ISOLATED ANTIBODY OR ITS ANTIGEN BINDING FRAGMENT, COMPOSITION UNDERSTANDING THE ANTIBODY ANTIBODY O

专利摘要:
ISOLATED ANTIBODY OR ITS ANTIGEN BINDING FRAGMENT, COMPOSITION UNDERSTANDING THE ANTIGEN ANTIBODY OR FRAGMENT, KIT, USE OF AN EFFECTIVE AMOUNT OF AN ANTIBODY OR ITS ANTIGEN BINDING FRAGMENT, THE METHOD OF HYGIENIC HYGIENIC LOSS and METHOD OF INHIBITING THE FORMATION OF ALPHA-TOXIN OLIGOMERS. The present invention relates to compositions, methods of production and methods of use pertaining to anti-alpha toxin antibodies and fragments.
公开号:BR112013020086B1
申请号:R112013020086-3
申请日:2012-02-07
公开日:2020-12-08
发明作者:Bret Sellman；Christine Tkaczyk；Lei Hua；Partha Chowdhury；Reena Varkey；Melissa Damschroder；Li Peng；Vaheh Oganesyan；Jamese Johnson Hilliard
申请人:Medimmune, Llc；
IPC主号:

专利说明:

[0001] Divided from BR112013020086-3, deposited on 02/07/2012. Field
[0002] The technology relates in part to antibodies and, in certain embodiments, to antibodies that specifically bind to the alpha toxin (alpha toxin) of Staphylococcus aureus. Background
[0003] Staphylococcus aureus is a gram-positive, facultatively aerobic, cluster-forming bacterium that commonly colonizes the nose and skin of healthy human beings. Approximately 20-30% of the population is colonized with S. aureus at any given time. Staphylococcus aureus bacteria are also sometimes referred to as "staph", "Staph. Aureus", or "S. aureus", are considered opportunistic pathogens that cause minor infections (eg, pimples, pimples) and systemic infections.
[0004] Mucous and epidermal barriers (skin) normally protect against S. aureus infections. The interruption of these natural barriers due to injuries (for example, burns, trauma, surgical procedures and the like) dramatically increases the risk of infection. Diseases that compromise the immune system (eg, diabetes, end-stage kidney disease, cancer and the like) also increase the risk of infection. Opportunistic S. aureus infections can become serious, causing various diseases or conditions, non-limiting examples of which include bacteremia, cellulite, eyelid infections, food poisoning, joint infections, skin infections, scalded skin syndrome, syndrome of toxic shock, pneumonia, osteomyelitis, endocarditis, meningitis and abscess formation.
[0005] S. aureust can also cause infections and diseases in animals. For example, S. aureus is often associated with bovine mastitis.
[0006] S. aureus has several virulence factors, including capsular polysaccharides and protein toxins. A virulence factor often associated with S. aureus infection that is the major cytotoxic agent is alpha-toxin (also known as alpha-hemolysin or Hla), a hemolytic exoprotein and that forms pores, produced by most pathogenic strains S. aureus. The toxin forms heptameric pores in membranes of susceptible cells such as white blood cells, platelets, erythrocytes, peripheral blood monocytes, macrophages, keratinocytes, fibroblasts and endothelial cells. The formation of alpha-toxin pores often leads to cell dysfunction or lysis. Brief description summary
[0007] In certain embodiments, a purified or isolated antibody, or an antigen-binding fragment, antibody or fragment thereof that immunospecifically binds to an alpha-toxin polypeptide of Staphylococcus aureus is provided. The terms "alpha-toxin polypeptide", "alpha-toxin monomer" and "alpha-toxin oligomers (e.g. heptamer)" are referred to herein as "AT," "AT monomer" and "AT oligomer", respectively . The term "variable heavy chain" is referred to herein as "VH". The term "variable light chain" is referred to herein as "VL".
[0008] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide includes (a) a VH CDR1 comprising an amino acid sequence identical to, or containing, 1 , 2, 3 amino acid residue substitutions for SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 8, 11, 14, 17, 70 or 75; and (c) a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71 , 72, 76 or 78.
[0009] In particular embodiments, the isolated antibody or its antigen binding fragment comprises a VH CDR1, VH CDR2 and VH CDR3 comprising amino acid sequences identical to, or containing, 1, 2, 3 amino acid residue substitutions in each relative CDR SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 13, 14 and 15; SEQ ID NOs: 7, 17 and 18; SEQ ID NOs: 7, 8 and 16; SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7, 8 and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs: 69, 70 and 71; SEQ ID NOs: 7, 8 and 72; SEQ ID NOs: 69, 75 and 71; SEQ ID NOs: 69, 75 and 76; or SEQ ID NOs: 69, 70 and 71.
[00010] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide includes (a) a VL CDR1 comprising an amino acid sequence identical to, or containing, 1 , 2, 3 substitutions of amino acid residues related to SEQ ID NO: 1 or 4; (b) a VL CDR2 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 2, 5, 73 or 77; and (c) a VL CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 3, 6, 64, 68 or 74.
[00011] In particular embodiments, the isolated antibody or its antigen-binding fragment comprises a VL CDR1, VL CDR2 and VL CDR3 comprising amino acid sequences identical to, or containing, 1, 2, 3 substitutions of amino acid residues in each relative CDR SEQ ID NOs: 1, 2 and 3; SEQ ID NOs: 4, 5 and 6; SEQ ID NOs: 1, 2 and 64; SEQ ID NOs: 1, 2 and 68; SEQ ID NOs: 1, 73 and 74; or SEQ ID NOs: 1, 77 and 74.
[00012] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide includes a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprising amino acid sequences identical to, or containing, 1, 2, 3 amino acid residue substitutions in each relative CDR: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70 or 75; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 4; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74.
[00013] In particular embodiments, the isolated antibody or its antigen binding fragment comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprising amino acid sequences identical to, or containing, 1, 2, 3 substitutions of amino acid residues in each CDR for SEQ ID NOs: 7, 8, 9, 1, 2 and 3; SEQ ID NOs: 10, 11, 12, 1, 2 and 3; SEQ ID NOs: 13, 14, 15, 4, 5 and 6; SEQ ID NOs: 7, 17, 18, 1, 2 and 3; SEQ ID NOs: 7, 8, 16, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 64; SEQ ID NOs; 7, 8, 66, 1, 2 and 64; SEQ ID NOs: 7, 8, 67, 1, 2 and 68; SEQ ID NOs: 7, 8, 67, 1, 2 and 64; SEQ ID NOs: 7, 8, 78, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 68; SEQ ID NOs: 69, 70, 71, 1, 2 and 68; SEQ ID NOs: 7, 8, 72, 1, 73 and 74; SEQ ID NOs: 69, 75, 71, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 77 and 74; SEQ ID NOs: 69, 70, 71, 1, 77 and 74.
[00014] In some embodiments, a composition is provided comprising an isolated antibody or its antigen binding fragment which (i) includes a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs; that (ii) immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide in which the three CDRs of the VH chain domain include (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70 or 75; and (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78. In particular embodiments, the VH CDR1, VH CDR2 and VH CDR3 corresponds to SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 13, 14 and 15; SEQ ID NOs: 7, 17 and 18; SEQ ID NOs: 7, 8 and 16; SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7, 8 and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs: 69, 70 and 71; SEQ ID NOs: 7, 8 and 72; SEQ ID NOs: 69, 75 and 71; SEQ ID NOs: 69, 75 and 76; or SEQ ID NOs: 69, 70 and 71.
[00015] There is also provided, in certain embodiments, a composition comprising an isolated antibody or antigen-binding fragment thereof (i) including a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs; whereas (ii) immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide in which the three CDRs of the VL chain domain include (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 4; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and (c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74. In particular embodiments, VL CDR1, VL CDR2 and VL CDR3 correspond to SEQ ID NOs: 1, 2 and 3; SEQ ID NOs: 4, 5 and 6; SEQ ID NOs: 1, 2 and 64; SEQ ID NOs: 1, 2 and 68; SEQ ID NOs: 1, 73 and 74; or SEQ ID NOs: 1, 77 and 74.
[00016] In some embodiments, compositions are also provided which include an isolated antibody or its antigen-binding fragment which (i) immunospecifically binds to the alpha-toxin polypeptide of Staphylococcus aureus, (ii) containing a heavy chain variable domain comprising at least 90% identity with the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and (iii) comprises a light chain variable domain comprising at least 90% identity with the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.
[00017] In some embodiments, the isolated antibody or its antigen binding fragment includes a heavy chain variable domain of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51 , 53, 55, 57, 79, 59, 61, or 62 and a light chain variable domain of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54 , 56, 58, 60 or 63.
[00018] In particular embodiments, the isolated antibody or its antigen-binding fragment comprises a VH and a VL, where the VH and VL are identical or have at least 90%, 95% or 98% identity with the sequences of amino acid VH and VL of SEQ ID NOs: 20 and 19; SEQ ID NOs: 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27 SEQ ID NOs: 47 and 48; SEQ ID SEQ ID NOs: 51 and 52; SEQ ID SEQ ID NOs: 55 and 56; SEQ ID SEQ ID NOs: 61 and 58; SEQ ID NOs: 62 and 58; SEQ ID NOs: 62 and 63; SEQ ID NOs: 79 and 63.
[00019] In some embodiments, an isolated antibody or its antigen-binding fragment binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide and has one or more of the selected characteristics of the group consisting of: (a) a constant affinity (KD) of about 13 nM or less for the Staphylococcus aureus alpha-toxin polypeptide; (b) inhibition of the oligomerization of the Staphylococcus aureus alpha-toxin polypeptide by at least 50%, 60%, 70%, 80%, 90%, or 95%; and (c) reduction of A549 lysis (pulmonary epithelial) or THP-1 (monocyte) and red cell lysis by at least 50%, 60%, 70%, 80%, 90%, or 95%, where the antibody isolated or its antigen-binding fragment and the S. aureus toxin are present in a molar ratio of about 1: 1.
[00020] In some embodiments, the erythrocyte is a blood cell. In certain embodiments, the blood cell is a red blood cell. In some embodiments, lysis is determined by an in vitro hemolytic assay. In other embodiments, lysis is determined by an in vitro lactate dehydrogenase release assay. These tests are described below or can be performed routinely by a specialist in the field.
[00021] In some embodiments, the isolated antibody or its antigen-binding fragment includes a diagnostic agent. In certain embodiments, the diagnostic agent includes an imaging agent. The diagnostic agent, in certain embodiments, includes a detectable marker. In certain embodiments, the isolated antibody or its antigen-binding fragment is linked to the diagnostic agent by means of a ligand.
[00022] In some embodiments, an alpha toxin polypeptide from Staphylococcus aureus is a native toxin polypeptide. An Staphylococcus aureus alpha-toxin polypeptide in some embodiments includes one or more amino acid deletions, additions and / or substitutions (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 insertions, deletions and / or amino acid substitutions) relative to the native toxin polypeptide. In some embodiments, an alpha-toxin polypeptide from Staphylococcus aureus is a native toxin polypeptide. In certain embodiments, the Staphylococcus aureus alpha-toxin polypeptide includes an amino acid sequence of SEQ ID NO: 39, or fragments thereof. In certain embodiments, the Staphylococcus aureus alpha-toxin polypeptide is an attenuated form of the polypeptide, such as H35L, in which the histidine at position 35 of the wild-type polypeptide is replaced by leucine, for example as represented by SEQ ID NO: 40.
[00023] In certain embodiments, the isolated antibody or its antigen binding fragment binds immunospecifically to an alpha toxin polypeptide from Staphylococcus aureus and includes a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 , 3 amino acid residue substitutions related to SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78, in which the antibody or an antigen-binding fragment neutralizes it the alpha-toxin polypeptide from Staphylococcus aureus. In certain embodiments, the isolated antibody or antigen-binding fragment of an isolated antibody binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide and includes a VL CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 , 3 amino acid residue substitutions for SEQ ID NO: 3, 6, 64, 68 or 74, wherein the antibody or an antigen-binding fragment neutralizes the Staphylococcus aureus alpha toxin polypeptide.
