. Exemplary techniques that can be used to make antibodies are described in U.S. patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as the use of libraries, are known in the art.
In this application, the term "cross-neutralizing" generally refers to neutralizing two or more toxins. The toxin may comprise a leukocidin. For example, the leukocidin may comprise hemolysin and/or PVL leukocidin. For example, the hemolysin may comprise gamma-hemolysin.
In the present application, the terms "polypeptide molecule" and "polypeptide" and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two moieties covalently linked together. Wherein each moiety may be a polypeptide having different properties. The property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two moieties may be linked by a single peptide bond or by a peptide linker.
In the present application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized synthetically.
In the present application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be used to transform, transduce or transfect a host cell so that the genetic material element it carries is expressed in the host cell. By way of example, the carrier may include: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyomavacuolium viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication origin. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.
In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been the recipient of a subject plasmid or vector, which includes a nucleic acid molecule of the invention or a vector of the invention. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. The cells can include cells transfected in vitro with a vector described herein. The cell can be a bacterial cell (e.g., E.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell.
In the present application, the term "immunoconjugate" generally refers to a conjugate of the additional agent (e.g., chemotherapeutic agent, radioactive element, cytostatic agent, and cytotoxic agent) conjugated (e.g., covalently linked by a linking molecule) to the antibody or antigen-binding fragment thereof, which conjugate can deliver the additional agent to a target cell by specific binding of the antibody or antigen-binding fragment thereof to an antigen on the target cell.
In the present application, 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 vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammal to which they are exposed at the dosages and concentrations employed. 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.
In the present application, 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 determine the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds a target (which may be an epitope) may be an antibody that binds the target with greater affinity, avidity, more readily, and/or for a longer duration than it binds other targets. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins of different species. In certain embodiments, specific binding may include, but is not required to be, exclusive binding. The term also applies, for example, to antigen binding proteins that are specific for a particular epitope that cross-reacts with multiple antigens, wherein the specific antibody binds to multiple antigens that carry the cross-reactive epitope. The binding sites of such antigen binding proteins and/or antigen binding proteins having specific binding cross-reactive epitopes are also referred to as multispecific or cross-specific binding site antigen binding proteins, respectively. For example, an antigen binding protein may have a multispecific binding site that specifically binds to an epitope that is cross-reactive with multiple different antigens.
In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.
In the present application, reference to protein, polypeptide and/or amino acid sequences is also to be understood as including at least the following ranges: variants or homologues having the same or similar function as said protein or polypeptide.
In the present application, the variant may be, for example, a protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to Staphylococcus aureus leukocidin). For example, the functional variant may comprise a protein or polypeptide which has been altered by at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to the alteration. For example, the substitution may be a conservative substitution.
In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof that specifically binds staphylococcus aureus leukocidin).
In the present application, homology generally refers to similarity, similarity or relatedness between two or more sequences. The "percentage of sequence homology" can be calculated by: the two sequences to be aligned are compared in a comparison window, the number of positions in the two sequences at which the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percentage of sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared or over a region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. The FASTA algorithm is described in "improved tools for biological sequence comparison" by w.r.pearson and d.j.lipman, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.), 85: 2444-2448, 1988; and "rapid and sensitive protein similarity search" by d.j.lipman and w.r.pearson, Science, 227: 1435-1441, 1989. See "an essential local contrast (alignment) search tool" by s.altschul, w.gish, w.miller, e.w.myers, and d.lipman, journal of molecular biology, 215: 403-410, 1990.
In this application, the term "comprising" is generally intended to mean including, summarizing, containing or including. In some cases, the meaning of "is", "consisting of … …" is also indicated.
In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.
Detailed Description
Isolated antigen binding proteins
The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be in contact with an antigen or an antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, consensus Kabat/Chothia, and the like. These coding systems are known in the art and can be found in particular, for example, http:// www.bioinf.org.uk/abs/index. One skilled in the art can determine the CDR regions using different coding systems based on the sequence and structure of the antibody. The CDR regions may differ using different coding systems. In the present application, the CDR encompasses CDR sequences divided according to any CDR division manner; variants thereof comprising substitution, deletion and/or addition of one or more amino acids of the amino acid sequence of the CDR are also contemplated. E.g., 1-30, 1-20, or 1-10, further e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and/or insertions; also encompassed are homologs thereof, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the CDR. In certain embodiments, the isolated antigen binding protein described herein is defined by the Kabat coding system.
In one aspect, the present application provides an isolated antigen binding protein that may comprise at least one CDR in an antibody heavy chain variable region VH, which VH may comprise the amino acid sequence set forth in SEQ ID No. 53. For example, the VH may comprise an amino acid sequence set forth in any one of SEQ ID NO 30, 31, 32, 33, 34, and 35. In the present application, the HCDR of the isolated antigen binding protein may be divided in any form, and any form of divided HCDR may fall within the scope of the present application, as long as the VH is identical to the amino acid sequence shown in any one of SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34 and SEQ ID NO 35.
In the present application, the isolated antigen binding protein may comprise HCDR3 and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 1.
In the present application, the isolated antigen binding protein may comprise HCDR2 and the HCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 2.
In the present application, the isolated antigen binding protein may comprise HCDR1 and the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 3.
In the present application, 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, the HCDR2 may comprise the amino acid sequence shown in SEQ ID No. 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 1.
In the present application, the isolated antigen binding protein may comprise H-FR1, the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 45.
In the present application, the H-FR1 may contain one or more amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO. 10. For example, the substitution may be at one or more positions selected from the group consisting of: 1 st bit, 5 th bit, 11 th bit, 12 th bit, 13 th bit, 14 th bit, 20 th bit and 23 th bit. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 10. For example, the H-FR1 can comprise the amino acid sequence shown in SEQ ID NO. 14. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 18.
In the present application, the isolated antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO. 46.
In the present application, the H-FR2 may contain one or more amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO. 11. For example, the substitution may be at one or more positions selected from the group consisting of: 3 rd bit, 5 th bit, 7 th bit, 13 th bit. For example, the substitution may be a conservative substitution of an amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 11. For example, the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 15. For example, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 19.
In the present application, 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 the amino acid sequence set forth in SEQ ID NO: 47.
In the present application, the H-FR3 may contain one or more amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO. 12. For example, the substitution may be at one or more positions selected from the group consisting of: 1 st bit, 2 nd bit, 10 th bit, 11 th bit, 15 th bit, 16 th bit, 19 th bit, 21 st bit, 23 rd bit and 29 th bit. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be a substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 12. For example, the H-FR3 can comprise the amino acid sequence shown in SEQ ID NO. 16. For example, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 20.
In the present application, the isolated antigen binding protein may comprise H-FR4, the N-terminus of H-FR4 is linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO. 48.
In the present application, the H-FR4 may contain one or more amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO. 13. For example, the substitution may be at one or more positions selected from the group consisting of: bit 6and bit 11. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 13. For example, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 17.
In the present application, the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:45, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:46, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:47, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 48. For example, the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO 10, SEQ ID NO 14 and SEQ ID NO 18, the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO 11, SEQ ID NO 15 and SEQ ID NO 19, the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO 12, SEQ ID NO 16 and SEQ ID NO 20, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO 13 or SEQ ID NO 17. For example, the H-FR1 may comprise the amino acid sequence shown by SEQ ID NO. 10, the H-FR2 may comprise the amino acid sequence shown by SEQ ID NO. 11, the H-FR3 may comprise the amino acid sequence shown by SEQ ID NO. 12, and the H-FR4 may comprise the amino acid sequence shown by SEQ ID NO. 13. For example, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 14, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 15, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 16, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 17. For example, the H-FR1 may comprise the amino acid sequence shown by SEQ ID NO. 18, the H-FR2 may comprise the amino acid sequence shown by SEQ ID NO. 15, the H-FR3 may comprise the amino acid sequence shown by SEQ ID NO. 16, and the H-FR4 may comprise the amino acid sequence shown by SEQ ID NO. 17. For example, the H-FR1 may comprise the amino acid sequence shown by SEQ ID NO. 18, the H-FR2 may comprise the amino acid sequence shown by SEQ ID NO. 19, the H-FR3 may comprise the amino acid sequence shown by SEQ ID NO. 16, and the H-FR4 may comprise the amino acid sequence shown by SEQ ID NO. 17. For example, the H-FR1 may comprise the amino acid sequence shown by SEQ ID NO. 18, the H-FR2 may comprise the amino acid sequence shown by SEQ ID NO. 15, the H-FR3 may comprise the amino acid sequence shown by SEQ ID NO. 20, and the H-FR4 may comprise the amino acid sequence shown by SEQ ID NO. 17. For example, the H-FR1 may comprise the amino acid sequence shown by SEQ ID NO. 18, the H-FR2 may comprise the amino acid sequence shown by SEQ ID NO. 19, the H-FR3 may comprise the amino acid sequence shown by SEQ ID NO. 20, and the H-FR4 may comprise the amino acid sequence shown by SEQ ID NO. 17.
In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR 4. For example, the HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 3, SEQ ID NO 2, SEQ ID NO 1, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, and SEQ ID NO 13, respectively. For example, the HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 3, SEQ ID NO 2, SEQ ID NO 1, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, and SEQ ID NO 17, respectively. For example, the HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 3, SEQ ID NO 2, SEQ ID NO 1, SEQ ID NO 18, SEQ ID NO 15, SEQ ID NO 16, and SEQ ID NO 17, respectively. For example, the HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 3, SEQ ID NO 2, SEQ ID NO 1, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 16, and SEQ ID NO 17, respectively. For example, the HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 3, SEQ ID NO 2, SEQ ID NO 1, SEQ ID NO 18, SEQ ID NO 15, SEQ ID NO 20, and SEQ ID NO 17, respectively. For example, the HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 3, SEQ ID NO 2, SEQ ID NO 1, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, and SEQ ID NO 17, respectively.
In the present application, the isolated antigen binding protein may comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 53.
In the present application, the VH may comprise one or more amino acid substitutions as compared to the amino acid sequence shown in SEQ ID NO 30. For example, the substitution may be at one or more positions selected from the group consisting of: 1 st, 5 th, 11 th, 12 th, 13 th, 14 th, 20 th, 23 th, 38 th, 40 th, 42 th, 48 th, 67 th, 68 th, 76 th, 77 th, 81 th, 82 th, 85 th, 87 th, 89 th, 95 th, 112 th and 117 th bits. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the VH may comprise an amino acid sequence set forth in any one of SEQ ID NO 30, 31, 32, 33, 34, and 35.
In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region. For example, the antibody heavy chain constant region may be derived from the heavy chain constant region of any one of the immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. For example, the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In the present application, the heavy chain constant region of the immunoglobulin may comprise a mutant thereof. In the present application, the antibody heavy chain constant region may be derived from the heavy chain constant region of any one of human IgG 1-4. For example, the isolated antigen binding protein may be derived from the human IgG1 heavy chain constant region. For example, the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 42.
In the present application, the isolated antigen binding protein may comprise a heavy chain, which may comprise a VH and a heavy chain constant region. For example, the VH of the heavy chain may comprise the amino acid sequence shown in SEQ ID NO 30 and the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO 42. For example, the VH of the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 31 and the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO. 42. For example, the VH of the heavy chain may comprise the amino acid sequence shown in SEQ ID NO 32 and the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO 42. For example, the VH of the heavy chain may comprise the amino acid sequence shown in SEQ ID NO 33 and the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO 42. For example, the VH of the heavy chain may comprise the amino acid sequence shown in SEQ ID NO 34 and the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO 42. For example, the VH of the heavy chain may comprise the amino acid sequence shown in SEQ ID NO 35 and the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO 42. For example, the heavy chain may comprise the amino acid sequence shown in any one of SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60 and SEQ ID NO 61.