[00024] In certain embodiments, the isolated antibody or its antigen-binding fragment binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide and includes a VH CDR3 comprising an amino acid sequence identical to, or including 1, 2, 3 amino acid residue substitutions related to SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78, where the antibody or an antigen-binding fragment thereof inhibits oligomerization of the Staphylococcus aureus alpha-toxin polypeptide. In some embodiments, the isolated antibody or its antigen-binding fragment binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide and includes a VL CDR3 comprising an amino acid sequence identical to, or including 1, 2, 3 substitutions of amino acid residues related to SEQ ID NO: 3, 6, 64, 68 or 74, wherein the antibody or an antigen binding fragment thereof inhibits the oligomerization of the Staphylococcus aureus alpha-toxin polypeptide. In certain embodiments, the inhibition of oligomerization is determined by an in vitro binding and electrophoretic mobility assay.
[00025] Also provided herein are kits, including (a) a composition comprising an isolated antibody or its antigen binding fragment, and (b) instructions for using the composition or indications for obtaining instructions for using the composition. In some embodiments, the antibody in the composition is attached to a solid support. In certain embodiments, the solid support is a sphere, and in some embodiments, the sphere is a sepharose sphere. In some embodiments, instructions for use include one or more of isolating, purifying, detecting and quantifying a Staphylococcus aureus alpha toxin polypeptide.
[00026] In certain embodiments, the kit includes a buffer and membrane suitable for a Western blot. In some embodiments, the kit includes a loading buffer and an elution buffer. In certain embodiments, the kit includes a buffer suitable for an enzyme linked immunosorbent assay (ELISA).
[00027] A method for preventing, treating or managing pneumonia in a patient is also provided here, including: administering a composition that includes an antibody or an antigen-binding fragment that immunospecifically binds to an alpha-toxin polypeptide of Staphylococcus aureus to a patient in need in an amount effective to prevent, treat or manage pneumonia.
[00028] In some modalities, the method prevents pneumonia. In certain embodiments, the antibody or an antigen-binding fragment thereof that immunospecifically binds to a linear or conformational epitope comprising one or more residues or a portion or fragment of amino acids 1 to 293 or amino acids 51 to 293 of a polypeptide of staphylococcus alpha-toxin. In certain embodiments, the antibody or its antigen-binding fragment binds to a fragment where the antibody or its antigen-binding fragment has contact residues in T261, T163, N264, K266 and K271 of SEQ ID NO: 39. In other embodiments, the antibody or an antigen-binding fragment binds to an alpha-toxin fragment where the antibody or an antigen-binding fragment has additional contact residues in N177, W179, G180, P181 , Y182, D183, D185, S186, W187, N188, P189, V190, Y191 and R200 of SEQ ID NO: 39.
[00029] In certain embodiments, the antibody or an antigen-binding fragment thereof has contact residues in N177, W179, G180, P181, Y182, D183, D185, S186, W187, N188, P189, V190, Y191, R200, T261, T163, N264, K266 and K271 of SEQ ID NO: 39. In other embodiments, the antibody or an antigen-binding fragment binds to a fragment comprising amino acids 261-272 of SEQ ID NO: 39. In others embodiments, the antibody or an antigen-binding fragment thereof binds to a fragment comprising amino acids 248-277 of SEQ ID NO: 39. In other embodiments, the antibody or an antigen-binding fragment binds to a fragment comprising amino acids 173-201 of SEQ ID NO: 39 and a fragment comprising amino acids 261-272 of SEQ ID NO: 39 or a fragment comprising amino acids 173-201 of SEQ ID NO: 39 and a fragment comprising amino acids 248-277 of SEQ ID NO: 39.
[00030] In certain embodiments, the antibody or an antigen-binding fragment binds to a fragment where the antibody or an antigen-binding fragment has contact residues in T261, T163, N264, K266 and K271 of SEQ ID NO: 40. In other embodiments, the antibody or an antigen-binding fragment binds to an alpha-toxin fragment where the antibody or an antigen-binding fragment has additional contact residues in N177, W179, G180, P181, Y182, D183, D185, S186, W187, N188, P189, V190, Y191 and R200 of SEQ ID NO: 40.
[00031] In certain embodiments, the antibody or an antigen-binding fragment thereof has contact residues in N177, W179, G180, P181, Y182, D183, D185, S186, W187, N188, P189, V190, Y191, R200, T261, T163, N264, K266 and K271 of SEQ ID NO: 40. In other embodiments, the antibody or an antigen-binding fragment binds to a fragment comprising amino acids 261-272 of SEQ ID NO: 40. In others embodiments, the antibody or an antigen-binding fragment thereof binds to a fragment comprising amino acids 248-277 of SEQ ID NO: 40. In other embodiments, the antibody or an antigen-binding fragment binds to a fragment comprising amino acids 173-201 of SEQ ID NO: 40 and a fragment comprising amino acids 261-272 of SEQ ID NO: 40.
[00032] In other embodiments, the antibody or an antigen-binding fragment thereof binds to a fragment comprising (1) amino acids 261-272, (2) amino acids 248-277 or (3) amino acids 173-201 and 261- 272, of a staphylococcus alpha-toxin polypeptide or variant of staphylococcus alpha-toxin polypeptide, where amino acids 261-272, amino acids 248-277 or amino acids 173-201 correspond to the same amino acid sequence of the corresponding zone in SEQ ID NO : 39, or contains substitutions within the fragment with the corresponding zone in SEQ ID NO: 39, where substitutions do not alter the ability of the antibody or an antigen-binding fragment to bind to the alpha toxin polypeptide.
[00033] In some embodiments, a method is provided to prevent, treat or manage a skin infection in a patient, including: administering a composition that includes an antibody or an antigen-binding fragment that immunospecifically binds to an alpha polypeptide Staphylococcus aureus toxin, according to the present invention, to a patient in need in an amount effective to prevent, treat or manage the skin infection condition. In certain embodiments, the condition of the skin infection is dermonecrosis. In some embodiments, the condition of the skin infection includes an infection of the skin by Staphylococcus aureus. In certain embodiments, the method prevents the condition of the skin infection.
[00034] In some embodiments, a method is provided to prevent, treat or manage an infection by Staphylococcus aureus, including: administering a composition that includes an antibody or an antigen-binding fragment that immunospecifically binds to an alpha-toxin polypeptide of Staphylococcus aureus according to the present invention to a patient in need in an amount effective to reduce oligomerization of the alpha-toxin polypeptide. In certain modalities, the method prevents the condition associated with infection by Staphylococcus aureus.
[00035] In some modalities, a method is provided to prevent, treat or manage an infection by S. aureus associated with the treatment of dialysis, high-risk surgery, pneumonia, pneumonia associated with mechanical ventilation (VAP) or reinfection after previous discharge from the hospital for prior treatment or surgery that includes administering to a patient in need a composition that includes an antibody or an antigen binding fragment thereof, which immunospecifically binds to an alpha toxin polypeptide from Staphylococcus aureus.
[00036] In some embodiments, a method is also provided to prevent, treat or manage a condition associated with infection by Staphylococcus aureus that includes administering a composition that includes an antibody or an antigen-binding fragment that immunospecifically binds to a polypeptide of Staphylococcus aureus alpha toxin to a needy patient in an amount effective to reduce cell lysis. In certain modalities, the method prevents a condition associated with infection by Staphylococcus aureus. In some embodiments, the cell is an erythrocyte from the blood or lung.
[00037] In some embodiments, the antibody or an antigen binding fragment binds immunospecifically to a linear or conformational epitope comprising one or more residues. In certain embodiments, the composition administered to the patient is in accordance with any of the compositions described herein.
[00038] In certain modalities a method is also provided that includes; administering a composition described herein to cells; and detecting the presence, absence or quantity of a biological effect associated with the administration of the composition to the cells. In certain embodiments, a method is also provided which includes: administering a composition described herein to a patient; and detecting the presence, absence or quantity of a biological effect on the patient associated with the administration of the composition. A method is also provided in certain embodiments which includes: administering a composition described herein to a patient and monitoring the condition of the patient.
[00039] Also provided in certain embodiments is a method for neutralizing an alpha-toxin polypeptide Staphylococcus aureus by administering to a needy patient an effective amount of any of the compositions described herein to neutralize the toxin polypeptide.
[00040] A method is also provided in certain modalities to prevent, treat or manage a condition mediated by Staphylococcus aureus infection in a needy patient, the method including administering to the patient an effective amount of any of the compositions described herein to prevent, treat or manage the condition. Also provided in certain embodiments is a method to treat, prevent or alleviate the symptoms of a disorder mediated by an alpha toxin of Staphylococcus aureus in a needy patient, including administering to the patient an effective amount of any of the compositions described herein to treat, prevent or relieve symptoms.
[00041] A method is also provided in certain modalities to diagnose a condition mediated by an alpha toxin of Staphylococcus aureus in a patient which includes selecting a patient in need of diagnosis and administering to the patient an effective dose from the point of view of diagnosing any one of the compositions described here. In some modalities the patient is a domestic animal and in certain modalities the patient is a human being.
[00042] Certain modalities are better described below in the description, examples, claims and figures that follow. Brief Description of the Figures
[00043] The figures illustrate modalities here and are not limiting. For clarity and ease of illustration, the figures are not to scale and in some cases some aspects may be represented in an exaggerated way or they may be larger to facilitate the understanding of particular modalities.
[00044] Figures 1A and 1B graphically illustrate the percent inhibition of red blood cell lysis by anti-alpha toxin antibodies. Details and experimental results are described in Example 3.
[00045] Figures 2A and 2B graphically illustrate the percentage inhibition of human cell lysis A549 and THP-1 by anti-toxin alpha antibodies. Details and experimental results are described in Example 3.
[00046] Figures 3A and 3B illustrate the results of passive immunization with inhibitory alpha anti-toxin S. aureus mAbs in a model of dermonecrosis. Groups of 5 BALB / c mice were passively immunized with 5 mg / kg of inhibitory mAbs and then infected with S. aureus Wood and the size of their lesions was monitored for 6 days. Figure 3A shows photographs of the lesion size 6 days after infection. Figure 3B graphically illustrates the reduction in the size of the lesion throughout the infection. Details and experimental results are described in Example 4.
[00047] Figures 4-7 graphically illustrate the survival of mice passively immunized with various mAbs described here in a model of pneumonia. Figure 4 illustrates the results of C57BL / 6J mice passively immunized with 5, 15 and 45 mg / kg of purified 12B8.19, 24 hours before infection by S. aureus USA300 (3 x 108 cfu).
[00048] Figure 5 illustrates the results of C57BL / 6J mice passively immunized with 5, 15 and 45 mg / kg of purified 2A3.1, 24 hours before infection by S. aureus USA300 (3 x 108 cfu).
[00049] Figure 6 illustrates the results of C57BL / 6J mice passively immunized with 5, 15 and 45 mg / kg of purified 28F6.1, 24 hours before infection by S. aureus USA300 (3 x 108 cfu).
[00050] Figure 7 illustrates the results of C57BL / 6J mice passively immunized with 5, 15 and 45 mg / kg of purified 10A7.5, 24 hours before infection by S. aureus USA300 (3 x 108 cfu). Details and experimental results are described in Example 5.
[00051] Figures 8A and 8B graphically illustrate the distribution of bacteria in the lung (Figure 8A) and kidney (Figure 8B) in mice passively immunized with a fully human version of mAb 2A3.1 (for example, 2A3hu) or isotype control (R347). C57BL / 6J mice were passively immunized with 2A3hu (15mg / kg) 24 hours before infection with USA300. Samples were also collected to measure cytokine levels and for histopathological analysis. Details and experimental results are described in Example 5.
[00052] Figure 9 graphically illustrates the reduction in inflammatory cytokine production following passive immunization with mAb 2A3hu. Results circled are for the 24 hour time point. Details and experimental results are described in Example 6.
[00053] Figure 10 shows representative photographs of lung histology in mice treated with the R347 control (see the top and bottom photos on the left in Figure 10) or treated with 2A3hu (see photos from the top and bottom right on the Figure 10). Details and experimental results are described in Example 6.
[00054] Figure 11 graphically illustrates that anti-AT (S. aureus alpha-toxin) mAbs described herein does not inhibit native alpha-toxin (nAT) from binding to receptors present in rabbit erythrocyte ghosts. Details and experimental results are described in Example 8.
[00055] Figure 12 is a representative western blot illustrating the inhibition of heptamer formation by antibodies described herein. Details and experimental results are described in Example 8.
[00056] Figures 13A and 13B illustrate representative western blots confirming the inhibition of oligomerization by the anti-AT mAbs described herein and further illustrating that the inhibition can be titrated. Details and experimental results are described in Example 8.
[00057] Figures 14-16 graphically illustrates the potency of fully human anti-AT antibodies in cell lysis inhibition assays. The fully human versions of the anti-AT lgG antibodies exhibit a potency similar to the corresponding chimeric anti-AT lgG antibodies. The inhibitory activity of fully human IgG antibodies was compared to chimeric IgG antibodies in rabbit RBC (red blood cell) lysis (Figure 14), A549 cell lysis (Figure 15) and THP-1 cell lysis (Figure 16). Details and experimental results are described in Example 9.
[00058] Figures 17A and 17B graphically illustrate the reduction in the size of the lesion over the time of infection after passive immunization with inhibitory S. aureus mAbs anti-toxin QD20, QD37, LC10, QD33, 2A3 and the R347 control in a model of dermonecrosis. Groups of 5 BALB / c mice were passively immunized with 1 mg / kg (Fig. 17A) and 0.5 mg / kg (Fig. 17B) of the inhibitory mAbs shown and then infected with S. aureus Wood and the size of their lesions was monitored for 6 days.
[00059] Figure 18 graphically illustrates the survival of mice passively immunized with mAbs QD20, QD37, LC10, QD33, 2A3 and the R347 control in a pneumonia model. Mice were passively immunized with 5 mg / kg of purified mAb, 24 hours before infection by S. aureus USA300 (~ 2 x 108 cfu).
[00060] Figure 19 graphically illustrates the ELISA characterization of the binding of LC10 YTE to alpha toxin and LukF-PV. The bacterial lysate containing His-tagged alpha-toxin or LukF-PV was coated on the surface of a 96-well plate. LC10 YTE or mouse anti-His mAb was added to the coated wells and incubated for one hour. The expression levels of alpha-toxin and LukF-PV were similar, as shown by the anti-His mAb binding signals, while LC10 YTE only significantly bound to alpha-toxin and not to LukF-PV.
[00061] Figure 20 shows sequence alignments of alpha toxin and LukF-PV proteins. The alpha-toxin shares 25% amino acid sequence identity with LukF-PV (UniProtKB / TrEMBL accession number B1Q018). Amino acid numbering is based on mature proteins. The alignment was performed using the Clustal W method. The aa 248-277 segment is highlighted by an underscore.
[00062] Figure 21 shows drawn representations of alpha-toxin structures. A) Drawing of the modeled structure of a soluble alpha-toxin monomer. The alpha-toxin modeled monomer structure was constructed with Maestro 9.1 (Schrodinger Inc) using the LukF-PV crystal structure as (input from Protein Data Bank 1PVL) (Pedelacq, Maveyraud et al. 1999). B) Drawn representation of the crystal structure of a hexamer alpha-toxin protomer (Protein Data Bank 7AHL entry) (Song, Hobaugh et al. 1996). aa 101-110 are shown in blue, aa 224-231 in orange and aa 248-277 in red.
[00063] Figure 22 is a strip diagram of the LC10 YTE Fab α-toxin complex. The alpha-toxin molecule is indicated by the strip at the top of the diagram. The heavy chain is indicated by the dark band at the bottom of the diagram, and the light chain is indicated by the light band at the bottom of the diagram.
[00064] Figure 23 is a drawn representation of heptameric and monomeric states of the α-toxin molecule. The. A mushroom-like heptameric assembly of the α-toxin molecule that creates a pore on the surface of the host cell. The gray and black zones correspond to LC10 YTE contact residues that are coated on the model. The contact residues present in the coated positions are consistent with the formation of LC10 blocking heptamers. B. Superimposed structures of α-toxin molecules before (light gray) and after (dark gray) the formation of pores.
[00065] Figure 24 graphically depicts the effectiveness of treating LC10 in a murine dermonecrosis model. Groups of 5 Balb / C mice were passively immunized with 15 mg / kg LC10 or R347 Balb / C mice were intranasally infected with 2 x 108S. aureus Wood. (A) 1 hour, (B) 3 hour or (C) 6 hour post-infection, the mice were then treated with 5, 15 or 45 mg / kg LC10 and the size of their lesions were monitored for 6 days. Groups of 5 who were administered 15 mg / kg LC10 or R347 24 hours before the bacteria challenge were included as controls.
[00066] Figure 25 graphically depicts the effectiveness of treating LC10 in a model of murine pneumonia. Groups of 10 C57BL / 6 mice were passively immunized with 15 mg / kg LC10 or R347 24 hours before the intranasal challenge with 2 x 108 S. aureus USA300. Groups of 10 C57BL / 6 mice were infected intranasally with 2 x 108S. aureus USA300. (A) 1 hour, (B) 3 hour or (C) 6 hour post-infection, the mice were then treated with 5, 15 or 45 mg / kg LC10 and survival was monitored for 7 days. Groups of 10 who were administered 15 mg / kg LC10 or R347 24 hours before the bacteria challenge were included as controls.
[00067] Figure 26 graphically depicts the effectiveness of treating LC10 in a model of murine pneumonia. Groups of 10 C57BL / 6 mice were infected intradermally with 2 x 108S. aureus USA300. One hour after infection, the animals received an intraperitoneal injection of LC-10 at (A) 15 mg / kg (B) 45 mg / kg. One group of cohort animals received 1 hour after subcutaneous vancomycin (VAN) infection. Additional treatments of VAN were given BID q 12 for a total of 6 treatments. A control group of 10 mice was treated with 15 mg / kg R347 one hour after infection. Survival was monitored for 7 days.
[00068] Figure 27 is a graphical representation of a synergy isobologram analysis in which N> 1: antagonism, N = 1: additive effect, N <1: synergy. Detailed Description
[00069] Antibodies are provided here, including human, humanized and / or chimeric forms, as well as fragments, derivatives / conjugates and their compositions that bind to Staphylococcus aureus alpha toxin. Such antibodies may be useful for detecting and / or visualizing alpha toxin and as such they may be useful in assays and diagnostic methods. Antibodies described herein also interfere with the formation of alpha-toxin heptamers, thereby inhibiting the formation of the active pore-forming complex and as such can be useful for therapeutic and prophylactic methods.
[00070] Staphylococcus aureus is a ubiquitous pathogen and is sometimes an etiologic agent for a variety of conditions, ranging in severity from moderate to fatal. S. aureus produces a large number of extracellular and cell-associated proteins, many of which are involved in the pathogenesis, such as alpha-toxin, beta-toxin, gamma-toxin, delta-toxin, leukocidin, toxic shock syndrome toxin ( TSST), enterotoxins, coagulase, protein A, fibrinogen, fibronectin binding protein and the like. Alpha-toxin (for example, encoded by the hla gene) is one of the virulence factors of Staphylococcus aureus and is produced by most pathogenic strains of S. aureus.
[00071] S. aureussion infections are relatively difficult to treat and invasive diseases and relapses can occur following antibiotic treatment. In addition, methicillin-resistant strains of S. aureus have become more prevalent in hospital settings (for example, associated with HA-MRSA or healthcare) and non-hospital settings (for example, associated with CA-MRSA or to the community), further complicating S. aureus infections. In many cases, methicillin-resistant strains of S. aureus are also resistant to one or more other antibiotics including aminoglycosides, tetracycline, chloramphenicol, macrolides and lincosamides.
[00072] Alpha-toxin is a pore-forming toxin that has cytolytic, hemolytic, dermonecrotic and lethal activities in humans as well as in animals. The alpha-toxin from stalifococci is secreted as a 293 amino acid water-soluble single-stranded polypeptide that is approximately 34 kilodaltons (kDa). Without being limited by theory, it is thought that there are two methods of alpha-toxin / target cell interaction; (i) the alpha-toxin binds to specific high affinity receptors (ADAM 10) on the surface of the human platelet cell, monocytes, endothelial cells, white blood cells, alveolar lung cells, macrophages, keratinocytes, fibroblasts, rabbit erythrocytes and other cells (see for example, Wilke et al., Proc. Natl. Acad. Sci. 107: 13473-13478 (2010)), or (ii) the alpha-toxin interacts non-specifically by absorption to lipid bilayers. In any of the toxin / cell interaction modes, seven alpha-toxin monomer molecules oligomerize to form a heptameric transmembrane channel 1-2 nm in diameter. The subsequent efflux of potassium and nucleotides and an influx of sodium and calcium leads to osmotic lysis and / or multiple secondary actions, including eicosanoid production, secretory processes, contractile dysfunction, apoptosis and cytokine release. It is considered that the disruption of cellular activities and cell lysis by alpha-toxin contributes to the conditions and diseases associated with infection by S. aureus.
[00073] Non-limiting examples of some common conditions caused by S. aureus infection include burns, cellulite, dermecrosis, eyelid infections, food poisoning, pneumonia, skin infections, surgical wound infection, scalded skin syndrome and shock syndrome toxic. In addition, it is a frequent pathogen in foreign body infections, such as intravascular lines, pacemakers, artificial heart valves and joint implants. Some of the conditions or diseases caused by S. aureussão described below. Some or all of the conditions and diseases described below may involve the direct action of the alpha toxin as a component of the infection or mediator of the condition or disease state, or some or all of the conditions may involve the indirect or secondary action of the alpha toxin (eg example, a primary virulence factor causes the main symptom or most of the symptoms associated with the condition, and alpha toxin acts to further advance the disease through its disruption of cell function and cell lysis activities). Burns
[00074] Burn wounds are often initially sterilized. However, moderate or severe burns usually compromise physical and immune barriers to infection (for example, blistering, cracking or peeling of the skin), causing a loss of fluid and electrolytes and resulting in local or general physiological dysfunction. Skin contact compromised with viable bacteria can sometimes result in mixed colonization at the wound site. The infection may be restricted to non-viable debris on the surface of the burn ("eschar") or colonization may progress to severe skin infection and invade viable tissue beneath the eschar. More serious infections can penetrate under the skin, enter the lymphatic system and / or blood circulation and develop septicemia. S. aureus is normally found among the pathogens that colonize burn wound infections. S. aureus can destroy granulation tissue and cause severe septicemia. Skin and soft tissue infections Cellulitis
[00075] Cellulite is an acute skin infection that often begins as a superficial infection that can spread down the skin layer. Cellulite is most often caused by a mixed infection of S. aureus together with S. pyogenes. Cellulite can lead to systemic infection. Cellulite is sometimes an aspect of synergistic bacterial gangrene. Synergistic bacterial gangrene is typically caused by a mixture of S. aureus and microaerophilic streptococci. Synergistic bacterial gangrene causes necrosis and treatment is limited to excision of necrotic tissue. The condition is often fatal. Dermonecrosis
[00076] Dermonecrosis is an infection of the skin and subcutaneous tissues that easily spreads along the fascial plane within the subcutaneous tissue. The condition causes the upper and / or lower layers of the skin to become necrotic and can spread to tissues below or around. Necrotic fasciitis
[00077] Necrotic fasciitis is referred to as "meat-eating disease" or "meat-eating bacteria syndrome". Necrotic fasciitis can be caused by a polymicrobial infection (for example, type I, caused by a mixed bacterial infection) or a monomicrobial infection (for example, type II, caused by a simple pathogenic strain of bacteria). Many types of bacteria can cause necrotic fasciitis; non-limiting examples include: group A streptococci (e.g., Streptococcus pyrogenes), Staphylococcus aureus, Vibrio vulnificus, Clostridium perfringens, and Bacteroides fragilis. Individuals with depressed or compromised immune systems are more likely to experience dermonecrosis (for example, necrotic fasciitis).