In the present application, the isolated antigen binding protein may comprise at least one CDR in the variable region VL of an antibody, which VL may comprise the amino acid sequence set forth in SEQ ID NO: 54. For example, the VL may comprise an amino acid sequence set forth in any one of SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, and SEQ ID NO 41. In the present application, the LCDR of the isolated antigen binding protein may be divided in any form, and the LCDR divided in any form may fall within the scope of the present application, as long as VL is identical to the amino acid sequence shown in any of SEQ ID NO. 54, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40 and SEQ ID NO. 41.
In the present application, the isolated antigen binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 4.
In the present application, the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 5.
In the present application, the isolated antigen binding protein may comprise LCDR1 and LCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 44.
In the present application, the L-CDR1 may comprise one or more amino acid substitutions as compared to the amino acid sequence set forth in SEQ ID NO. 6. For example, the substitution may be at one or more positions selected from the group consisting of: 8th bit, 11 th bit and 12 th bit. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO. 6. For example, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 7. For example, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 8. For example, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 9.
In the present application, the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 44, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 4.
In the present application, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR 3. For example, 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, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 4. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 7, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 4. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 8, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 4. For example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No.9, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 4.
In the present application, the isolated antigen binding protein may comprise L-FR1, the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 49.
In the present application, the L-FR1 may contain one or more amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 21. For example, the substitution may be at one or more positions selected from the group consisting of: 2 nd, 11 th, 14 th, 17 th, 14 th and 18 th bits. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 21. For example, the L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO. 25. For example, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 29.
In the present application, the isolated antigen binding protein may comprise L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 50.
In the present application, the L-FR2 may contain one or more amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 22. For example, the substitution may be at one or more positions selected from the group consisting of: 3 rd bit, 5 th bit, 9 th bit and 11 th bit. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 22. For example, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 26.
In the present application, 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 the amino acid sequence set forth in SEQ ID NO. 51.
In the present application, the L-FR3 may contain one or more amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO. 23. For example, the substitution may be at one or more positions selected from the group consisting of: 11 th bit, 12 th bit, 27 th bit and 31 th bit. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 23. For example, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 27.
In the present application, the isolated antigen binding protein may comprise L-FR4, the N-terminus of L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO. 52.
In the present application, the L-FR4 may contain one or more amino acid substitutions as compared with the amino acid sequence shown in SEQ ID NO: 24. For example, the substitution may be at position 3. For example, the substitution may be a conservative substitution of the amino acid. For example, the conservative substitution may be the replacement of an amino acid residue with an amino acid residue having a similar side chain. For example, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 24. For example, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 28.
In the present application, the isolated antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 49, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 50, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 51, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 52. For example, the L-FR1 may comprise the amino acid sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:25 and SEQ ID NO:29, the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:26, the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:27, and the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:24 or SEQ ID NO: 28. For example, the L-FR1 may comprise the amino acid sequence shown by SEQ ID NO. 21, the L-FR2 may comprise the amino acid sequence shown by SEQ ID NO. 22, the L-FR3 may comprise the amino acid sequence shown by SEQ ID NO. 23, and the L-FR4 may comprise the amino acid sequence shown by SEQ ID NO. 24; for example, the L-FR1 may comprise the amino acid sequence shown by SEQ ID NO. 25, the L-FR2 may comprise the amino acid sequence shown by SEQ ID NO. 26, the L-FR3 may comprise the amino acid sequence shown by SEQ ID NO. 27, and the L-FR4 may comprise the amino acid sequence shown by SEQ ID NO. 28; for example, the L-FR1 may comprise the amino acid sequence shown by SEQ ID NO. 29, the L-FR2 may comprise the amino acid sequence shown by SEQ ID NO. 26, the L-FR3 may comprise the amino acid sequence shown by SEQ ID NO. 27, and the L-FR4 may comprise the amino acid sequence shown by SEQ ID NO. 28.
In the present application, the isolated antigen binding protein may comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR 4. For example, the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO 6, SEQ ID NO 5, SEQ ID NO 4, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24, respectively, in that order. For example, the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO 7, SEQ ID NO 5, SEQ ID NO 4, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, respectively, in that order. For example, the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 8, SEQ ID NO 5, SEQ ID NO 4, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, and SEQ ID NO 28, respectively. For example, the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 9, SEQ ID NO 5, SEQ ID NO 4, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, and SEQ ID NO 28, respectively. For example, the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 8, SEQ ID NO 5, SEQ ID NO 4, SEQ ID NO 29, SEQ ID NO 26, SEQ ID NO 27, and SEQ ID NO 28, respectively. For example, the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3, and L-FR4 of the isolated antigen binding protein may comprise, in order, the amino acid sequences shown in SEQ ID NO 9, SEQ ID NO 5, SEQ ID NO 4, SEQ ID NO 29, SEQ ID NO 26, SEQ ID NO 27, and SEQ ID NO 28, respectively.
In the present application, the isolated antigen binding protein may comprise a VL comprising the amino acid sequence set forth in SEQ ID NO. 54.
In the present application, the VL may comprise one or more amino acid substitutions as compared to the amino acid sequence shown in SEQ ID NO: 36. For example, the substitution may be at one or more positions selected from the group consisting of: 2 nd, 11 th, 14 th, 17 th, 18 th, 31 st, 34 th, 35 th, 42 th, 44 th, 48 th, 50 th, 72 th, 88 th, 92 th and 115 th bits. For example, the VL may comprise the amino acid sequence set forth in SEQ ID NO: 36. For example, the VL may comprise the amino acid sequence set forth in SEQ ID NO 37. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO 38. For example, the VL may comprise the amino acid sequence set forth in SEQ ID NO: 39. For example, the VL may comprise the amino acid sequence shown in SEQ ID NO. 40. For example, the VL may comprise the amino acid sequence set forth in SEQ ID NO 41.
In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region. For example, the light chain constant region may be derived from a human antibody light chain constant region. For example, the light chain constant region may be derived from a human Ig kappa constant region. For example, the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 43.
In the present application, the isolated antigen binding protein may include a light chain, which may include a VL and a light chain constant region. For example, the VL of the light chain may comprise the amino acid sequence set forth in SEQ ID NO. 36 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO. 43. For example, the VL of the light chain may comprise the amino acid sequence set forth in SEQ ID NO 37 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 43. For example, the VL of the light chain may comprise the amino acid sequence set forth in SEQ ID NO. 38 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO. 43. For example, the VL of the light chain may comprise the amino acid sequence set forth in SEQ ID NO 39 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 43. For example, the VL of the light chain may comprise the amino acid sequence set forth in SEQ ID NO. 40 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO. 43. For example, the VL of the light chain may comprise the amino acid sequence set forth in SEQ ID NO 41 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 43. For example, the light chain may comprise an amino acid sequence shown in any one of SEQ ID NO 56, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65 and SEQ ID NO 66.
In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR 3. For example, the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID No. 3, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 1, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 44, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 5, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 4.
In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3. For example, 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, the HCDR3 can comprise the amino acid sequence set forth in SEQ ID No. 1, the LCDR1 can comprise the amino acid sequence set forth in any one of SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No.9, the LCDR2 can comprise the amino acid sequence set forth in SEQ ID No. 5, and the LCDR3 can comprise the amino acid sequence set forth in SEQ ID No. 4.
In the present application, the isolated antigen binding protein may comprise a VH and a VL. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO. 53 and the VL may comprise the amino acid sequence shown in SEQ ID NO. 54. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 30 and the VL comprises the amino acid sequence shown in SEQ ID NO. 36. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 30 and the VL comprises the amino acid sequence shown in SEQ ID NO. 37. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 30 and the VL comprises the amino acid sequence shown in SEQ ID NO. 38. For example, the VH comprises the amino acid sequence set forth in SEQ ID NO. 30 and the VL comprises the amino acid sequence set forth in SEQ ID NO. 39. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 30 and the VL comprises the amino acid sequence shown in SEQ ID NO. 40. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 30 and the VL comprises the amino acid sequence shown in SEQ ID NO. 41. For example, the VH comprises the amino acid sequence set forth in SEQ ID NO. 31 and the VL comprises the amino acid sequence set forth in SEQ ID NO. 36. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 31 and the VL comprises the amino acid sequence shown in SEQ ID NO. 37. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 31 and the VL comprises the amino acid sequence shown in SEQ ID NO. 38. For example, the VH comprises the amino acid sequence shown as SEQ ID NO. 31 and the VL comprises the amino acid sequence shown as SEQ ID NO. 39. For example, the VH comprises the amino acid sequence shown as SEQ ID NO. 31 and the VL comprises the amino acid sequence shown as SEQ ID NO. 40. For example, the VH comprises the amino acid sequence shown as SEQ ID NO. 31 and the VL comprises the amino acid sequence shown as SEQ ID NO. 41. For example, the VH comprises the amino acid sequence shown in SEQ ID NO:32 and the VL comprises the amino acid sequence shown in SEQ ID NO: 36. For example, the VH comprises the amino acid sequence shown in SEQ ID NO:32 and the VL comprises the amino acid sequence shown in SEQ ID NO: 37. For example, the VH comprises the amino acid sequence shown in SEQ ID NO:32 and the VL comprises the amino acid sequence shown in SEQ ID NO: 38. For example, the VH comprises the amino acid sequence shown in SEQ ID NO:32 and the VL comprises the amino acid sequence shown in SEQ ID NO: 39. For example, the VH comprises the amino acid sequence shown in SEQ ID NO:32 and the VL comprises the amino acid sequence shown in SEQ ID NO: 40. For example, the VH comprises the amino acid sequence shown as SEQ ID NO. 32 and the VL comprises the amino acid sequence shown as SEQ ID NO. 41. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 33 and the VL comprises the amino acid sequence shown in SEQ ID NO. 36. For example, the VH comprises the amino acid sequence set forth in SEQ ID NO. 33 and the VL comprises the amino acid sequence set forth in SEQ ID NO. 37. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 33 and the VL comprises the amino acid sequence shown in SEQ ID NO. 38. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 33 and the VL comprises the amino acid sequence shown in SEQ ID NO. 39. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 33 and the VL comprises the amino acid sequence shown in SEQ ID NO. 40. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 33 and the VL comprises the amino acid sequence shown in SEQ ID NO. 41. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 34 and the VL comprises the amino acid sequence shown in SEQ ID NO. 36. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 34 and the VL comprises the amino acid sequence shown in SEQ ID NO. 37. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 34 and the VL comprises the amino acid sequence shown in SEQ ID NO. 38. For example, the VH comprises the amino acid sequence shown as SEQ ID NO. 34 and the VL comprises the amino acid sequence shown as SEQ ID NO. 39. For example, the VH comprises the amino acid sequence shown as SEQ ID NO. 34 and the VL comprises the amino acid sequence shown as SEQ ID NO. 40. For example, the VH comprises the amino acid sequence shown as SEQ ID NO. 34 and the VL comprises the amino acid sequence shown as SEQ ID NO. 41. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 35 and the VL comprises the amino acid sequence shown in SEQ ID NO. 36. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 35 and the VL comprises the amino acid sequence shown in SEQ ID NO. 37. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 35 and the VL comprises the amino acid sequence shown in SEQ ID NO. 38. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 35 and the VL comprises the amino acid sequence shown in SEQ ID NO. 39. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 35 and the VL comprises the amino acid sequence shown in SEQ ID NO. 40. For example, the VH comprises the amino acid sequence shown in SEQ ID NO. 35 and the VL comprises the amino acid sequence shown in SEQ ID NO. 41.