[00078] Historically, group A streptococci have been diagnosed as the cause of most cases of type II dermonecrotic infections. However, since 2001, methicillin-resistant Staphylococcus aureus (MRSA) has been observed more frequently as the cause of monomicrobial necrotic fasciitis. The infection starts locally, sometimes at the trauma site, which can be severe (for example, as a result of surgery), minor or even not visible. Patients usually complain of severe pain that may seem excessive given the external appearance of the skin. As the disease progresses, the tissue becomes swollen, often within hours. Diarrhea and vomiting are also common symptoms.
[00079] The signs of inflammation may not be visible in the initial stage of infection, if the bacteria are very much inside the tissue. If the bacteria are not very deep, the signs of inflammation, such as redness, swelling or hot skin, are quickly visible. The color of the skin may evolve to purple, and blisters may form, with subsequent necrosis (for example, death) of the subcutaneous tissues. Patients with necrotic fasciitis usually have a fever and look very sick. The mortality rate has been recorded at levels that reach 73%, if left untreated. Without adequate medical assistance, the infection progresses quickly and eventually leads to death. Pneumonia
[00080] S. aureust has also been diagnosed as the cause of staphylococcus pneumonia. Staphylococcus pneumonia causes inflammation and swelling of the lung, which in turn causes fluid to accumulate in the lung. The fluid accumulated in the lung can prevent oxygen from entering the bloodstream. People with the flu are at risk of developing bacterial pneumonia. Staphylococcus aureus is the most common cause of bacterial pneumonia in people who already have the flu. Common symptoms of staphylococcal pneumonia include coughing, breathing difficulties and fever. Additional symptoms include fatigue, yellow or bloody mucus, and chest pain that worsens when breathing. Methicillin-resistant S. aureus (MRSA) is increasingly diagnosed as the strain identified in staphylococcal pneumonia. Surgical wound infections
[00081] Surgical wounds often penetrate deeply into the body. Infections from such wounds pose a serious risk to the patient if the wound becomes infected. S. aureus is often a causative agent of infections in surgical wounds. S. aureus is unusually adept at invading surgical wounds, sutured wounds can be infected by many fewer S. aureus cells than are necessary to cause infections in normal skin. Invasion of surgical wounds can lead to severe S. aureus septicemia. The invasion of the bloodstream by S. aureus can lead to the spread and infection of internal organs, in particular heart valves and bone, causing systemic diseases such as endocarditis and osteomyelitis. Scalded skin syndrome
[00082] S. aureus is probably a major causative agent, if not the causative agent of "scalded skin syndrome", also referred to as "staphylococcal scalded skin syndrome", "toxic epidermal necrosis", "localized bullous impetigo", "disease of Ritter "and" Lyell's disease ". Scalded skin syndrome often occurs in older children, typically in outbreaks caused by the flowering of S. aureus strains that produce epidermolytic exotoxins (eg, exfoliatin A and B, sometimes referred to as scalded skin syndrome toxin), which causes separation within the epidermal layer of one of the exotoxins is encoded by the bacterial chromosome and the other is encoded by a plasmid. Exotoxins are proteases that cleave desmoglein-1 that normally holds the granular and prickly layers together.
[00083] The bacteria can initially infect only a minor lesion, however, the toxin destroys intercellular connections, widens the epidermal layer and allows the infection to penetrate the outer layer of the skin, producing the peeling that characterizes the disease. The peeling of the outer layer of the skin usually reveals normal skin underneath, but the fluid lost in the process can cause serious damage in young children, if not treated properly. Toxic shock syndrome
[00084] Toxic shock syndrome (TSS) is caused by strains of S. aureus that produce the so-called "toxic shock syndrome toxin". The disease can be caused by infection with S. aureus in any location, but it is often wrongly seen as a disease exclusively for women who use tampons. The disease involves toxemia and septicemia and can be fatal.
[00085] The symptoms of toxic shock syndrome vary depending on the underlying cause. TSS resulting from infection with the bacteria Staphylococcus aureus typically manifests itself in otherwise healthy individuals through high fevers, along with hypotension, malaise and confusion, which can quickly progress to lethargy, coma and multiple organ failure. Skin rashes often seen in the course of the disease resemble sunburn, and can involve any area of the body, including the lips, mouth, eyes, palms and soles. In patients who survive the initial attack of the infection, the rashes flake off or the skin eventually falls off after 10-14 days.
[00086] As mentioned above, due to the increase in strains of S. aureus resistant to multiple drugs, an increasing number of antibiotics commonly used to treat infections S. aureusjá does not control or eliminate infections of methicillin-resistant Staphylococcus aureus and multiple drugs. The antibodies against the S. aureus alpha toxin described herein can help to reduce the severity of the infection and can also help to eliminate, prevent (prophylactically) or reduce S. aureuspatogenic from an infected host. Antibodies.
[00087] As used herein, the terms "antibody", "antibodies" (also known as immunoglobulins) and "antigen binding fragments" comprise monoclonal antibodies (including integral monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope-binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single chain Fvs (scFv), single chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F (ab ') 2 fragments, antibody fragments that exhibit the desired biological activity (for example the antigen binding portion), disulfide-bound Fvs (dsFv) and anti-idiotypic (anti-ld) antibodies (including for example anti -ld to antibodies provided herein), intrabodies and bound fragments of epitopes of any of the above. In particular antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, that is, molecules that contain at least one antigen binding point. Immunoglobulin molecules can be of any isotype (for example, IgG, IgE, IgM, IgD, IgA and IgY), sub-type (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or allotype (for example, Gm , for example G1m (f, z, a or x), G2m (n), G3m (g, b, or c), Am, Em, and Km (1, 2 or 3)). Antibodies can be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice and the like, or other animals such as birds (for example, chickens).
[00088] Native antibodies are generally heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide connections varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains (CH). Each light chain has a variable domain at one end (VL) and a constant domain (CL) at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Light chains are classified as lambda chains or kappa chains based on the amino acid sequence of the constant zone of the light chain. The variable domain of a kappa light chain can also be referred to here as VK.
[00089] The antibodies provided herein include integral or intact antibody, antibody fragments, native sequence antibody or amino acid variants, humanized, humanized, post-translationally modified, chimeric or fusion antibodies, immunoconjugates and their functional fragments. Antibodies can be modified in the Fc zone, and certain modifications can provide the desired effector functions or serum half-life.
[00090] Antibodies with one or more biological characteristics (for example, potency, alpha-toxin affinity, effector function, orthologically binding affinity, neutralization, inhibition of alpha-toxin heptomer formation, and the like) of the present antibodies to alpha-toxin and fragments are also contemplated. Anti-alpha-toxin antibodies and fragments can be used to diagnose and / or treat and / or alleviate and / or prevent one or more symptoms associated with Staphylococcus aureus in a mammal, as described above.
[00091] A composition comprising an anti-alpha-toxin antibody or fragment and a carrier is provided herein. For the purposes of treating diseases associated with S. aureus, compositions can be administered to a patient in need of such treatment, wherein the composition may comprise one or more anti-alpha-toxin antibodies and / or fragments thereof. Formulations comprising an anti-alpha-toxin antibody or fragment thereof as provided herein and a carrier are also provided. In some embodiments, the formulation is a prophylactic or therapeutic formation comprising a pharmaceutically acceptable carrier.
[00092] In certain embodiments, the methods provided here are useful for treating a disease or condition and / or an alpha toxin associated with Staphylococcus aureus and / or preventing and / or alleviating one or more symptoms of the disease or condition in a mammal, comprising administering a therapeutically effective amount of an anti-alpha toxin antibody or fragment to a mammal. Prophylactic or therapeutic antibody compositions can be administered in the short term (acute) or in a chronic or intermittent manner, as directed by the physician.
[00093] In certain embodiments, production articles comprise at least one anti-alpha toxin antibody or fragment, such as in sterile dosage form and / or in a kit. A kit containing an anti-alpha toxin antibody or fragment can be used, for example, for S. aureus cell elimination assays, for purification or immunoprecipitation of cell alpha-toxin. For example, for isolation and purification of the alpha-toxin, a kit may contain an anti-alpha-toxin antibody or fragment attached to strands (for example, Sepharose strands). A kit can contain an antibody for detection and quantification of S. aureus and / or alpha-toxin in vitro, for example and an ELISA or Western blot. Such an antibody useful for detection can be provided with a marker, such as a fluorescent or radiolabel. Terminology
[00094] It should be understood that the method provided here is not limited to specific compositions or process steps, as these may vary. It should be noted that, as used in this report and the appended claims, the singular forms "one", "one" and "o / a and os" include singular and plural referents unless the context clearly indicates otherwise.
[00095] An isolated antibody or its antigen-binding fragment, which specifically binds an alpha-toxin polypeptide (e.g., alpha-toxin monomer, is referred to here as a "anti-alpha-toxin antibody or fragment" in the singular and as "anti-alpha-toxin antibodies and fragments" in the plural). Alpha-toxin polypeptides are sometimes referred to as alpha hemosiline. The alpha toxin forms pores on cell membranes after oligomerizing into a heptamer, where oligomerized polypeptides are sometimes referred to collectively as an "alpha toxin pore" or "alpha toxin heptamer".
[00096] Amino acids are normally referred to here by three-letter symbols or by one-letter symbols recommended by the IUPAC-IUB Committee on Nomenclature in Chemistry. Nucleotides, likewise, are often referred to by commonly accepted one-letter codes.
[00097] The amino acid numbering in the variable domain, complementarity determining zone (CDR) and structure (FR) zones of an antibody follow, unless otherwise stated, the Kabat definition as defined in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Maryland. (1991). Using this system, the linear amino acid sequence acts may contain fewer or more amino acids corresponding to a decrease in or insertion in the FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b and 82c, etc. according to Kabat) after FR heavy chain residue 82. The Kabat numbering of residues can be determined for a given antibody by aligning on homology zones of the antibody sequence with a "standard" numbered Kabat sequence. The maximum alignment of structure residues often requires the insertion of "spacer" residues in the numbering system, to be used in the Fv zone. In addition, the identity of certain individual residues at any number of Kabat sites can vary from antibody chain to antibody chain due to interspecies or allelic divergences. Anti-alpha toxin antibodies and fragments
[00098] In certain embodiments, an anti-alpha-toxin antibody or fragment is isolated and / or purified and / or free of pyrogens. The term "purified" as used herein refers to a molecule of interest that has been identified and separated and / or recovered from a component of its natural environment. Thus, in some embodiments, the antibody provided is a purified antibody in which it has been separated from one or more components of its natural environment. The term "isolated antibody" as used herein refers to an antibody substantially free of other antibody molecules with different antigen specificities (for example an isolated antibody that specifically binds alpha toxin is substantially free of antibodies that specifically bind antigens other than alpha-toxin). A bi or multispecific antibody molecule is an antibody isolated when substantially free of other antibody molecules. Thus, in some embodiments, the antibodies provided are antibodies that have been separated from antibodies with a different specificity. An isolated antibody can be a monoclonal antibody. An isolated antibody that specifically binds to an epitope, isoform or variant of the S. aureus alpha-toxin may, however, have trans-reactivity towards other related antigens, for example from other species (for example, homologs of staph species). An isolated antibody is provided that can be substantially free of one or more other cellular materials. In some embodiments, a combination of "isolated" monoclonal antibodies is provided and belongs to antibodies with different specificities and combined in a defined composition. Methods for producing and purifying / isolating an anti-alpha toxin antibody or fragment are described in greater detail here.