In the present application, the isolated antigen binding protein may comprise a VH, a heavy chain constant region, a VL, and a light chain constant region. For example, the VH may be selected from the amino acid sequence set forth in any one of SEQ ID NO 30, 31, 32, 33, 34 and 35, the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 42, the VL may be selected from the amino acid sequence set forth in any one of SEQ ID NO 36, 37, 38, 39, 40 and 41, and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 43.
In the present application, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof. In the present application, the antigen binding fragment may include Fab, Fab ', Fv fragment, F (ab') 2 ，F(ab) 2 scFv, di-scFv and/or dAb. In the present application, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody and/or a fully human antibodyAn antibody. For example, the VH of the humanized antigen binding protein may comprise an amino acid sequence set forth in any one of SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, and SEQ ID NO 35. For example, the VL of the humanized antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40 and SEQ ID NO 41.
In addition, it is contemplated that the isolated antigen binding proteins described herein may comprise heavy and/or light chain sequences with one or more conservative sequence modifications thereto. By "conservative sequence modifications" is meant amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the isolated antigen binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in a CDR region of an isolated antigen binding protein described herein can be replaced with other amino acid residues of the ipsilateral chain set. One skilled in the art will recognize that some conservative sequence modifications will not abolish antigen binding, as seen in particular, for example, in 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.10:341-6and Beers et al, (2000) Clin.Can.Res.6: 2835-43.
The staphylococcus aureus leukocidin antigen-binding proteins described herein can be identified, screened or characterized by various assays known in the art.
For example, the antigen binding activity of the antigen binding proteins of the present application can be tested by known methods such as enzyme linked immunosorbent assay (ELISA), immunoblotting (e.g., western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like.
In the present application, the isolated antigen binding protein is capable of specifically binding to staphylococcus aureus toxin. In the present application, the staphylococcus aureus toxin may comprise a leukocidin. For example, the leukocidin may comprise HlgA, HlgB, LukD and/or LukF. For example, the isolated antigen binding proteins are capable of specifically binding to staphylococcus aureus HlgA, HlgB, LukD, and LukF. For example, the isolated antigen binding protein is capable of specifically binding to staphylococcus aureus HlgA and LukD. For example, the isolated antigen binding protein is capable of specifically binding to staphylococcus aureus HlgA and LukF. In the present application, the isolated antigen binding protein is capable of specifically binding a bi-component toxin, which can be selected from the group consisting of an F component and an S component, such as HlgA and HlgB, such as HlgA and LukD, such as HlgA and LukF.
In the present application, the binding of the isolated antigen binding protein to staphylococcus aureus leukocidin can be detected by ELISA. For example, an antigen binding protein described herein can be present in an EC of less than or equal to about 0.1 μ g/mL, less than or equal to about 0.095 μ g/mL, less than or equal to about 0.09 μ g/mL, less than or equal to about 0.085 μ g/mL, less than or equal to about 0.08 μ g/mL, less than or equal to about 0.075 μ g/mL, less than or equal to about 0.06 μ g/mL, less than or equal to about 0.05 μ g/mL, less than or equal to about 0.04 μ g/mL, less than or equal to about 0.034 μ g/mL, less than or equal to about 0.03 μ g/mL, less than or equal to about 0.02 μ g/mL, and less than or equal to about 0.01 μ g/mL 50 Values bind to staphylococcus aureus HlgA. In certain embodiments, the EC in which the antigen binding protein described herein binds to HlgA 50 The value was 0.076. mu.g/mL. In certain embodiments, the EC in which the antigen binding protein described herein binds to HlgA 50 The value was 0.034. mu.g/mL. For example, an antigen binding protein described herein can be present in an EC of less than or equal to about 0.1 μ g/mL, less than or equal to about 0.09 μ g/mL, less than or equal to about 0.08 μ g/mL, less than or equal to about 0.07 μ g/mL, less than or equal to about 0.06 μ g/mL, less than or equal to about 0.055 μ g/mL, less than or equal to about 0.05 μ g/mL, less than or equal to about 0.045 μ g/mL, less than or equal to about 0.04 μ g/mL, less than or equal to about 0.035 μ g/mL, and less than or equal to about 0.02 μ g/mL 50 Values bind to staphylococcus aureus HlgB. In certain embodiments, the EC in which the antigen binding protein described herein binds to HlgB 50 The value was 0.051. mu.g/mL. In certain embodiments, the EC in which the antigen binding protein described herein binds to HlgB 50 The value was 0.055. mu.g/mL. For example, an antigen binding protein described herein can be present in an EC of less than or equal to about 0.08 μ g/mL, less than or equal to about 0.07 μ g/mL, less than or equal to about 0.06 μ g/mL, less than or equal to about 0.05 μ g/mL, less than or equal to about 0.045 μ g/mL, less than or equal to about 0.04 μ g/mL, less than or equal to about 0.035 μ g/mL, less than or equal to about 0.034 μ g/mL, less than or equal to about 0.03 μ g/mL, less than or equal to about 0.02 μ g/mL, and less than or equal to about 0.01 μ g/mL 50 The values bind to the LukD of Staphylococcus aureus. In certain embodiments, the EC of the antigen binding protein described herein that binds to LukD 50 The value was 0.038. mu.g/mL. In certain embodiments, the EC of the antigen binding protein described herein that binds to LukD 50 The value was 0.036. mu.g/mL. For example, an antigen binding protein described herein can be administered with an EC of less than or equal to about 0.08 μ g/mL, less than or equal to about 0.07 μ g/mL, less than or equal to about 0.06 μ g/mL, less than or equal to about 0.05 μ g/mL, less than or equal to about 0.045 μ g/mL, less than or equal to about 0.04 μ g/mL, less than or equal to about 0.035 μ g/mL, less than or equal to about 0.034 μ g/mL, less than or equal to about 0.03 μ g/mL, less than or equal to about 0.02 μ g/mL, and less than or equal to about 0.01 μ g/mL 50 Value and golden grapeCoccal LukF binding. In certain embodiments, the EC to which an antigen binding protein described herein binds LukF 50 The value was 0.053. mu.g/mL. In certain embodiments, the EC to which an antigen binding protein described herein binds LukF 50 The value was 0.043. mu.g/mL.
In the present application, the isolated antigen binding proteins are capable of neutralizing the activity of Staphylococcus aureus leukocytin. For example, rabbit erythrocytes can be added to the leukocidin and antigen-binding protein of Staphylococcus aureus, and their anti-hemolytic activity can be detected using a microplate reader. For example, the isolated frontal antigen binding protein can have a complete neutralization effect on 4 μ g of the staphylococcus aureus bi-component toxin HlgAB, HlgA + LukD, and/or HlgA + LukF at less than or equal to about 5 μ g, less than or equal to about 4 μ g, less than or equal to about 3 μ g, less than or equal to about 2 μ g, less than or equal to about 1 μ g.
In the present application, the isolated antigen binding protein is capable of preventing and/or treating a disease and/or disorder. In the present application, the disease and/or condition may be caused or mediated by staphylococcus aureus. In the present application, the disease and/or condition may comprise a complication of the disease and/or condition caused or mediated by staphylococcus aureus. In certain embodiments, the disease and/or disorder and its complications may include sepsis and/or bacteremia.
Polypeptide molecules, nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions
In another aspect, the present application provides polypeptide molecules that can comprise an isolated antigen binding protein described herein.
In the present application, the polypeptide molecule may comprise a fusion protein. In the present application, the polypeptide molecule may be a fusion protein. In the present application, the fusion protein may comprise a multispecific antibody.
In another aspect, the present application provides isolated nucleic acid molecules that can encode the isolated antigen binding proteins described herein. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g., by Polymerase Chain Reaction (PCR); (ii) produced by clonal recombination; (iii) purified, e.g., fractionated by enzymatic cleavage and gel electrophoresis; or (iv) synthetic, for example by chemical synthesis.
In another aspect, the present application provides a vector, which may comprise a nucleic acid molecule as described herein. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may contain expression control elements that allow for the proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, among others. The vector may be used to express the genetic material element carried by the vector in a host cell by transformation, transduction or transfection of the host cell. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may include components that assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.
In another aspect, the present application provides a cell that can comprise a nucleic acid molecule described herein or a vector described herein. In certain embodiments, each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cell, e.g., a eukaryotic cell, such as a plant-derived cell, a fungal or yeast cell, and the like. In certain embodiments, the cell can be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, 293T cell, COS-1 cell, SP2/0 cell, NS0 cell, or myeloma cell. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.
In another aspect, the present application also provides immunoconjugates that can comprise the isolated antigen binding proteins described herein.
In certain embodiments, 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 linkage may be through one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of linkers (which may be known in the art) may be used to form immunoconjugates. In addition, the immunoconjugate may be provided in the form of a fusion protein, which may be expressed from a polynucleotide encoding the immunoconjugate. The immunoconjugate may also comprise, for example, an antibody-drug conjugate (ADC). In an ADC, the antibody and therapeutic agent may be cross-linked by a linker that is cleavable, for example, a peptide linker, a disulfide linker, or a hydrazone linker.
In another aspect, the present application also provides pharmaceutical compositions that can comprise an isolated antigen binding protein described herein, a polypeptide molecule described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable carrier.
In certain embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, as well as clinical parameters of the subject.
In certain embodiments, the pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with pharmaceutical administration, generally safe and non-toxic.
In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flow into the patient, as described in WO 2015/036583.
Preparation method
In another aspect, 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. For example, these methods can be performed by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like, which are known to those of ordinary skill in the art.
Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application. For example, animals may be immunized with a linked or naturally occurring staphylococcal leukocidin or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used. In certain embodiments, isolated antigen binding proteins against staphylococcus aureus leukocidin can be screened and enriched by extracting immune alpaca peripheral blood lymphocytes, extracting nucleic acid fragments cloned into vectors, and screening by phage surface display systems.
Any suitable form of Staphylococcus aureus leukocidin may be used as an immunogen (antigen) for the production of non-human antibodies specific for Staphylococcus aureus leukocidin, which antibodies are screened for biological activity. For example, the challenge immunogen may be a full-length staphylococcus aureus leukocidin, including a native homodimer, or a peptide containing a single/multiple epitope. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art.
Method and use
In another aspect, the present application provides the use of the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition in the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.
In another aspect, the present application also provides methods of preventing and/or treating a disease and/or disorder, which may comprise administering to a subject in need thereof an isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition described herein.
In the present application, the administration can be carried out in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
In another aspect, the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition described herein may be used for the prevention and/or treatment of a disease and/or disorder.
In the present application, the disease and/or condition may be caused or mediated by staphylococcus aureus.
In the present application, the disease and/or condition may be a complication of a disease and/or condition caused or mediated by staphylococcus aureus.
In the present application, the disease and/or disorder may include a bacterial infection.
In the present application, the disease and/or condition may comprise bacteremia and/or sepsis.
In the present application, the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition may be used alone or in combination with other drugs for the prevention and/or treatment of diseases and/or disorders. In certain embodiments, the other drug may be any drug currently known to have an antibacterial effect. The disease and/or condition may be caused by infection with staphylococcus aureus or may be related diseases caused by other infections.
In another aspect, the present application also provides a method of detecting staphylococcus aureus leukocidin in a sample, the method comprising administering the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition.
In the present application, the method for detecting the leukocidin of staphylococcus aureus in the sample can be an in vitro method. For example, isolated antigen binding proteins described herein are contacted with a sample ex vivo to detect the presence and/or amount of staphylococcus aureus leukocidin in the sample. In some cases, the method of detecting Staphylococcus aureus leukocidin in a sample is for non-therapeutic purposes. In some cases, the method of detecting staphylococcus aureus leukocidin in a sample is not a diagnostic method.
In another aspect, the present application also provides a reagent or a kit for detecting staphylococcus aureus leukocidin in a sample, which comprises the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell, the immunoconjugate and/or the pharmaceutical composition.
In another aspect, the present application also provides the use of the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition in the preparation of a kit for detecting the presence and/or amount of staphylococcus aureus leukocidin in a sample.
The present application also encompasses the following specific embodiments:
1. an isolated antigen binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence set forth in SEQ ID NO: 53.
2. The isolated antigen binding protein of embodiment 1, comprising HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 1.
3. The isolated antigen binding protein of any of embodiments 1-2, comprising HCDR2, the HCDR2 comprising the amino acid sequence set forth in SEQ ID No. 2.
4. The isolated antigen binding protein of any of embodiments 1-3, comprising HCDR1, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO. 3.
5. The isolated antigen binding protein of any of embodiments 1-4, comprising HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence set forth in SEQ ID No. 3, said HCDR2 comprising the amino acid sequence set forth in SEQ ID No. 2, and said HCDR3 comprising the amino acid sequence set forth in SEQ ID No. 1.
6. The isolated antigen binding protein of any of embodiments 4-5, comprising H-FR1, the C-terminus of H-FR1 being linked directly or indirectly to the N-terminus of HCDR1, and the H-FR1 comprising the amino acid sequence set forth in SEQ ID NO: 45.
7. The isolated antigen binding protein of embodiment 6, wherein said H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO 10, SEQ ID NO 14, and SEQ ID NO 18.
8. The isolated antigen binding protein of any one of embodiments 4-7, comprising H-FR2, the H-FR2 being located between the HCDR1 and the HCDR2, and the H-FR2 comprising the amino acid sequence set forth in SEQ ID NO 46.
9. The isolated antigen binding protein of embodiment 8, wherein said H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO. 11, SEQ ID NO. 15, and SEQ ID NO. 19.
10. The isolated antigen binding protein of any one of embodiments 3-9, comprising H-FR3, the H-FR3 being located between the HCDR2 and the HCDR3, and the H-FR3 comprising the amino acid sequence set forth in SEQ ID NO: 47.
11. According to the amino acid sequence set forth in embodiment 10, said H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO 12, SEQ ID NO 16 and SEQ ID NO 20.
12. The isolated antigen binding protein of any one of embodiments 2-11, comprising H-FR4, the N-terminus of H-FR4 being linked to the C-terminus of HCDR3, and the H-FR4 comprising the amino acid sequence set forth in SEQ ID No. 48.
13. The isolated antigen binding protein of embodiment 12, wherein H-FR4 comprises the amino acid sequence set forth in SEQ ID NO 13 or SEQ ID NO 17.
14. The isolated antigen binding protein of any one of embodiments 1-13, comprising H-FR1, H-FR2, H-FR3, and H-FR4, said H-FR1 comprising the amino acid sequence set forth in SEQ ID NO 45, said H-FR2 comprising the amino acid sequence set forth in SEQ ID NO 46, said H-FR3 comprising the amino acid sequence set forth in SEQ ID NO 47, and said H-FR4 comprising the amino acid sequence set forth in SEQ ID NO 48.
15. The isolated antigen binding protein of any one of embodiments 1-14, comprising H-FR1, H-FR2, H-FR3, and H-FR4, said H-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NO 10, SEQ ID NO 14, and SEQ ID NO 18, said H-FR2 comprising the amino acid sequence set forth in any one of SEQ ID NO 11, SEQ ID NO 15, and SEQ ID NO 19, said H-FR3 comprising the amino acid sequence set forth in any one of SEQ ID NO 12, SEQ ID NO 16, and SEQ ID NO 20, and said H-FR4 comprising the amino acid sequence set forth in SEQ ID NO 13 or SEQ ID NO 17.
16. The isolated antigen binding protein of any one of embodiments 1-15, comprising H-FR1, H-FR2, H-FR3, and H-FR4 selected from the group consisting of:
1) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 10, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 11, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 12, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 13;
2) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 14, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 15, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 16, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 17;
3) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 18, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 15, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 16, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 17;
4) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 18, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 19, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 16, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 17;
5) the H-FR1 comprises an amino acid sequence shown as SEQ ID NO. 18, the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 15, the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 20, and the H-FR4 comprises an amino acid sequence shown as SEQ ID NO. 17; and
6) the H-FR1 comprises the amino acid sequence shown by SEQ ID NO. 18, the H-FR2 comprises the amino acid sequence shown by SEQ ID NO. 19, the H-FR3 comprises the amino acid sequence shown by SEQ ID NO. 20, and the H-FR4 comprises the amino acid sequence shown by SEQ ID NO. 17.
17. The isolated antigen binding protein of any one of embodiments 1-16, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 53.
18. The isolated antigen binding protein of embodiment 17, wherein said VH comprises an amino acid sequence set forth in any one of SEQ ID NO 30, 31, 32, 33, 34, and 35.
19. The isolated antigen binding protein of any one of embodiments 1-18, comprising an antibody heavy chain constant region.
20. The isolated antigen binding protein of embodiment 19, wherein the heavy chain constant region is derived from a human IgG constant region.
21. The isolated antigen binding protein of any one of embodiments 19-20, wherein the heavy chain constant region is derived from a human IgG1 heavy chain constant region.
22. The isolated antigen binding protein of any one of embodiments 19-21, wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID No. 42.
23. The isolated antigen binding protein of any one of embodiments 1-21, comprising an antibody heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, and SEQ ID NO 61.
24. The isolated antigen binding protein of any of embodiments 1-23, comprising at least one CDR in an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID No. 54.
25. The isolated antigen binding protein of any one of embodiments 1-24, comprising LCDR3, the LCDR3 comprising the amino acid sequence set forth in SEQ ID No. 4.
26. The isolated antigen binding protein of any one of embodiments 1-25, comprising LCDR2, the LCDR2 comprising the amino acid sequence set forth in SEQ ID No. 5.
27. The isolated antigen binding protein of any one of embodiments 1-26, comprising LCDR1, the LCDR1 comprising the amino acid sequence set forth in SEQ ID No. 44.
28. The isolated antigen binding protein of embodiment 27, wherein the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9.
29. The isolated antigen binding protein of any one of embodiments 1-28, comprising LCDR1, LCDR2 and LCDR3, said LCDR1 comprising the amino acid sequence set forth in SEQ ID No. 44, said LCDR2 comprising the amino acid sequence set forth in SEQ ID No. 5, and said LCDR3 comprising the amino acid sequence set forth in SEQ ID No. 4.
30. The isolated antigen binding protein of any one of embodiments 1-29, comprising LCDR1, LCDR2 and LCDR3, said LCDR1 comprising the amino acid sequence set forth in any one of SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No.9, said LCDR2 comprising the amino acid sequence set forth in SEQ ID No. 5, and said LCDR3 comprising the amino acid sequence set forth in SEQ ID No. 4.
31. The isolated antigen binding protein of any one of embodiments 27-30, comprising L-FR1, the C-terminus of L-FR1 being linked directly or indirectly to the N-terminus of LCDR1, and the L-FR1 comprising the amino acid sequence set forth in SEQ ID No. 49.
32. The isolated antigen binding protein of embodiment 31, wherein said L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:25, and SEQ ID NO: 29.
33. The isolated antigen binding protein of any one of embodiments 27-32, comprising L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and wherein the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO 50.
34. The isolated antigen binding protein of embodiment 33, wherein said L-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 22 or SEQ ID NO. 26.
35. The isolated antigen binding protein of any one of embodiments 26-34, comprising L-FR3, wherein the L-FR3 is located between the LCDR2 and LCDR3, and wherein the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 51.
36. The isolated antigen binding protein of embodiment 35, wherein said L-FR3 comprises the amino acid sequence set forth in SEQ ID NO. 23 or SEQ ID NO. 27.
37. The isolated antigen binding protein of any one of embodiments 1-36, comprising L-FR4, the N-terminus of L-FR4 being linked directly or indirectly to the C-terminus of LCDR3, and the L-FR4 comprising the amino acid sequence set forth in SEQ ID NO 52.
38. The isolated antigen binding protein of embodiment 37, wherein said L-FR4 comprises the amino acid sequence set forth in SEQ ID NO. 24 or SEQ ID NO. 28.
39. The isolated antigen binding protein of any one of embodiments 1-38, comprising L-FR1, L-FR2, L-FR3, and L-FR4, said L-FR1 comprising the amino acid sequence set forth in SEQ ID NO 49, said L-FR2 comprising the amino acid sequence set forth in SEQ ID NO 50, said L-FR3 comprising the amino acid sequence set forth in SEQ ID NO 51, and said L-FR4 comprising the amino acid sequence set forth in SEQ ID NO 52.
40. The isolated antigen binding protein of any one of embodiments 1-39, comprising L-FR1, L-FR2, L-FR3, and L-FR4, said L-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:25, and SEQ ID NO:29, said L-FR2 comprising the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:26, said L-FR3 comprising the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:27, and said L-FR4 comprising the amino acid sequence set forth in SEQ ID NO:24 or SEQ ID NO: 28.
41. The isolated antigen binding protein of any one of embodiments 1-40, comprising L-FR1, L-FR2, L-FR3, and L-FR4 selected from any one of the group consisting of:
1) the L-FR1 comprises an amino acid sequence shown as SEQ ID NO. 21, the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 22, the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 23, and the L-FR4 comprises an amino acid sequence shown as SEQ ID NO. 24;
2) the L-FR1 comprises an amino acid sequence shown as SEQ ID NO. 25, the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 26, the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 27, and the L-FR4 comprises an amino acid sequence shown as SEQ ID NO. 28; and
3) the L-FR1 comprises an amino acid sequence shown as SEQ ID NO. 29, the L-FR2 comprises an amino acid sequence shown as SEQ ID NO. 26, the L-FR3 comprises an amino acid sequence shown as SEQ ID NO. 27, and the L-FR4 comprises an amino acid sequence shown as SEQ ID NO. 28.
42. The isolated antigen binding protein of any one of embodiments 1-41, comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 54.
43. The isolated antigen binding protein of embodiment 42, wherein the VL comprises an amino acid sequence set forth in any one of SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, and SEQ ID NO 41.
44. The isolated antigen binding protein of any one of embodiments 1-43, comprising an antibody light chain constant region.
45. The isolated antigen binding protein of embodiment 44, wherein the light chain constant region is derived from a human Ig kappa constant region.
46. The isolated antigen binding protein of any one of embodiments 44-45, wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 43.
47. The isolated antigen binding protein of any one of embodiments 1-45, comprising an antibody light chain comprising the amino acid sequence set forth in any one of SEQ ID NO 56, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, and SEQ ID NO 66.