[00099] The isolated antibodies shown comprise antibody amino acid sequences disclosed herein that can be encoded by any suitable polynucleotide. Isolated antibodies are sometimes provided in formulated form. In some embodiments, an anti-alpha toxin antibody or fragment binds S. aureus alpha toxin and thus partially or substantially alters at least one biological activity of the alpha toxin, for example the oligomerization in the active heptamer complex.
[000100] The anti-alpha toxin antibody or fragment often binds to one or more specific epitopes of the alpha-toxin protein, peptide, subunit, fragment portion, oligomers or any combination thereof and generally does not specifically bind to other polypeptides. The term "oligomers" or "alpha-toxin oligomers" refers to an association of alpha-toxin monomers (for example 2 monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers or 7 monomers) to form a functional pore ( for example 7 alpha-toxin monomers). An epitope can comprise at least one antibody binding zone that comprises at least a portion of the alpha-toxin protein. The term "epitope" as used herein refers to a protein determinant capable of binding an antibody. Epitopes generally include surface clusters of chemically active molecules such as amino acid and / or sugar side chains and generally have specific structural characteristics of three dimensions, as well as specific chemical characteristics (e.g. charge, polarity, basic, acidity, hydrophobicity and the like). Conformational and non-conformational epitopes are distinguished in that the bond to the former but not to the latter is lost in the presence of denatured solvents. In some embodiments, the recognized epitope interferes with the formation of the active heptamer (for example, it inhibits the oligomerization of alpha-toxin monomers in an active heptamer complex).
[000101] In certain embodiments, an epitope consists of at least a portion of alpha-toxin protein, which is involved in the form of an alpha-toxin heptamer complex. A specified epitope may comprise a combination of at least one amino acid sequence of at least 3 amino acid residues for the entire specified portion of contiguous amino acids of the alpha toxin protein. In some embodiments, the epitope is at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues or at least 15 amino acid residues for the entire specified portion of contiguous amino acid from the alpha toxin protein. In certain other embodiments, the epitope comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acid residues. In other embodiments, amino acid residues within the epitope are involved in the formation of alpha-toxin heptamer complexes. In certain embodiments, the contact residues comprise T261, T263, N264, K266 and K271. In other embodiments, the contact residues comprise N177, W179, G180, P181, Y182, D183, D185, S186, W187, N188, P189, V190, Y191 and R200 of SEQ ID NO: 39. In other embodiments, the contact residues contacts comprise N177, W179, G180, P181, Y182, D183, D185, S186, W187, N188, P189, V190, Y191, R200, T261, T263, N264, K266 and K271 of SEQ ID NO: 39. In certain embodiments, the portion of the alpha toxin in contact with the antibody or an antigen-binding fragment thereof comprises amino acids 261-272 of SEQ ID NO: 39. In other embodiments, the portion of the alpha-toxin in contact with the antibody or a fragment thereof antigen binding agent comprises amino acids 248-277 of SEQ ID NO: 39. In other embodiments, the alpha toxin portion in contact with the antibody or an antigen binding fragment thereof comprises amino acids 173-201 and 261-272 of SEQ ID NO: 39.
[000102] Thus, in specific embodiments, anti-alpha toxin antibodies and fragments bind immunospecifically to a molecule comprising the amino acid sequence according to SEQ ID NO: 39 and / or to a molecule comprising the amino acid sequence according to SEQ ID NO: 40. In certain embodiments, the anti-toxin antibodies and fragments also bind to alpha-toxin homologues or orthologs of different species or to variants of the amino acid sequence of SEQ ID NO: 39, where the histidine in position 35 is replaced by leucine, or replaced by other amino acids corresponding to H35 mutations known to those skilled in the art. Variable zones
[000103] In certain embodiments, an anti-alpha toxin antibody or fragment of a major antibody is prepared. In some embodiments, an anti-alpha toxin antibody or fragment within the main antibody is comprised. As used herein, the term "main antibody" refers to the antibody encoded by an amino acid sequence used for the preparation of the variant or derivative, defined herein. A major polypeptide can comprise a native antibody sequence (for example, a naturally occurring variant, including a naturally occurring allelic variant) or an antibody sequence with pre-existing amino acid sequence modifications (such as insertions, deletion and / or substitutions) of a sequence occurring naturally. A major antibody can be a humanized antibody or a human antibody. In specific embodiments, anti-alpha toxin antibodies and fragments are variants of the main antibody. As used herein, the term "variant" refers to an anti-alpha toxin antibody or fragment that differs in the amino acid sequence from an amino acid sequence to an anti-alpha toxin antibody or major fragment via addition, elimination and / or replacing one or more amino acid residues in the main antibody sequence.
[000104] The antigen-binding portion of an antibody comprises one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., alpha-toxin). It has been shown that the antigen binding function of an antibody can be performed by fragments of an integral antibody. Examples of binding fragments covered by the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the articulation zone; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain; and (vi) an isolated complementarity determination zone (CDR). Although the two domains of the Fv fragment VL and VH are often encoded by two separate genes they can be joined using recombinant methods, by a synthetic linker that allows them to be made as a single protein chain in which the VL and VH zones pair for form monovalent molecules [known as single chain Fv (scFv)]. Such single chain antibodies are also covered by the terms "antibody" and "antigen-binding portion" of an antibody. These antibody fragments can be obtained using known techniques and the fragments can be evaluated by screening for binding activity in the same way that intact antibodies are. Antigen-binding moieties can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
[000105] The present anti-toxin antibodies and fragments comprise at least one antigen binding domain. In some embodiments, an anti-toxin antibody or fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55 , 57, 79, 59, 61, or 62. In certain embodiments, an anti-toxin antibody or fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44 , 46, 48, 50, 52, 54, 56, 58, 60 or 63. In yet other embodiments, an anti-alpha toxin antibody or fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 20, 22, 24 , 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and a VL comprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. See example 11, table 7 for a representation of VH and VL sequences as shown here that can be present in any combination to form an anti-alpha toxin antibody or fragment or present in a combination to form a mAb of the invention. In some embodiments, the VH is selected from SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In several modalities, the VL is selected from SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. Certain nucleotide sequences are presented VH and VL in example 11, table 8.
[000106] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a VH and a VL in which the VH and VL have amino acid sequences represented by SEQ ID NOs: 20 and 19; SEQ ID NOs; 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID SEQ ID NOs: 41 and 42; SEQ ID SEQ ID NOs: 47 and 48; SEQ ID SEQ ID NOs: 51 and 52; SEQ ID SEQ ID NOs: 55 and 56; SEQ ID SEQ ID NOs: 61 and 58; SEQ ID NOs: 62 and 58; SEQ ID NOs: 62 and 63; SEQ ID NOs: 79 and 63.
[000107] Tables 1-7 of example 11 provide heavy chain variable zones (VH), light chain variable zones (VL) and complementarity determination zones (CDR) for certain modalities of antibodies and fragments presented here. In certain embodiments, the anti-toxin antibodies and fragments comprise a VH and / or VL that has a certain percentage of identity for at least one of the VH and / or VL sequences disclosed in table 7. As used herein, the term "identity percentage sequence (%) "also including" homology "is defined as the percentage of amino acid or nucleotide residues in a candidate sequence that are identical to the amino acid or nucleotide residues in the reference sequences, such as the main antibody sequence, after aligning the sequences and introducing intervals, if necessary, to achieve maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. The optimal alignment of the sequences for comparison can be carried out, in addition to manually, by means of local homology algorithms known in the art or by means of computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).
[000108] In some embodiments, an anti-alpha toxin antibody or fragment comprises a VH amino acid sequence comprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95% identity with, or comprising 100% identity with the amino acid sequence of SEQ ID NOs: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 In some embodiments, an anti-toxin antibody or fragment includes a VH amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical to, the amino acid sequence of SEQ ID NOs: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In certain embodiments, an anti-alpha toxin antibody or fragment comprises 1-10 conservative substitutions in the amino acid sequence of SEQ ID NOs: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In certain embodiments, an anti-alpha toxin antibody or fragment comprising a VH amino acid sequence with a certain percentage of identity with SEQ ID NOs: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 have one or more characteristics (described in more detail below) selected from the group consisting of: (a) affinity constant (KD) for alpha toxin of about 13 nM or less; (b) binds to alpha-toxin monomers, but does not inhibit the binding of the alpha-toxin to the alpha-toxin receptor; (c) inhibits the formation of alpha-toxin oligomers by at least 50%, 60%, 70%, 80%, 90% or 95%; (d) reduces the alpha-toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (for example, as determined by hemolysis cell lysis assays); (e) reduces cell infiltration and release of pro-inflammatory cytokine (for example, in a model of animal pneumonia).
[000109] In some embodiments, an isolated antibody or its antigen-binding fragment binds an antigen (eg, alpha toxin) with an affinity characterized by a dissociation constant (KD) in the range of 0.01 nM to about 50 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30nM, or 40 nM.
[000110] In certain embodiments, an anti-toxin antibody or fragment comprises a VL amino acid sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95% identical to, or 100% identical to the amino acid sequence of SEQ ID NOs: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In some embodiments, an anti-alpha antibody toxin or fragment includes a VL amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical to the amino acid of SEQ ID NOs: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In various embodiments, the anti-alpha toxin antibody or fragment comprises 1-10 conservative substitutions in the amino acid sequence of SEQ ID NOs: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In certain embodiments , the anti-alpha toxin antibody or fragment comprising a VL amino acid sequence with a certain percentage identity with SEQ ID NOs: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63 and has one or more characteristics (described in more detail below) selected from the group consisting of: (a) affinity constant (KD) for alpha toxin of about 13 nM or less; (b) binds to alpha-toxin monomers, but does not inhibit the binding of alpha-toxin to the alpha-toxin receptor: (c) inhibits the formation of alpha-toxin oligomers by at least 50%, 60%, 70 %, 80%, 90% or 95%; (d) reduces the alpha-toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (for example, as determined by hemolysis cell lysis assays); (e) reduces cell infiltration and pro-inflammatory cytokine release (for example, in animal pneumonia models).
[000111] In some embodiments, an isolated antibody or its antigen-binding fragment binds an antigen (for example, alpha-toxin) with an affinity characterized by a dissociation constant (KD) in the range of 0.01 nM to about 50 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30nM, or 40 nM.