48. The isolated antigen binding protein of any one of embodiments 1-47, comprising any one of the group of VH and VL selected from:
1) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
2) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
3) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
4) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
5) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
6) the VH comprises an amino acid sequence shown as SEQ ID NO. 30, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
7) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
8) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
9) the VH comprises an amino acid sequence shown in SEQ ID NO. 31, and the VL comprises an amino acid sequence shown in SEQ ID NO. 38;
10) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
11) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
12) the VH comprises an amino acid sequence shown as SEQ ID NO. 31, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
13) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
14) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
15) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
16) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
17) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
18) the VH comprises an amino acid sequence shown as SEQ ID NO. 32, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
19) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
20) the VH comprises an amino acid sequence shown in SEQ ID NO. 33, and the VL comprises an amino acid sequence shown in SEQ ID NO. 37;
21) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
22) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
23) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
24) the VH comprises an amino acid sequence shown as SEQ ID NO. 33, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
25) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
26) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
27) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
28) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
29) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40;
30) the VH comprises an amino acid sequence shown as SEQ ID NO. 34, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41;
31) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 36;
32) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 37;
33) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 38;
34) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 39;
35) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 40; and
36) the VH comprises an amino acid sequence shown as SEQ ID NO. 35, and the VL comprises an amino acid sequence shown as SEQ ID NO. 41.
49. The isolated antigen binding protein of any one of embodiments 1-48, comprising an antibody or antigen binding fragment thereof.
50. The isolated antigen binding protein of embodiment 49, wherein said antigen binding fragment comprises a Fab, Fab ', Fv fragment, F (ab')2, F (ab)2, scFv, di-scFv, and/or dAb.
51. The isolated antigen binding protein of any one of embodiments 49-50, wherein the antibody is selected from one or more of the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
52. The isolated antigen binding protein of any one of embodiments 1-51, which is capable of specifically binding to Staphylococcus aureus toxin.
53. The isolated antigen binding protein of embodiment 52, wherein said Staphylococcus aureus toxin comprises a leukocidin.
54. The isolated antigen binding protein of any one of embodiments 52-53, wherein the Staphylococcus aureus toxin is selected from one or more of the group consisting of: HlgA, HlgB, LukD and LukF.
55. The isolated antigen binding protein of any one of embodiments 1-54, which is capable of neutralizing the activity of one or several S.aureus virulence factors selected from the group consisting of: HlgA, HlgB, LukD and LukF.
56. The isolated antigen binding protein of any one of embodiments 1-55, which is capable of treating a disease and/or condition caused by Staphylococcus aureus.
57. The isolated antigen binding protein of embodiment 56, wherein said disease and/or disorder comprises a complication of a disease and/or disorder caused by Staphylococcus aureus.
58. A polypeptide molecule comprising the isolated antigen binding protein of any one of embodiments 1-57.
59. The polypeptide molecule of embodiment 58 comprising a fusion protein.
60. An immunoconjugate comprising the isolated antigen binding protein of any one of embodiments 1-57.
61. A nucleic acid molecule encoding the isolated antigen binding protein of any one of embodiments 1-57 or the polypeptide molecule of any one of embodiments 58-59.
62. A vector comprising the nucleic acid molecule of embodiment 61.
63. A cell comprising the nucleic acid molecule of embodiment 61 or the vector of embodiment 62.
64. A pharmaceutical composition comprising an isolated antigen binding protein of any one of embodiments 1-57, a polypeptide molecule of any one of embodiments 58-59, an immunoconjugate of embodiment 60, a nucleic acid molecule of embodiment 61, a vector of embodiment 62, and/or a cell of embodiment 63, and optionally a pharmaceutically acceptable carrier.
65. A method of making the isolated antigen binding protein of any one of embodiments 1-57, the method comprising culturing the cell of embodiment 63 under conditions such that the antigen binding protein is expressed.
66. Use of an isolated antigen binding protein of any one of embodiments 1-57, a polypeptide molecule of any one of embodiments 58-59, an immunoconjugate of embodiment 60, a nucleic acid molecule of embodiment 61, a vector of embodiment 62, a cell of embodiment 63, and/or a pharmaceutical composition of embodiment 64 in the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.
67. The use according to embodiment 65, wherein the disease and/or condition and its complications are caused or mediated by Staphylococcus aureus.
68. The use according to any one of embodiments 66-67, wherein the disease and/or condition comprises a complication of a disease and/or condition caused or mediated by Staphylococcus aureus.
69. The use according to any one of embodiments 66-68, wherein the disease and/or condition comprises sepsis and/or bacteremia.
70. The isolated antigen binding protein of any one of embodiments 1-57, the polypeptide molecule of any one of embodiments 58-59, the immunoconjugate of embodiment 60, the nucleic acid molecule of embodiment 61, the vector of embodiment 62, the cell of embodiment 63, and/or the pharmaceutical composition of embodiment 64, alone or in combination with other drugs.
71. A method of detecting staphylococcus aureus leukocidin in a sample, the method comprising administering an isolated antigen-binding protein of any one of embodiments 1-57, a polypeptide molecule of any one of embodiments 58-59, an immunoconjugate of embodiment 60, a nucleic acid molecule of embodiment 61, a vector of embodiment 62, a cell of embodiment 63, and/or a pharmaceutical composition of embodiment 64.
72. A kit for detecting staphylococcus aureus leukocidin in a sample, the kit comprising administering an isolated antigen-binding protein of any one of embodiments 1-57, a polypeptide molecule of any one of embodiments 58-59, an immunoconjugate of embodiment 60, a nucleic acid molecule of embodiment 61, a vector of embodiment 62, a cell of embodiment 63, and/or a pharmaceutical composition of embodiment 64.
73. Use of the isolated antigen binding protein of any one of embodiments 1-57, the polypeptide molecule of any one of embodiments 58-59, the immunoconjugate of embodiment 60, the nucleic acid molecule of embodiment 61, the vector of embodiment 62, the cell of embodiment 63, and/or the pharmaceutical composition of embodiment 64 in the preparation of a kit for detecting the presence and/or amount of staphylococcus aureus leukocidin in a sample.
Without wishing to be bound by any theory, the following examples are only intended to illustrate the antigen binding proteins, preparation methods and uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.
Examples
EXAMPLE 1 recombinant expression of Staphylococcus aureus leukocidin
Constructing plasmids of recombinant staphylococcus aureus HlA (Uniport KB-P0A074, 30-309, His label is added at C end), staphylococcus aureus HlB (Uniport KB-P0A077, 26-325, His label is added at C end), staphylococcus aureus LukD (Uniport KB-O54082, 27-327, His label is added at C end) and staphylococcus aureus LukF (UniProt KB-P31715, 26-323, His label is added at C end), transforming the recombinant plasmids into BL21(DE3) pLysS competent cells, performing protein expression by using an escherichia coli expression system, and purifying supernatant after bacterial liquid is subjected to ultrasonic lysis by Ni-NTA affinity chromatography to obtain HlGA, HlgB, LukD and LukF proteins.
Example 2 Mixed immunization of Balb/C mice with HlgA and LukF antigens
The prepared HlgA and LukF proteins are adopted to carry out subcutaneous multipoint immunization on 5 Balb/C mice by a subcutaneous multipoint immunization method.
The immunization protocol is as follows:
Figure BDA0003510874290000441
example 3 fusion of splenocytes from immunized mice with myeloma cells and screening for antigen binding proteins with Cross-affinities
Myeloma cells P3X63Ag8.653 were fused with splenocytes from immunized HlgA, LukF mice using PEG 3350. The fused hybridoma cells were inoculated into a 96-well plate and cultured. After 24 and 48 hours, HAT medium and HT medium were added to the cells, respectively, to select hybridoma cells. After 12 days of culture, 100. mu.L of cell culture supernatant was taken. Hybridoma clone cells capable of producing anti-HlgA, LukF antigen binding protein were screened by ELISA assay. Positive hybridoma cell masses were added to a 96-well plate containing mouse spleen cells by the limiting dilution method, and monoclonal cells were labeled. Monoclonal cells capable of secreting anti-HlgA, LukF antigen binding proteins were further screened by ELISA.
Example 4 acquisition of murine antigen binding protein sequences with Cross-affinity
Extracting 1 × 10 with RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.) 6 The cell secretes monoclonal cell total RNA against the HlgA, LukF antigen binding protein. And the total RNA was inverted into cDNA using a reverse transcription kit (Takara). Amplifying cDNA fragments by using primers (Anke Krebber.1997), purifying PCR products by using a DNA product purification kit (Beijing Tiangen Biotechnology Co., Ltd.), connecting the PCR products to a T vector by using a connection kit (Beijing Optimus department Biotechnology Co., Ltd.), transforming the T vector into competent cells, and entrusting Shanghai's work to perform sequence determination to finally obtain the sequences of the heavy chain variable region and the light chain variable region of the murine antigen binding protein 17C 7.
17C7 heavy chain variable region (17C7Hv) SEQ ID NO 30
EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENDI GEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYFCNALWGGVLDYWGQGTSVTVSA
17C7 light chain variable region (17C7Lv) SEQ ID NO 36
DVVMTQTPLSLPVSLGDQASISCRSSQSLVNSNGNTYLHWYLQKPGQSPNLLIYRVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKR
Wherein the underlined parts indicate the CDR regions defined using the Kabat method.
Example 5 detection of binding of antigen binding proteins to various Staphylococcus aureus leukocidins by ELISA
Leukocidin was diluted to 1. mu.g/mL with PBS buffer and 100. mu.L per well was coated in a 96-well plate (Thermo) and incubated overnight at 4 ℃; the next day the 96-well plate was removed and washed with PBST (containing 0.5% PBS) and the residual water was spun off thoroughly after 1min of each soak. Respectively adding 200 mu L of PBST containing 5% BSA into sample wells, and sealing at 37 ℃ for 1 h; the plates were then washed with PBST and the wells were spin dried. 100. mu.L of each sample to be tested was added to each 96-well plate and incubated overnight at 4 ℃. After the 96-well plate was taken out and washed with PBST, a secondary antibody of 100. mu.L of HRP antibody (Beijing ancient Biotech Co., Ltd.) was added to each well, and incubated at 37 ℃ for 1 hour. Washing with PBST for 5 times, adding 100 μ L of Substrate Solution (Invitrogen) per well, and incubating at 37 deg.C for 10 min; after the reaction was terminated by adding 50. mu.L of 1mol of sulfuric acid to each well, absorbance was measured at a wavelength of 450nm using a microplate reader (Multiskcin FC, Thermo).
The results are shown in FIG. 1, and show that the antigen-binding proteins described herein can bind to leukocidins HlgA, HlgB, LukD and LukF. EC in combination with HlgA 50 EC with a value of 0.076. mu.g/mL, bound to HlgB 50 EC binding to LukD with a value of 0.051. mu.g/mL 50 EC with LukF binding with a value of 0.038. mu.g/mL 50 The value was 0.053. mu.g/mL.
EXAMPLE 6 in vitro neutralization of various Staphylococcus aureus leukocidin Activity assays
The leukocidin was diluted stepwise by 5. mu.g/mL and used as a stock solution. Mu.g of the leukocidin combination (HlgAB, HlgA + LukD, HlgA + LukF) was added to a 96-well plate with leukocidin antigen-binding protein (0.25-4. mu.g), PBS and 5% rabbit red blood cells and mixed well. The whole was then incubated at 37 ℃ for 1h and centrifuged to obtain a supernatant. Transfer 100. mu.l of supernatant to a new 96-well plate and measure OD405 absorbance with a microplate reader.
The results are shown in FIG. 2, and show that 4. mu.g of 17C7 antigen-binding protein has a complete neutralizing effect on 4. mu.g of the combined bi-component toxins HlgAB (A), HlgA + LukD (B), and HlgA + LukF (C).
Example 7 protective Effect of murine antigen binding protein 17C7 on mice infected with Staphylococcus aureus
Taking out a methicillin-resistant staphylococcus aureus USA300 seed bank, activating for 2 generations on a TSA solid culture medium plate, and inoculating into a TSB liquid culture medium for overnight culture. The cells were collected by centrifugation and resuspended in PBS for use.The C57BL/6J mice were randomly divided into a model group and an antigen-binding protein drug treatment group according to body weight. Each group of mice was infected via tail vein with USA 3006X 10 7 CFU/mouse, and corresponding drug treatment was given 2h after infection; the dose of antigen-binding protein and control antibody AR-301 administered was 15mg/kg (tail vein injection). Among them, AR-301 is a fully human antibody drug targeting Staphylococcus aureus alpha-toxin (Salvecin, tosatoxumab) developed by Aridis pharmaceutical company. The survival of each group of mice was observed and recorded.
According to the experimental result (figure 3) of the bacteremia caused by the staphylococcus aureus given to the mice, the antigen binding protein 17C7 has a remarkable protective effect on a mouse bacteremia model caused by the staphylococcus aureus and is superior to the control antibody AR-301.
Example 8 humanization of murine antigen binding protein 17C7
Through NCBI database comparison (https:// www.ncbi.nlm.nih.gov/igblast /), selecting a germline sequence closest to the 17C7 antigen binding protein as a template, humanizing the 17C7, and optimizing post-translational modification sites of the antigen binding protein to obtain humanized sequences as follows.
Humanized antigen binding protein 17C7 heavy chain variable region:
17C7H3v SEQ ID NO:31
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWIGWIDPENDIGEYAPKFQGRATMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSS
17C7H4v SEQ ID NO:32
EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWIGWIDPENDIGEYAPKFQGRATMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSS
17C7H12v SEQ ID NO:33
EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWMGWIDPENDIGEYAPKFQGRATMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSS
17C7H13v SEQ ID NO:34
EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWIGWIDPENDIGEYAPKFQGRVTMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSS
17C7H17v SEQ ID NO:35
EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWMGWIDPENDIGEYAPKFQGRVTMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSS
humanized antigen binding protein 17C7 light chain variable region:
17C7L4v SEQ ID NO:37
DVVMTQTPLSSPVTLGQPASISCRSSQSLVASNGNTYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKR
17C7L5v SEQ ID NO:38
DVVMTQTPLSSPVTLGQPASISCRSSQSLVASNRNTYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKR
17C7L6v SEQ ID NO:39
DVVMTQTPLSSPVTLGQPASISCRSSQSLVASNRATYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKR
17C7L7v SEQ ID NO:40
DIVMTQTPLSSPVTLGQPASISCRSSQSLVASNRNTYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKR
17C7L8v SEQ ID NO:41
DIVMTQTPLSSPVTLGQPASISCRSSQSLVASNRATYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKR
wherein, underlined parts indicate CDR regions defined using the Kabat method.
Example 917 recombinant expression and purification of humanized antigen binding protein of C7
The C-terminal of the 17C7 heavy chain variable region was ligated to the N-terminal of the constant region of human immunoglobulin γ 1(IgG1) and constructed into pcDNA3.4 vector to obtain the 17C7 chimeric antibody heavy chain expression vector pcDNA3.4-17C 7H. The C-terminus of the 17C7 light chain variable region was ligated to the N-terminus of the light chain kappa constant region and constructed into pcDNA3.4 vector to obtain the 17C7 chimeric antibody light chain expression vector pcDNA3.4-17C 7L.
17C7 chimeric antibody heavy chain (17C7H) sequence SEQ ID NO:55
EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENDIGEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYFCNALWGGVLDYWGQGTSVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
17C7 chimeric antibody light chain (17C7L) sequence SEQ ID NO:56
DVVMTQTPLSLPVSLGDQASISCRSSQSLVNSNGNTYLHWYLQKPGQSPNLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
The C-terminal of the 17C7 humanized heavy chain variable region 17C7H3v, 17C7H4v, 17C7H12v,17C7H 13v and 17C7H17v were linked to the N-terminal of the constant region of human immunoglobulin gamma 1(IgG1), respectively, and constructed into pcDNA3.4 vector to obtain humanized heavy chain expression vectors pcDNA3.4-17C7H3, pcDNA3.4-17C7H4, pcDNA3.4-17C7H12, pcDNA3.4-17C7H13 and pcDNA3.4-17C7H 17.
Humanized antigen binding protein 17C7 heavy chain sequence:
17C7H3 SEQ ID NO:57
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWIGWIDPENDIGEYAPKFQGRATMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
17C7H4 SEQ ID NO:58
EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWIGWIDPENDIGEYAPKFQGRATMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
17C7H12 SEQ ID NO:59
EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWMGWIDPENDIGEYAPKFQGRATMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
17C7H13 SEQ ID NO:60
EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWIGWIDPENDIGEYAPKFQGRVTMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
17C7H17 SEQ ID NO:61
EVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQGLEWMGWIDPENDIGEYAPKFQGRVTMTADTSISTAYMELSRLRSDDTAVYYCNALWGGVLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
respectively connecting the C ends of the 17C7 humanized light chain variable region 17C7L4v, 17C7L5v, 17C7L6v, 17C7L7v and 17C7L8v with the N end of a light chain kappa constant region and constructing the same into a pcDNA3.4 vector to obtain humanized light chain expression vectors pcDNA3.4-17C7L4, pcDNA3.4-17C7L5, pcDNA3.4-17C7L6, pcDNA3.4-17C7L7 and pcDNA3.4-17C7L 8;
humanized antigen binding protein 17C7 light chain sequence:
17C7L4 SEQ ID NO:62
DVVMTQTPLSSPVTLGQPASISCRSSQSLVASNGNTYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
17C7L5 SEQ ID NO:63
DVVMTQTPLSSPVTLGQPASISCRSSQSLVASNRNTYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
17C7L6 SEQ ID NO:64
DVVMTQTPLSSPVTLGQPASISCRSSQSLVASNRATYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
17C7L7 SEQ ID NO:65
DIVMTQTPLSSPVTLGQPASISCRSSQSLVASNRNTYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
17C7L8 SEQ ID NO:66
DIVMTQTPLSSPVTLGQPASISCRSSQSLVASNRATYLHWYQQRPGQPPRLLIYRVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
the heavy chain expression vector pcDNA3.4-17C7H, pcDNA3.4-17C7H3, pcDNA3.4-17C7H4, pcDNA3.4-17C7H12, pcDNA3.4-17C7H13 or pcDNA3.4-17C7H17 and the light chain expression vector pcDNA3.4-17C7L, pcDNA3.4-17C7L4, pcDNA3.4-17C7L5, pcDNA3.4-17C7L6, pcDNA3.4-17C7L7 or pcDNA3.4-17C7L8 are co-transfected with expi293F cell to express magnetic beads and purified by ProteinA to obtain purified chimeric antibody 17C7HL and humanized antibody. For example, after the heavy chain expression vector pcDNA3.4-17C7H and the light chain expression vector pcDNA3.4-17C7L5 are co-transfected with cells for expression and purification, the humanized antibody 17C7HL5 is obtained; for example, after the heavy chain expression vector pcDNA3.4-17C7H and the light chain expression vector pcDNA3.4-17C7L6 are co-transfected with cells for expression and purification, the humanized antibody 17C7HL6 is obtained; for example, after the heavy chain expression vector pcDNA3.4-17C7H and the light chain expression vector pcDNA3.4-17C7L7 are co-transfected and cell is expressed and purified, the humanized antibody 17C7HL7 is obtained; for example, after the heavy chain expression vector pcDNA3.4-17C7H and the light chain expression vector pcDNA3.4-17C7L8 are co-transfected and cell is expressed and purified, the humanized antibody 17C7HL8 is obtained; for example, after the heavy chain expression vector pcDNA3.4-17C7H4 and the light chain expression vector pcDNA3.4-17C7L are co-transfected and cell is expressed and purified, the humanized antibody 17C7H4L is obtained; for example, after the heavy chain expression vector pcDNA3.4-17C7H12 and the light chain expression vector pcDNA3.4-17C7L are co-transfected and cell is expressed and purified, the humanized antibody 17C7H12L is obtained; for example, after the heavy chain expression vector pcDNA3.4-17C7H13 and the light chain expression vector pcDNA3.4-17C7L are co-transfected and cell is expressed and purified, the humanized antibody 17C7H13L is obtained; for example, after the heavy chain expression vector pcDNA3.4-17C7H17 and the light chain expression vector pcDNA3.4-17C7L are co-transfected and cell is expressed and purified, the humanized antibody 17C7H17L is obtained; for example, after the cell cotransfected by the heavy chain expression vector pcDNA3.4-17C7H13 and the light chain expression vector pcDNA3.4-17C7L8 is expressed and purified, the humanized antibody 17C7H13L8 is obtained; for example, after the cell cotransfected by the heavy chain expression vector pcDNA3.4-17C7H17 and the light chain expression vector pcDNA3.4-17C7L8 is expressed and purified, the humanized antibody 17C7H17L8 is obtained; for example, after the cell cotransfected by the heavy chain expression vector pcDNA3.4-17C7H4 and the light chain expression vector pcDNA3.4-17C7L8 is expressed and purified, the humanized antibody 17C7H4L8 is obtained; for example, after the cell cotransfected by the heavy chain expression vector pcDNA3.4-17C7H12 and the light chain expression vector pcDNA3.4-17C7L8 is expressed and purified, the humanized antibody 17C7H12L8 is obtained; .
Example 10 detection of the affinity of the heavy and light chains of the different 17C7 humanized antigen binding proteins to the leukocidin HlgB
The recombinant leukocidin HlgB was diluted to 1. mu.g/mL with PBS buffer and 100. mu.L per well was coated in a 96-well plate (Thermo) and incubated overnight at 4 ℃; the next day the 96-well plate was removed and washed with PBST (containing 0.5% PBS) and the residual water was spun off thoroughly after 1min of each soak. Adding 200 μ l PBST containing 5% BSA into sample wells, and sealing at 37 deg.C for 1 h; the plates were then washed with PBST and the wells were spin dried. 100 μ L of 17C 74 ℃ was added to each 96-well plate and incubated overnight. After the 96-well plate was taken out and washed with PBST, 100. mu.L of HRP antibody (Beijing Dingguo Biotech Co., Ltd.) was added to each well, and incubated at 37 ℃ for 1 hour. Washing with PBST for 5 times, adding 100 μ L of Substrate Solution (Invitrogen) per well, and incubating at 37 deg.C for 10 min; after the reaction was terminated by adding 50. mu.L of 1mol of sulfuric acid to each well, absorbance was measured at a wavelength of 450nm using a microplate reader (Multiskcin FC, Thermo).