[000112] In some specific embodiments, an antibody or antibody fragment binds immunospecifically to the alpha toxin and comprises a heavy chain variable domain comprising at least 90% identity with the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and comprises a light chain variable domain comprising at least 90% identity with the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63, in which the antibody has the activity of inhibiting the binding of one or more alpha-toxin monomers to each other (for example, inhibits oligomerization). Complementarity determination zones
[000113] While the variable domain (VH and VL) comprises the antigen binding zone, the variability is not evenly distributed across the variable domains of the antibodies. It is concentrated in segments called Complementarity Determination Zones (CDRs), both in the variable domains of the light chain (VL or VK) and the heavy chain (VH). The most highly conserved portions of the variable domains are called the structure zones (FR). The variable domains of heavy and light chains each comprise four FRs, largely adopting a β-leaf configuration, joined by three CRDs, forming cycles that link to, and in some cases form part of, the beta-leaf structure. The CDRs of each chain are held together in close proximity by the FR and, with the CDRs of the other chain, contribute to the formation of the antibody antigen binding site (see Kabat et al., Supra). The three heavy chain CDRs are designated VH-CDR1, VH CDR2, and VH-CDR-3, and the three light chain CDRs are designated VL-CDR1, VL-CDR2, and VL-CDR3. The Kabat numbering system is used here. As such, VH-CDR1 starts at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids and ends at the next serine residue. VH-CDR2 starts at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids and ends at the next glycine residue. VH-CDR3 begins at approximately the thirteenth amino acid residue after the end of VH-CDR2; includes approximately 13-15 amino acids; and ends in the sequence M-D-V. VL-CDR1 starts at approximately residue 24 (ie, after the cysteine residue): includes approximately 10-15 residues; and ends with the sequence Y-V-S. VL-CDR2 begins at approximately the sixteenth residue after the end of VL-CDR1 and includes approximately 7 residues. VL-CDR3 begins at approximately the thirty-third residue after the end of VH-CDR2; includes approximately 7-11 residues and ends in the sequence T-I-L. Note that CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology to Kabat consensus sequences).
[000114] The anti-alpha toxin antibodies and fragments present comprise at least one antigen binding domain that includes at least one complementarity determining zone (CDR1, CDR2 or CDR3). In some embodiments, an anti-alpha toxin antibody or fragment comprises a VH that includes at least one VH CDR (for example, CDR-H1, CDR-H2 or CDR-H3). In some embodiments, an anti-alpha toxin antibody or fragment comprises a VL that includes at least one VL CDR (for example, CDR-L1, CDR-L2 or CDR-L3).
[000115] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide includes (a) a VH CDR1 comprising an amino acid sequence identical to, or containing, 1 , 2 or 3 amino acid residue substitutions for SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions relating to SEQ ID NO: 8, 11, 14, 17, 70 or 75; and (c) a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78.
[000116] In particular embodiments, the isolated antibody or its antigen-binding fragment comprises a VH CDR1, VH CDR2 and VH CDR3 comprising amino acid sequences identical to, or containing, 1, 2 or 3 amino acid residue substitutions in each CDR relating to SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 13, 14 and 15; SEQ ID NOs: 7, 17 and 18; SEQ ID NOs: 7, 8 and 16; SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7, 8 and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs: 69, 70 and 71; SEQ ID NOs: 7, 8 and 72; SEQ ID NOs: 69, 75 and 71; SEQ ID NOs: 69, 75 and 76; or SEQ ID NOs: 69, 70 and 71.
[000117] In some embodiments, the isolated antibody or its antigen-binding fragment that immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide includes (a) a VL CDR1 comprising an amino acid sequence identical to, or containing, 1 , 2, 3 substitutions of amino acid residues related to SEQ ID NO: 1 or 4; (b) a VL CDR2 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions relating to SEQ ID NO: 2, 5, 73 or 77; and (c) a VL CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 3, 6, 64, 68 or 74.
[000118] In particular embodiments, the isolated antibody or its antigen-binding fragment comprises a VL CDR1, VL CDR2 and VL CDR3 comprising amino acid sequences identical to, or containing, 1, 2 or 3 amino acid residue substitutions in each CDR relating to SEQ ID NOs: 1, 2 and 3; SEQ ID NOs: 4, 5 and 6; SEQ ID NOs: 1, 2 and 64; SEQ ID NOs: 1, 2 and 68; SEQ ID NOs: 1, 73 and 74; or SEQ ID NOs: 1, 77 and 74.
[000119] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide includes a VL CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprising amino acid sequences identical to, or containing, 1, 2, 3 amino acid residue substitutions in each relative CDR: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70 or 75; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 4; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74.
[000120] In particular embodiments, the isolated antibody or its antigen-binding fragment comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprising amino acid sequences identical to, or containing, 1, 2 or 3 amino acid residue substitutions in each CDR for SEQ ID NOs: 7, 8, 9, 1, 2 and 3; SEQ ID NOs: 10, 11, 12, 1, 2 and 3; SEQ ID NOs: 13, 14, 15, 4, 5 and 6; SEQ ID NOs: 7, 17, 18, 1, 2 and 3; SEQ ID NOs: 7, 8, 16, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 64; SEQ ID NOs; 7, 8, 66, 1, 2 and 64; SEQ ID NOs: 7, 8, 67, 1, 2 and 68; SEQ ID NOs: 7, 8, 67, 1, 2 and 64; SEQ ID NOs: 7, 8, 78, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 68; SEQ ID NOs: 69, 70, 71, 1, 2 and 68; SEQ ID NOs: 7, 8, 72, 1, 73 and 74; SEQ ID NOs: 69, 75, 71, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 77 and 74; SEQ ID NOs: 69, 70, 71, 1, 77 and 74.
[000121] In some embodiments, a composition is provided comprising an isolated antibody or its antigen binding fragment which (i) includes a VL chain domain comprising three CDRs and a VL chain domain comprising three CDRs; that (ii) immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide in which the three CDRs of the VH chain domain include (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70 or 75; and (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78. In particular embodiments, the VH CDR1, VH CDR2 and VH CDR3 corresponds to SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 13, 14 and 15; SEQ ID NOs: 7, 17 and 18; SEQ ID NOs: 7, 8 and 16; SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7, 8 and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs: 69, 70 and 71; SEQ ID NOs: 7, 8 and 72; SEQ ID NOs: 69, 75 and 71; SEQ ID NOs: 69, 75 and 76; or SEQ ID NOs: 69, 70 and 71.
[000122] Also provided, in certain embodiments, is a composition comprising an isolated antibody or its antigen binding fragment which (i) includes a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs; whereas (ii) immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide in which the three CDRs of the VL chain domain include (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 4; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and (c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74. In particular embodiments, VL CDR1, VL CDR2 and VL CDR3 correspond to SEQ ID NOs: 1, 2 and 3; SEQ ID NOs: 4, 5 and 6; SEQ ID NOs: 1, 2 and 64; SEQ ID NOs: 1, 2 and 68; SEQ ID NOs: 1, 73 and 74; or SEQ ID NOs: 1, 77 and 74.
[000123] In certain embodiments, an anti-alpha toxin antibody or fragment binds immunospecifically to a S. aureus alpha toxin and comprises (a) a VH CDR1 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions relating to SEQ ID NO: 8, 11, 14, 17, 70 or 75; and (c) a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 4; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74 and has one or more characteristics (described in more detail below) selected from the group consisting of: (a) constant of affinity (KD) for alpha toxin of about 13 nM or less; (b) binds to alpha-toxin monomers, but does not inhibit the binding of the alpha-toxin to the alpha-toxin receptor; . (c) inhibits the formation of alpha-toxin oligomers by at least 50%, 60%, 70%, 80%, 90% or 95%; (d) reduces the alpha-toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (for example, as determined by hemolysis cell lysis assays); (e) reduces cell infiltration and pro-inflammatory cytokine release (for example, in animal pneumonia models).
[000124] In some embodiments, an isolated antibody or its antigen-binding fragment binds an antigen (eg, alpha-toxin) with an affinity characterized by a dissociation constant (KD) in the range of 0.01 nM to about 50 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30nM, or 40 nM.
[000125] Example 11, Tables 1-7 provide sequences for VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 for antibodies of the present invention. Table 9 provides a summary of VH and VL CDRs. These zones can be combined in various combinations, as each CDR zone can be independently selected for a given antibody. Table 7 illustrates different sequences that can be selected for each zone. In certain embodiments, the VL CDR3 sequences can be present in any combination to form an anti-alpha toxin antibody or fragment present. In certain embodiments, VH CDR1 is selected from SEQ ID NO: 7, 10, 13 or 69, VH CDR2 is selected from SEQ ID NO: 8, 11, 14, 17, 70 or 75 and VH CDR3 is selected from SEQ ID NO: SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78, as shown in table 9. In some embodiments, VL CDR1 is selected from SEQ ID NO : 1 or 4, VL CDR2 is selected from SEQ ID NO: 2, 5, 73, or 77 and VL CDR3 is selected from SEQ ID NO: 3, 6, 64, 68 or 74 as shown in table 9.
[000126] The VH CDR3 and VL CDR3 domains play a role in the binding specificity / affinity of an antibody to an antigen. Accordingly, in some embodiments, an anti-alpha toxin antibody or fragment or its antigen binding fragment comprises a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78. In various embodiments, an anti-toxin antibody or fragment comprises a VL CDR3 comprising a amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 3, 6, 64, 68 or 74. The remaining portions of anti-toxin antibody and fragments (for example CDR1 , CDR2, VH, VL and the like) may comprise specific sequences disclosed herein or sequences known to the extent that anti-alpha toxin antibodies and fragments bind immunospecifically to S. aureus alpha toxin.
[000127] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to an alpha toxin and comprises a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 residue substitutions of amino acids related to SEQ ID NO: SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78, where the antibody or antigen binding fragment inhibits alpha oligomerization -toxin.
[000128] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to an alpha toxin and comprises a VL CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 residue substitutions of amino acids relating to SEQ ID NO: 3, 6, 64, 68 or 74, wherein the antibody or antigen binding fragment inhibits alpha-toxin oligomerization.
[000129] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to an alpha toxin and comprises a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 residue substitutions of amino acids related to SEQ ID NO: SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78, where the antibody or antigen binding fragment reduces or inhibits release of cytokines.
[000130] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to an alpha toxin and comprises a VL CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 residue substitutions of amino acids relative to SEQ ID NO: 3, 6, 64, 68 or 74, wherein the antibody or antigen binding fragment reduces or inhibits cytokine release.
[000131] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to an alpha-toxin and comprises a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 residue substitutions of amino acids related to SEQ ID NO: SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78, where the antibody or antigen binding fragment alleviates or eliminates dermonecrosis.
[000132] In some embodiments, the isolated antibody or its antigen-binding fragment that binds immunospecifically to an alpha-toxin and comprises a VL CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 residue substitutions of amino acids relative to SEQ ID NO: 3, 6, 64, 68 or 74, wherein the antibody or antigen binding fragment alleviates or eliminates dermonecrosis.
[000133] Anti-alpha toxin antibodies and fragments often comprise one or more amino acid sequences substantially the same as the amino acid sequence described above. Amino acid sequences that are substantially the same include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and / or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid with a second amino acid that has chemical and / or physical properties (e.g. charge, structure, polarity, hydrophobicity / hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacing one amino acid with another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Structure zones
[000134] The variable domains of the heavy and light chains each comprise four structure zones (in general FR1, FR2, FR3, FR4 or alternatively FW1, FW2, FW3, FW4), which are the most highly conserved portions of the variable domains. The four structure zones of the heavy chain are designated here VH-FW1, VH-FW2, VH-FW3 and VH-FW4, and the four structure zones of the light chain are here designated VL-FW1, VL-FW2, VL-FW3 and VH-FW4. The Kabat numbering system is used here and as such VH-FW1 starts at position 1 and ends at approximately amino acid 30, VH-FW2 is approximately amino acid 36 to 49, VH-FW3 is approximately amino acid 66 to 94 and VH-FW4 is approximately from amino acid 103 to 113. Vl-FW1 begins at amino acid 1 and ends at approximately amino acid 23, VL-FW2 is approximately from amino acid 35 to 49, VL-FW3 is approximately from amino acid 57 to 88 and VL-FW4 is approximately from amino acid 98 to 107. In certain embodiments, the structure zones contain substitutions according to the Kabat numbering system, for example inserts in 106A in VL-FW1. In addition to naturally occurring substitutions, one or more changes (for example substitutions) or FR residues can also be introduced into an anti-alpha toxin antibody or fragment. In certain embodiments, these changes result in an improvement or optimization in the binding affinity of the antibody to the anti alpha toxin. Non-limiting examples of structure zone residues that can be modified include those that non-covalently bind antigen directly, interact with / effect the formation of a CDR and / or participate in the VL-VH interface.