The results of affinity measurements of antigen-binding protein 17C7 and light chain humanized antigen-binding proteins 17C7HL5, 17C7HL6, 17C7HL7, 17C7HL8 (in which the heavy chain variable region is selected from the group consisting of 17C7 murine sequences and the light chain variable regions are respectively selected from the group consisting of L5, L6, L7, L8 sequences in example 8) and leukocidin HlgB are shown in fig. 4, and indicate that leukocidin HlgB has strong affinity with light chain humanized monoclonal antibodies 17C7HL5, 17C7HL6, 17C7HL7, 17C7HL8 in which leukocidin HlgB binds to EC 17C7HL5 50 EC with a value of 0.009. mu.g/mL, in combination with 17C7HL6 50 EC with a value of 0.012. mu.g/mL, in combination with 17C7HL7 50 EC with value of 0.016. mu.g/mL, combined with 17C7HL8 50 The value was 0.038. mu.g/mL.
The results of the affinity detection of antigen-binding protein 17C7 and heavy chain humanized antigen-binding proteins 17C7H4L, 17C7H12L, 17C7H13L, 17C7H17L and leukocidin HlgB are shown in fig. 5, and indicate that leukocidin HlgB has a strong affinity with light chain humanized monoclonal antibodies 17C7H4L, 17C7H12L, 17C7H13L, 17C7H17L, wherein leukocidin HlgB binds to 17C7H4LEC of (1) 50 EC with a value of 0.014. mu.g/mL, binding to 17C7H12L 50 EC with a value of 0.03. mu.g/mL, binding to 17C7H13L 50 EC with a value of 0.02. mu.g/mL, in combination with 17C7H17L 50 The value was 0.17. mu.g/mL.
The results of the affinity assays for antigen binding protein 17C7 and humanized antigen binding proteins 17C7H4L8, 17C7H12L8, 17C7H13L8, 17C7H17L8 and leukocidin HlgB are shown in fig. 6, and indicate that leukocidin HlgB has a strong affinity for light chain humanized monoclonal antibodies 17C7H4L8, 17C7H12L8, 17C7H13L8, 17C7H17L8, wherein leukocidin HlgB binds to EC 17C7H4L8 50 EC with a value of 0.01. mu.g/mL, binding to 17C7H12L8 50 EC with a value of 0.019. mu.g/mL in combination with 17C7H13L8 50 EC value of 0.024 μ g/mL in combination with 17C7H17L8 50 The value was 0.035. mu.g/mL.
Example 1117C 7 affinity assay for humanized antigen binding proteins and various Staphylococcus aureus leukocidins
Leukocidin was diluted to 1. mu.g/mL with PBS buffer and 100. mu.L per well was coated in a 96-well plate (Thermo) and incubated overnight at 4 ℃; the next day the 96-well plate was removed and washed with PBST (containing 0.5% PBS) and the residual water was spun off thoroughly after 1min of each soak. Adding 200 μ l PBST containing 5% BSA into sample wells, and sealing at 37 deg.C for 1 h; the plates were then washed with PBST and the wells were spin dried. 100 μ L of 17C 74 ℃ was added to each 96-well plate and incubated overnight. After the 96-well plate was taken out and washed with PBST, a secondary antibody of 100. mu.L of HRP antibody (Beijing ancient Biotech Co., Ltd.) was added to each well, and incubated at 37 ℃ for 1 hour. Washing with PBST for 5 times, adding 100 μ L of Substrate Solution (Invitrogen) per well, and incubating at 37 deg.C for 10 min; after the reaction was terminated by adding 50. mu.L of 1mol of sulfuric acid to each well, absorbance was measured at a wavelength of 450nm using a microplate reader (Multiskcin FC, Thermo).
The results of the detection of the binding of antigen-binding protein 17C7 and humanized antigen-binding protein 17C7H17L8 to four leukocidins, HlgA (A), HlgB (B), LukD (C) and LukF (D), are shown in FIG. 7, and indicate that the four antigens have strong affinity with humanized monoclonal antibody 17C7H17L8, wherein the EC bound to HlgA 50 EC with a value of 0.034. mu.g/mL, in combination with HlgB 50 EC with a value of 0.055. mu.g/mL, binding to LukD 50 EC with a value of 0.036. mu.g/mL, in combination with LukF 50 The value was 0.043. mu.g/mL.
Example 12 Staphylococcus aureus leukocidin HlgAB causes mouse sepsis model replication and evaluation of the pharmacodynamic effects of the humanized antigen-binding protein 17C7H17L8
Different doses of antigen binding protein were previously injected into C57BL/6 mice, and 10. mu.g of Staphylococcus aureus leukocidin HlgAB (containing 5. mu.g of HlgA and 5. mu.g of HlgB) was injected into the tail vein after 30min, using human IgG as a control. Mice were observed for survival time.
The results of testing the therapeutic effect of the humanized antigen binding protein 17C7H17L8 using a mouse model of sepsis caused by s.aureus α -toxin are shown in fig. 8. The results indicate that the humanized antigen binding protein 17C7H17L8(1.5mg/kg) described herein has full protective effect.
Example 13 Staphylococcus aureus infection of mice results in bacteremia model replication and evaluation of the pharmacodynamic effects of the humanized antigen-binding protein 17C7H17L8
Staphylococcus aureus USA300 was activated from a-80 ℃ refrigerator, activated for two passages in TSA medium, and inoculated to 2ml TSB for 12 h. After 3 washes with PBS, mice were infected with 6X 10 of the tail vein 7 And (4) CFU. The antigen binding protein was injected 2h after infection and mice were observed for survival using human IgG as a control.
The results of evaluating the anti-infective pharmacodynamic effects of the humanized antigen-binding protein 17C7H17L8 using a mouse bacteremia model caused by staphylococcus aureus are shown in fig. 9. The result shows that the humanized antigen binding protein 17C7H17L8 has a remarkable protective effect on a mouse bacteremia model caused by staphylococcus aureus.
Sequence listing
<110> Saji biological (Suzhou) Co., Ltd
<120> antigen binding proteins cross neutralizing staphylococcus aureus toxins and uses thereof
<130> 0193-PA-008
<160> 66
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 1
Leu Trp Gly Gly Val Leu Asp Tyr
1 5
<210> 2
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 2
Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe Gln
1 5 10 15
Gly
<210> 3
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 3
Asp Tyr Tyr Met His
1 5
<210> 4
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 4
Ser Gln Ser Thr His Val Pro Tyr Thr
1 5
<210> 5
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 5
Arg Val Ser Asn Arg Phe Ser
1 5
<210> 6
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L LCDR1
<400> 6
Arg Ser Ser Gln Ser Leu Val Asn Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210> 7
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L4 LCDR1
<400> 7
Arg Ser Ser Gln Ser Leu Val Ala Ser Asn Gly Asn Thr Tyr Leu His
1 5 10 15
<210> 8
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L5、17C7L7 LCDR1
<400> 8
Arg Ser Ser Gln Ser Leu Val Ala Ser Asn Arg Asn Thr Tyr Leu His
1 5 10 15
<210> 9
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L6、17C7L8 LCDR1
<400> 9
Arg Ser Ser Gln Ser Leu Val Ala Ser Asn Arg Ala Thr Tyr Leu His
1 5 10 15
<210> 10
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H H-FR1
<400> 10
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys
20 25 30
<210> 11
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H H-FR2
<400> 11
Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly
1 5 10
<210> 12
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H H-FR3
<400> 12
Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln
1 5 10 15
Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys Asn Ala
20 25 30
<210> 13
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H H-FR4
<400> 13
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ala
1 5 10
<210> 14
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H3 H-FR1
<400> 14
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys
20 25 30
<210> 15
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H3、17C7H4、17C7H13 H-FR2
<400> 15
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly
1 5 10
<210> 16
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H3、17C7H4、17C7H12 H-FR3
<400> 16
Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu
1 5 10 15
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Asn Ala
20 25 30
<210> 17
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H3、17C7H4、17C7H12、17C7H13、17C7H17 H-FR4
<400> 17
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 18
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H4、17C7H12、17C7H13、17C7H17 H-FR1
<400> 18
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys
20 25 30
<210> 19
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H12、17C7H17 H-FR2
<400> 19
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
1 5 10
<210> 20
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H13、17C7H17 H-FR3
<400> 20
Arg Val Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu
1 5 10 15
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Asn Ala
20 25 30
<210> 21
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L L-FR1
<400> 21
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys
20
<210> 22
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L L-FR2
<400> 22
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Asn Leu Leu Ile Tyr
1 5 10 15
<210> 23
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L L-FR3
<400> 23
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
20 25 30
<210> 24
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L L-FR4
<400> 24
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
1 5 10
<210> 25
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L4、17C7L5、17C7L6 L-FR1
<400> 25
Asp Val Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys
20
<210> 26
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L4、17C7L5、17C7L6、17C7L7、17C7L8 L-FR2
<400> 26
Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr
1 5 10 15
<210> 27
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L4、17C7L5、17C7L6、17C7L7、17C7L8 L-FR3
<400> 27
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr
1 5 10 15
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
20 25 30
<210> 28
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L4、17C7L5、17C7L6、17C7L7、17C7L8 L-FR4
<400> 28
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
1 5 10
<210> 29
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L7、17C7L8 L-FR1
<400> 29
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys
20
<210> 30
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7Hv VH
<400> 30
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ala
115
<210> 31
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H3v VH
<400> 31
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 32
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H4v VH
<400> 32
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 33
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H12v VH
<400> 33
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 34
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H13v VH
<400> 34
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 35
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H17v VH
<400> 35
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 36
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7Lv VL
<400> 36
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Asn Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Asn Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 37
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L4v VL
<400> 37
Asp Val Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 38
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L5v VL
<400> 38
Asp Val Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 39
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L6v VL
<400> 39
Asp Val Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Arg Ala Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 40
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L7v VL
<400> 40
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 41
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L8v VL
<400> 41
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Arg Ala Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 42
<211> 330
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IgG1 heavy chain constant region
<400> 42
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 43
<211> 106
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Ig kappa light chain constant region
<400> 43
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 44
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L LCDR1; 17C7L4 LCDR1; 17C7L5、17C7L7 LCDR1; 17C7L6、17C7L8 LCDR1
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa = Ala or Asn
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Gly or Arg
<220>
<221> misc_feature
<222> (12)..(12)
<223> Xaa = Ala or Asn
<400> 44
Arg Ser Ser Gln Ser Leu Val Xaa Ser Asn Xaa Xaa Thr Tyr Leu His
1 5 10 15
<210> 45
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H H-FR1; 17C7H3 H-FR1; 17C7H4、17C7H12、17C7H13、17C7H17 H-FR1
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa = Glu or Gln
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa = Gln or Val
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Leu or Val
<220>
<221> misc_feature
<222> (12)..(12)
<223> Xaa = Lys or Val
<220>
<221> misc_feature
<222> (13)..(13)
<223> Xaa = Lys or Arg
<220>
<221> misc_feature
<222> (14)..(14)
<223> Xaa = Pro or Ser
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa = Leu or Val
<220>
<221> misc_feature
<222> (23)..(23)
<223> Xaa = Lys or Thr
<400> 45
Xaa Val Gln Leu Xaa Gln Ser Gly Ala Glu Xaa Xaa Xaa Xaa Gly Ala
1 5 10 15
Ser Val Lys Xaa Ser Cys Xaa Ala Ser Gly Phe Asn Ile Lys
20 25 30
<210> 46
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H H-FR2; 17C7H3、17C7H4、17C7H13 H-FR2; 17C7H12、17C7H17 H-FR2
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa = Lys or Arg
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa = Ala or Arg
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa = Glu or Gly
<220>
<221> misc_feature
<222> (13)..(13)
<223> Xaa = Ile or Met
<400> 46
Trp Val Xaa Gln Xaa Pro Xaa Gln Gly Leu Glu Trp Xaa Gly
1 5 10
<210> 47
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H H-FR3; 17C7H3、17C7H4、17C7H12 H-FR3; 17C7H13、17C7H17 H-FR3
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa = Lys or Arg
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa = Ala or Val
<220>
<221> misc_feature
<222> (10)..(10)
<223> Xaa = Ile or Ser
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Asn or Ser
<220>
<221> misc_feature
<222> (15)..(15)
<223> Xaa = Leu or Met
<220>
<221> misc_feature
<222> (16)..(16)
<223> Xaa = Glu or Gln
<220>
<221> misc_feature
<222> (19)..(19)
<223> Xaa = Arg or Ser
<220>
<221> misc_feature
<222> (21)..(21)
<223> Xaa = Arg or Thr
<220>
<221> misc_feature
<222> (23)..(23)
<223> Xaa = Asp or Glu
<220>
<221> misc_feature
<222> (29)..(29)
<223> Xaa = Phe or Tyr
<400> 47
Xaa Xaa Thr Met Thr Ala Asp Thr Ser Xaa Xaa Thr Ala Tyr Xaa Xaa
1 5 10 15
Leu Ser Xaa Leu Xaa Ser Xaa Asp Thr Ala Val Tyr Xaa Cys Asn Ala
20 25 30
<210> 48
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H H-FR4; 17C7H3、17C7H4、17C7H12、17C7H13、17C7H17 H-FR4
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa = Leu or Ser
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Ala or Ser
<400> 48
Trp Gly Gln Gly Thr Xaa Val Thr Val Ser Xaa
1 5 10
<210> 49
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L L-FR1; 17C7L4、17C7L5、17C7L6 L-FR1; 17C7L7、17C7L8 L-FR1
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa = Ile or Val
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Leu or Ser
<220>
<221> misc_feature
<222> (14)..(14)
<223> Xaa = Ser or Thr
<220>
<221> misc_feature
<222> (17)..(17)
<223> Xaa = Asp or Gln
<220>
<221> misc_feature
<222> (18)..(18)
<223> Xaa = Pro or Gln
<400> 49
Asp Xaa Val Met Thr Gln Thr Pro Leu Ser Xaa Pro Val Xaa Leu Gly
1 5 10 15
Xaa Xaa Ala Ser Ile Ser Cys
20
<210> 50
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L4、17C7L5、17C7L6、17C7L7、17C7L8 L-FR2; 17C7L L-FR2
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa = Leu or Gln
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa = Lys or Arg
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa = Pro or Ser
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Asn or Arg
<400> 50
Trp Tyr Xaa Gln Xaa Pro Gly Gln Xaa Pro Xaa Leu Leu Ile Tyr
1 5 10 15
<210> 51
<211> 32
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L L-FR3; 17C7L4、17C7L5、17C7L6、17C7L7、17C7L8 L-FR3
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Ala or Ser
<220>
<221> misc_feature
<222> (27)..(27)
<223> Xaa = Leu or Val
<220>
<221> misc_feature
<222> (31)..(31)
<223> Xaa = Phe or Tyr
<400> 51
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Xaa Gly Thr Asp Phe Thr
1 5 10 15
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Xaa Gly Val Tyr Xaa Cys
20 25 30
<210> 52
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L L-FR4; 17C7L4、17C7L5、17C7L6、17C7L7、17C7L8 L-FR4
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa = Gly or Gln
<400> 52
Phe Gly Xaa Gly Thr Lys Leu Glu Ile Lys Arg
1 5 10
<210> 53
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H VH; 17C7H3 VH; 17C7H4 VH; 17C7H12 VH; 17C7H13 VH; 17C7H17 VH
<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa = Glu or Gln
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa = Gln or Val
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Leu or Val
<220>
<221> misc_feature
<222> (12)..(12)
<223> Xaa = Lys or Val
<220>
<221> misc_feature
<222> (13)..(13)
<223> Xaa = Lys or Arg
<220>
<221> misc_feature
<222> (14)..(14)
<223> Xaa = Pro or Ser
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa = Leu or Val
<220>
<221> misc_feature
<222> (23)..(23)
<223> Xaa = Lys or Thr
<220>
<221> misc_feature
<222> (38)..(38)
<223> Xaa = Lys or Arg
<220>
<221> misc_feature
<222> (40)..(40)
<223> Xaa = Ala or Arg
<220>
<221> misc_feature
<222> (42)..(42)
<223> Xaa = Glu or Gly
<220>
<221> misc_feature
<222> (48)..(48)
<223> Xaa = Ile or Met
<220>
<221> misc_feature
<222> (67)..(67)
<223> Xaa = Lys or Arg
<220>
<221> misc_feature
<222> (68)..(68)
<223> Xaa = Ala or Val
<220>
<221> misc_feature
<222> (76)..(76)
<223> Xaa = Ile or Ser
<220>
<221> misc_feature
<222> (77)..(77)
<223> Xaa = Asn or Ser
<220>
<221> misc_feature
<222> (81)..(81)
<223> Xaa = Leu or Met
<220>
<221> misc_feature
<222> (82)..(82)
<223> Xaa = Glu or Gln
<220>
<221> misc_feature
<222> (85)..(85)
<223> Xaa = Arg or Ser
<220>
<221> misc_feature
<222> (87)..(87)
<223> Xaa = Arg or Thr
<220>
<221> misc_feature
<222> (89)..(89)
<223> Xaa = Asp or Glu
<220>
<221> misc_feature
<222> (95)..(95)
<223> Xaa = Phe or Tyr
<220>
<221> misc_feature
<222> (112)..(112)
<223> Xaa = Leu or Ser
<220>
<221> misc_feature
<222> (117)..(117)
<223> Xaa = Ala or Ser
<400> 53
Xaa Val Gln Leu Xaa Gln Ser Gly Ala Glu Xaa Xaa Xaa Xaa Gly Ala
1 5 10 15
Ser Val Lys Xaa Ser Cys Xaa Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Xaa Gln Xaa Pro Xaa Gln Gly Leu Glu Trp Xaa
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Xaa Xaa Thr Met Thr Ala Asp Thr Ser Xaa Xaa Thr Ala Tyr
65 70 75 80
Xaa Xaa Leu Ser Xaa Leu Xaa Ser Xaa Asp Thr Ala Val Tyr Xaa Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Xaa
100 105 110
Val Thr Val Ser Xaa
115
<210> 54
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L VL; 17C7L4 VL; 17C7L5 VL; 17C7L6 VL; 17C7L7 VL; 17C7L8 VL
<220>
<221> misc_feature
<222> (2)..(2)
<223> Xaa = Ile or Val
<220>
<221> misc_feature
<222> (11)..(11)
<223> Xaa = Leu or Ser
<220>
<221> misc_feature
<222> (14)..(14)
<223> Xaa = Ser or Thr
<220>
<221> misc_feature
<222> (17)..(17)
<223> Xaa = Asp or Gln
<220>
<221> misc_feature
<222> (18)..(18)
<223> Xaa = Pro or Gln
<220>
<221> misc_feature
<222> (31)..(31)
<223> Xaa = Ala or Asn
<220>
<221> misc_feature
<222> (34)..(34)
<223> Xaa = Gly or Arg
<220>
<221> misc_feature
<222> (35)..(35)
<223> Xaa = Ala or Asn
<220>
<221> misc_feature
<222> (42)..(42)
<223> Xaa = Leu or Gln
<220>
<221> misc_feature
<222> (44)..(44)
<223> Xaa = Lys or Arg
<220>
<221> misc_feature
<222> (48)..(48)
<223> Xaa = Pro or Ser
<220>
<221> misc_feature
<222> (50)..(50)
<223> Xaa = Asn or Arg
<220>
<221> misc_feature
<222> (72)..(72)
<223> Xaa = Ala or Ser
<220>
<221> misc_feature
<222> (88)..(88)
<223> Xaa = Leu or Val
<220>
<221> misc_feature
<222> (92)..(92)
<223> Xaa = Phe or Tyr
<220>
<221> misc_feature
<222> (105)..(105)
<223> Xaa = Gly or Gln
<400> 54
Asp Xaa Val Met Thr Gln Thr Pro Leu Ser Xaa Pro Val Xaa Leu Gly
1 5 10 15
Xaa Xaa Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Xaa Ser
20 25 30
Asn Xaa Xaa Thr Tyr Leu His Trp Tyr Xaa Gln Xaa Pro Gly Gln Xaa
35 40 45
Pro Xaa Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Xaa Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Xaa Gly Val Tyr Xaa Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Xaa Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 55
<211> 447
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7 chimeric antibody heavy chain (17C7H)
<400> 55
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 56
<211> 219
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7 chimeric antibody light chain (17C7L)
<400> 56
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Asn Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Asn Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 57
<211> 446
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H3 heavy chain
<400> 57
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 58
<211> 446
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H4 heavy chain
<400> 58
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 59
<211> 445
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H12 heavy chain
<400> 59
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 60
<211> 446
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H13 heavy chain
<400> 60
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 61
<211> 446
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7H17 heavy chain
<400> 61
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asp Pro Glu Asn Asp Ile Gly Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Leu Trp Gly Gly Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 62
<211> 219
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L4 light chain
<400> 62
Asp Val Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 63
<211> 219
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L5 light chain
<400> 63
Asp Val Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 64
<211> 219
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L6 light chain
<400> 64
Asp Val Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Arg Ala Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 65
<211> 219
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L7 light chain
<400> 65
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 66
<211> 219
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 17C7L8 light chain
<400> 66
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ala Ser
20 25 30
Asn Arg Ala Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro
35 40 45
Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215

Claims (10)

1. An isolated antigen binding protein comprising at least one CDR in the variable region VH of an antibody heavy chain, said VH comprising the amino acid sequence set forth in SEQ ID NO: 53.
2. The isolated antigen binding protein of claim 1, comprising HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence set forth in SEQ ID No. 3, said HCDR2 comprising the amino acid sequence set forth in SEQ ID No. 2, and said HCDR3 comprising the amino acid sequence set forth in SEQ ID No. 1.
3. The isolated antigen binding protein of any of claims 1-2, comprising at least one CDR in an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID No. 54.
4. The isolated antigen binding protein of any of claims 1-3, comprising LCDR1, LCDR2 and LCDR3, said LCDR1 comprising the amino acid sequence set forth in SEQ ID NO 44, said LCDR2 comprising SEQ ID NO
The amino acid sequence shown in ID NO. 5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 4.
5. A polypeptide molecule comprising the isolated antigen binding protein of any one of claims 1-4.
6. A nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-4 or the polypeptide molecule of claim 5.
7. A vector comprising the nucleic acid molecule of claim 6.
8. A cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.
9. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-4, the polypeptide molecule of claim 5, the nucleic acid molecule of claim 6, the vector of claim 7, and/or the cell of claim 8, and optionally a pharmaceutically acceptable carrier.
10. Use of the isolated antigen binding protein of any one of claims 1-4, the polypeptide molecule of claim 5, the nucleic acid molecule of claim 6, the vector of claim 7, the cell of claim 8, and/or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.
CN202210152322.2A 2021-02-20 2022-02-18 Antigen binding proteins that cross-neutralize staphylococcus aureus toxins and uses thereof Pending CN114957460A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110192894 2021-02-20
CN2021101928949 2021-02-20

Publications (1)

Publication Number Publication Date
CN114957460A true CN114957460A (en) 2022-08-30

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Country Status (1)

Country Link
CN (1) CN114957460A (en)

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