[000135] In certain embodiments, a structure zone can comprise one or more amino acid changes with the aim of making a "germ line". For example, the amino acid sequences of selected heavy and light antibody chains are compared to heavy and light germline chain amino acid sequences and in which certain structure residues of the selected VL and / or VH chains differ from the germline configuration (for example as a result of the somatic mutation of the immunoglobulin genes used to prepare the phage library), it may be desirable to "re-mutate" the altered structure residues of the selected antibodies to the germline configuration (i.e., change the sequences of the selected antibodies frame to be the same as the germline frame amino acid sequences). Such "retromutation" (or "germ line") of structure residues can be achieved by standard molecular biological methods to introduce specific mutations (eg, site-directed mutagenesis; PCR-mediated mutagenesis and the like). In some embodiments, light and / or heavy variable chain structure residues are reassembled. In certain embodiments, the heavy variable chain of an isolated antibody or its displayed antigen-binding fragment is retromuted. In certain embodiments, the heavy variable chain of an isolated antibody or its antigen-binding fragment comprises at least one, at least two, at least three, at least four or more retromutations.
[000136] In certain embodiments, the VH of an anti-alpha toxin antibody or fragment shown herein may comprise FR1, FR2, FR3 and / or FR4 that has an amino acid sequence identity with the corresponding structure zones (i.e., FR1 of antibody X as compared to FR1 of antibody Y) within SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61 , or 62 that is between about 65% to about 100%. In some embodiments, an anti-toxin antibody or fragment comprises a VH FR amino acid sequence (FR1, FR2, FR3 and / or FR4) at least 65%, 70%, 75%, 80%, 85%, 90%, 95% identical to, or 100% identical to the corresponding FR of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In certain embodiments, an anti-alpha toxin antibody or fragment comprises a VH FR amino acid sequence (FR1, FR2, FR3 and / or FR4) at least 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical to the corresponding FR of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62.
[000137] In certain embodiments, an anti-alpha toxin antibody or fragment may comprise a VH FR (FR1, FR2, FR3 and / or FR4) comprising an amino acid sequence identical to, or containing, 1, 2 or 3 residue substitutions of amino acids relative to the corresponding FR of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In particular VH FR1, FR2, FR3 or FR4 can each have an amino acid sequence identical to or comprising 1, 2 or 3 amino acid substitutions relative to the corresponding FR1, FR2, FR3 or FR4 of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62.
[000138] In certain embodiments, the VL of an anti-alpha toxin antibody or fragment provided herein may comprise FR1, FR2, FR3 and / or FR4 that has an amino acid sequence identity with the corresponding structure zones (i.e., FR1 antibody X as compared to antibody Y FR1) within the FR of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63 (for example, from about 65% to about 100% identical sequence). In some embodiments, an anti-toxin antibody or fragment comprises a VL FR amino acid sequence (FR1, FR2, FR3 and / or FR4) at least 65%, 70%, 75%, 80%, 85%, 90%, 95% identical to, or 100% identical to the corresponding FR of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In certain embodiments, an anti-toxin antibody or fragment comprises a VL FR amino acid sequence (FR1, FR2, FR3 and / or FR4) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical to the corresponding FR of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54 , 56, 58, 60 or 63.
[000139] In certain embodiments, an anti-alpha toxin antibody or fragment comprises a VL FR (FR1, FR2, FR3 and / or FR4) comprising an amino acid sequence identical to, or containing, 1, 2 or 3 residue substitutions amino acids relative to the corresponding FR of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In particular FR1, FR2, FR3 or VL FR4 can each have an amino acid sequence identical to or comprising 1, 2 or 3 amino acid substitutions relative to the corresponding FR1, FR2, FR3 or FR4 of VH SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.
[000140] In certain embodiments, an isolated antibody or its antigen-binding fragment binds immunospecifically to the alpha toxin and comprises a VH FR (FR1, FR2, FR3 and / or FR4) comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for the corresponding VH FR SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and / or VL FR (FR1, FR2, FR3 and / or FR4) comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions relative to the corresponding FR of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63 where the antibody has one or more characteristics (described below in more detail) selected from the group consisting of: (a) affinity constant (KD) for alpha toxin of about 13 nM or less; (b) binds to alpha-toxin monomers, but does not inhibit the binding of the alpha-toxin to the alpha-toxin receptor; . (c) inhibits the formation of alpha-toxin oligomers by at least 50%, 60%, 70%, 80%, 90% or 95%; (d) reduces the alpha-toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (for example, as determined by hemolysis cell lysis assays); (e) reduces cell infiltration and pro-inflammatory cytokine release (for example, in animal pneumonia models).
[000141] In some embodiments, an isolated antibody or its antigen-binding fragment binds an antigen (eg, alpha toxin) with an affinity characterized by a dissociation constant (KD) in the range of 0.01 nM to about 50 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30nM, or 40 nM. Nucleotide sequences encoding anti-alpha toxin antibodies and fragments
[000142] In addition to the amino acid sequences described above, nucleotide sequences corresponding to the amino acid sequences and coding of human, humanized and / or chimeric antibodies disclosed herein are also provided. In some embodiments, polynucleotides comprise a nucleotide sequence encoding an anti-alpha toxin antibody or fragment described herein or fragments thereof. These include, but are not limited to, nucleotide sequences that encode the aforementioned amino acid sequences. Nucleotide sequences are provided in example 11, table 8. Thus, polynucleotide sequences encoding VH and VL structure zones including CDRs and FRs of antibodies described herein are also provided, as well as expression vectors for their efficient expression in cells (for example, mammalian cells). The following describes methods of making an anti-alpha toxin antibody or fragment using polynucleotides.
[000143] Also included are polynucleotides that hybridize under stringent or less stringent hybridization conditions, for example as defined herein for polynucleotides that encode an anti-toxin antibody or fragment. The term "stringency" as used herein refers to experimental conditions (for example, temperature and salt concentration) of a hybridization experiment to denote the degree of homology between two nucleic acids; the higher the stringency, the greater the percentage of homology between the two nucleic acids.
[000144] Strict hybridization conditions include, but are not limited to, hybridization to filter DNA in 6X sodium chloride / sodium citrate (SSC) at about 45 degrees Celsius 0.2X SSC / 0.1% SDS at about 50-65 degrees Celsius, highly stringent conditions such as hybridization to filter DNA in 6X SSC at about 45 degrees Celsius followed by one or more washes in 0.1X SSC / 0.2% SDS at about 65 degrees Celsius, or any other known stringent hybridization conditions.
[000145] In certain embodiments, a nucleic acid or fragment thereof can encode an anti-alpha toxin antibody or fragment and hybridize under stringent conditions to a nucleic acid including a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.
[000146] In certain embodiments, a polynucleotide sequence may comprise a nucleotide sequence encoding an anti-alpha toxin antibody or fragment at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% , or 100% identical to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In some embodiments, a polynucleotide sequence may comprise a nucleotide sequence encoding an anti-alpha toxin antibody or fragment at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% , or 100% identical to a nucleotide sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37 or 38. In some embodiments, an anti-toxin antibody or fragment includes a nucleotide sequence comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a nucleotide sequence encoding the amino acid sequence SEQ ID NO: 19, 2 1, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.
[000147] In certain embodiments, a polynucleotide sequence may comprise a nucleotide sequence encoding an anti-alpha toxin antibody or fragment at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% , or 100% identical to a VH nucleotide sequence encoding the VH amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57 , 79, 59, 61, or 62. In some embodiments, a polynucleotide sequence may comprise a nucleotide sequence encoding an anti-alpha toxin antibody or fragment at least about 65%, 70%, 75%, 80%, 85 %, 90%, 95%, or 100% identical to a VH nucleotide sequence of SEQ ID NO: 30, 32, 34, 36 or 38. In some embodiments, an anti-toxin antibody or fragment is encoded by a sequence nucleotide encoding VH comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a nucleotide sequence that encodes the sequence of amino acid of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62.
[000148] In certain embodiments, a polynucleotide sequence may comprise a nucleotide sequence encoding an anti-alpha toxin antibody or fragment at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% , or 100% identical to a VL nucleotide sequence encoding the VL amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58 , 60 or 63. In some embodiments, a polynucleotide sequence may comprise a nucleotide sequence encoding an anti-alpha toxin antibody or fragment at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a VL nucleotide sequence of SEQ ID NO: 29, 31, 33, 35 or 37. In some embodiments, an anti-toxin antibody or fragment is encoded by a VL encoding nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a nucleotide sequence encoding the SEQ amino acid sequence ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.
[000149] In particular embodiments, a polynucleotide sequence may comprise a nucleotide sequence encoding an anti-alpha toxin antibody or fragment at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH amino acid sequence and a nucleotide sequence encoding an anti-toxin antibody or fragment at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence VL, where the sequences VH and VL are represented by SEQ ID NOs: 20 and 19; SEQ ID NOs; 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27; SEQ ID SEQ ID NOs: 45 and 46; SEQ ID SEQ ID NOs: 49 and 50; SEQ ID SEQ ID NOs: 53 and 54; SEQ ID SEQ ID NOs: 59 and 60; SEQ ID SEQ ID NOs: 62 and 63; SEQ ID NOs: 79 and 63.
[000150] Substantially identical sequences can be polymorphic sequences, that is, alternative sequences or alleles in a population. An allelic difference can be as small as a base pair. Substantially identical sequences can also comprise mutagenized sequences, including sequences comprising silent mutations. A mutation can comprise one or more changes in residues, an elimination of one or more residues or an insertion of one or more additional residues.
[000151] Polynucleotides can be obtained and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. For example, if the nucleotide sequence of the antibody is known, an encoding of the antibody polynucleotide can be assembled from chemically synthesized oligonucleotides which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, tempering and binding these oligonucleotides and then amplify the PCR-linked oligonucleotides.
[000152] An antibody polynucleotide encoding can also be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (for example, a cDNA library antibody , or a cDNA library generated from, or nucleic acid, sometimes polyA + RNA, isolated from any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using hybridizable primers for the ends 3 'and 5' of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to be identified, for example a cDNA clone of a cDNA library encoding the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method known in the art.
[000153] Once the nucleotide sequence and the corresponding amino acid sequence of the antibody have been determined, the nucleotide sequence of the antibody can be manipulated using methods known in the art for manipulating nucleotide sequences, for example recombinant DNA techniques, targeted mutagenesis, PCR and the like, to generate antibodies with a different amino acid sequence, for example to create amino acid substitutions, deletions and / or insertions.
[000154] As used herein, the term "stringent conditions" refers to conditions for hybridization and washing. Methods for optimizing the hybridization reaction temperature conditions are known to those skilled in the art. Aqueous and non-aqueous methods are described in this regard and any one of them can be used. Non-limiting examples of stringent hybridization conditions are hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50 degrees Celsius. Another example of stringent hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55 degrees Celsius . Another example of stringent hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 degrees Celsius. Often, stringent hybridization conditions are hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65 degrees Celsius. Even more often, stringent conditions are 0.5M sodium phosphate, 7% SDS at 65 degrees Celsius, followed by one or more washes at 0.2X SSC, 1% SDS at 65 degrees Celsius. The stringent hybridization temperatures can also be changed (for example, lowered) with the addition of certain organic solvents, for example, formamide. Organic solvents such as formamide reduce the thermal stability of double-stranded polynucleotides so that hybridization can be performed at lower temperatures, while maintaining stringent conditions and extending the life of nucleic acids that may be unstable at temperature .
[000155] As used herein, the phrase "hybridize" or its grammatical variations refers to the attachment of a first nucleic acid molecule to a second nucleic acid molecule under low, medium or high stringency conditions, or under synthetic conditions nucleic acid. Hybridization can include cases where a first nucleic acid molecule binds to a second nucleic acid molecule, where the first and second nucleic acid molecules are complementary. As used herein, "specifically hybridizes" refers to preferred hybridization under conditions of nucleic acid synthesis of a primer, for a nucleic acid molecule with a sequence complementarity to the primer compared to hybridization with a nucleic acid molecule that it does not have a sequence of complementarity. For example, specific hybridization includes hybridizing a primer to a target nucleic acid sequence that is complementary to the primer.
[000156] In some embodiments, primers may include a nucleotide substrate that can be complementary to a solid phase nucleic acid primer hybridization sequence or substantially complementary to a solid phase nucleic acid primer hybridization sequence (e.g. example, about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% identical to the primer hybridization sequence complement when aligned). A primer may contain a non-complementary or substantially non-complementary nucleotide substrate to a solid phase nucleic acid primer hybridization sequence (for example, at the 3 'or 5' end of the nucleotide substrate in the primer complementary to or substantially complementary to solid phase primer hybridization sequence).
[000157] A primer, in certain embodiments, may contain a modification such as inosines, abasic sites, blocked nucleic acids, minor slot ligands, duplex stabilizers (for example acridine, spermidine) modifiers Tm or any modifier that alters the binding properties of primers or probes.
[000158] A primer, in certain embodiments, may contain a detectable molecule or identity (for example, a fluorophore, radioisotope, colorimetric agent, particle, enzyme and the like). When desired, the nucleic acid can be modified to include a detectable marker using any method known to those skilled in the art. The marker can be incorporated as part of the synthesis, or added before using the initiator in any of the processes described here. The incorporation of the marker can be carried out in both the liquid and the solid phase. In some embodiments, the detectable marker can be useful for target detection. In some embodiments, the detectable marker may be useful for the quantification of target nucleic acids (for example, to determine the number of copies of a particular sequence or species of nucleic acid). Any detectable marker suitable for detecting an interaction or biological activity in a system can be appropriately selected and used by the expert. Examples of detectable markers are fluorescent markers such as fluorescein, rhodamine and others (for example, Anantha, et al., Biochemistry (1998) 37: 2709 2714; and Qu & Chaires, Methods Enzymol. (2000) 321: 353 369); radioactive isotopes (e.g. 125I, 131I, 35S, 31P, 32P, 33P, 14C, 3H, 7Be, 28Mg, 57Co, 65Zn, 67Cu, 68Ge, 82Sr, 83Rb, 95Tc, 96Tc, 103Pd, 109Cd, and 127Xe); light scattering markers (e.g., U.S. Patent No. 6,214,560, and marketed by Genicon Sciences Corporation, California); chemiluminescent markers and enzyme substrates (for example, dioxetanes and acridinium esters), enzymatic or protein markers (for example, fluorescent green protein (GFP) a color variant, luciferase, peroxidase); other chromogenic markers or dyes (for example, cyanine), and other cofactors or biomolecules such as digoxygenin, strepavidin, biotin (for example, members of a binder pair such as biotin and avidin, for example), affinity capture fractions and the like. In some embodiments, a primer can be tagged with an affinity capture fraction. Also included in the detectable markers are markers useful for mass modification for detection with mass spectrometry (for example, matrix assisted laser ionization / desorption (MALDI) electrospray mass spectrometry (ES)).
[000159] A primer can also refer to a polynucleotide sequence that hybridizes to a substrate of a target nucleic acid or other primer and facilitates the detection of a primer, a target nucleic acid or masks, as with molecular signals, for example. The term "molecular signal" as used herein refers to a detectable molecule in which the molecule's detectable property is detectable only under certain specific conditions, thus allowing it to function as a specific and informative signal. Non-limiting examples of detectable properties are optical properties, electrical properties, magnetic properties, chemical properties and time or speed through an opening of known size.
[000160] In some embodiments, a molecular signal can be a single-stranded oligonucleotide capable of forming a loop structure, where the cycle sequence can be complementary to a target nucleic acid sequence of interest and is flanked by short complementary arms that can form a loop. The oligonucleotide can be labeled at one end with a fluorophore and at the other end with a suppressor molecule. In loop conformation, the energy of the excited fluorophore is transferred to the suppressor, through long-range dipole-dipole coupling similar to that seen in the transfer of fluorescent resonance energy, or FRET, and released as heat instead of light. When the cycle sequence is hybridized to a specific target sequence, the two ends of the molecule are separated and the excited fluorophore energy is emitted as light, generating a detectable signal. Molecular signals offer the additional advantage that excessive sonar removal is unnecessary due to the self-suppressing nature of the unhybridized probe. In some embodiments, molecular signal probes can be designed to discriminate or tolerate mismatches between the cycle and target sequences by modulating the relative forces of the target-cycle hybridization and loop formation. As referred to herein, the term "mismatched nucleotide" or "mismatch" refers to a nucleotide that is not complementary to the target sequence at that or those positions. A probe can have at least one mismatch, but it can also have 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides. Biological characteristics of anti-toxin antibodies and fragments
[000161] An antibody can have one or more characteristics identical or similar to an antibody described herein, and often has one or more biological characteristics that distinguish it from other antibodies that bind the same antigen, alpha-toxin. As used herein, "biological characteristics" of an antibody refers to one or more functional and binding biochemical characteristics that can be used to select antibodies for therapeutic, research and diagnostic uses. For example, anti-alpha toxin antibodies and fragments can be the same or different with respect to epitope binding, bleaching, affinity, neutralization and inhibition of oligomerization or complex formation of heptamer pores, for example.
[000162] The biochemical characteristics of an anti-alpha toxin antibody or fragment include, but are not limited to, isoelectric point (pl) and melting temperature (Tm). The binding characteristics of an anti-alpha toxin antibody or fragment include but are not limited to the specificity of binding; dissociation constant (Kd), or its inverse, association constant (Ka), or its kon or koff component rates; epitope to which it binds; ability to distinguish between various forms and / or preparations of alpha-toxin (for example, recombinant, native, acetylated) and ability to bind a soluble and / or immobilized antigen. Functional characteristics of an antibody presented herein include, but are not limited to, inhibition of alpha-toxin receptor binding, inhibition of alpha-toxin oligomerization, inhibition of gene expression induced by cascade reactions reacting to the expression or activity of alpha- toxin, depletion of cells expressing alpha-toxin, inhibition of cell growth expressing alpha-toxin, inhibition of the location of alpha-toxin, and protection of one or more diseases or disorders related to S. aureus, alpha-toxin or S. aureus and alpha-toxin. Described herein are characteristics of anti-toxin antibodies and fragments and methods for modifying and improving these characteristics. Methods for measuring antibody characteristics are known in the art, some of which are now described. Connection characteristics
[000163] As described above, an anti-alpha toxin antibody or fragment immunospecifically binds to at least one epitope or antigenic determinants of the protein, peptide, subunit, fragment, portion or any combination thereof exclusively or preferably in relation to other polypeptides. The term "epitope" or "antigenic determinant" as used herein refers to a protein determinant capable of binding an antibody in which the term "binding" here often refers to a specific binding. These protein determinants or epitopes often include surface clusters of chemically active molecules such as amino acids or sugar side chains, often having specific three-dimensional structural characteristics and often having specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the bond to the former but not to the latter is lost in the presence of denatured solvents. The term "discontinuous epitope" as used herein, refers to a conformational epitope on a protein antigen of at least two separate zones in the primary protein sequence.
[000164] In certain embodiments, an anti-alpha-toxin antibody or fragment binds immunospecifically to the alpha-toxin of Staphylococcus aureus and antigenic fragments associated with its alpha-toxin oligomerization. In some embodiments, an anti-toxin antibody or fragment binds immunospecifically to the alpha-toxin or at least any three contiguous amino acids of SEQ ID NO: 39 or 40. In some embodiments, the epitope is at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues or at least 9 amino acid residues for the entire specified portion of the contiguous amino acid of the alpha toxin protein. In certain embodiments, the contact residues comprise T261, T263, N264, K266 and K271. In other embodiments, the contact residues comprise N177, W179, G180, P181, Y182, D183, D185, S186, W187, N188, P189, V190, Y191 and R200 of SEQ ID NO: 39. In other embodiments, the contact residues contacts comprise N177, W179, G180, P181, Y182, D183, D185, S186, W187, N188, P189, V190, Y191, R200, T261, T263, N264, K266 and K271 of SEQ ID NO: 39. In certain embodiments, the portion of the alpha toxin in contact with the antibody or an antigen-binding fragment thereof comprises amino acids 261-272 of SEQ ID NO: 39. In other embodiments, the portion of the alpha-toxin in contact with the antibody or a fragment thereof antigen binding agent comprises amino acids 248-277 of SEQ ID NO: 39. In other embodiments, the alpha toxin portion in contact with the antibody or an antigen binding fragment thereof comprises amino acids 173-201 and 261-272 of SEQ ID NO: 39.
[000165] In several embodiments, an anti-toxin antibody or fragment associated with its alpha-toxin oligomerization immunospecifically binds an alpha-toxin polypeptide or antigenic fragment thereof, having at least 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 39 or 40. An anti-toxin antibody or fragment can sometimes bind immunospecifically to an alpha toxin polypeptide or antigenic fragment thereof, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, or 100 % identity to the amino acid sequence of SEQ ID NO: 39 or 40.
[000166] In certain embodiments, an anti-alpha toxin antibody or fragment may bind to a conserved epitope across species. In some embodiments, an anti-toxin antibody or fragment binds to S. aureus alpha-toxin and alpha-toxin homologues or orthologs from other bacterial species and their antigenic fragments. In some embodiments, an anti-toxin antibody or fragment may bind to one or more orthologs and / or alpha-toxin isoforms. In a specific embodiment, an anti-alpha toxin antibody or fragment binds to antigenic and alpha-toxin fragments associated with its alpha-toxin oligomerization of one or more species of bacteria having alpha-toxin homologues or orthologs. In certain embodiments, anti-alpha toxin antibodies or fragments may bind to an epitope within staphylococcus or other closely related bacteria over homologues and / or isoforms and / or conformational variants and / or alpha-toxin subtypes.
[000167] The interactions between antigens and antibodies are often the same as for other non-covalent protein-protein interactions. In general, there are four types of binding interactions between antigens and antibodies: (i) hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forces between Lewis acids and Lewis bases, and (iv) hydrophobic interactions. Hydrophobic interactions are a significant driving force for antibody-antigen interactions and are based on the repulsion of water by non-polar groups rather than the attraction of molecules. However, certain physical forces also contribute to antigen-antibody binding, for example, fitting or complementing epitope forms with different antibody binding sites. Other materials and antigens may exhibit trans-reactivity with an antibody, thus competing for available free antibodies.
[000168] The measurement of a constant affinity and specificity of binding between antigen and antibody is often an element in determining the effectiveness of therapeutic, diagnostic and research methods using alpha-toxin antibodies and fragments. "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site on a molecule (for example, an antibody) and its binding partner (for example, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (for example, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (Kd), which is calculated by the koff / kon ratio. Affinity can be measured by standard methods known in the art, including those described and exemplified here (for example, BiaCore methods). Low-affinity antibodies generally bind antigens slowly and tend to dissociate quickly, while high-affinity antibodies generally bind antigens more quickly and tend to stay on longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present technology.
[000169] An anti-toxin antibody or fragment sometimes has a binding affinity to an alpha-toxin epitope characterized by a dissociation constant (Kd) of 1x10-2M or less, 1x10-3M or less, 1x10-4M or less, 1x10-5M or less, 1x10-6M or less, 1xio — 7M or less, 1x10-8M or less, 1x10-9M or less, 1x10-10M or less, 1x10-11M or less, 1x10-12M or less, 1x10-13M or less, 1x10-14M or less or 1x10-15M or less. For example, the Kd can be from 1x10-15M to 1x10-2M, from 1x10-14M to 1x10-10M, from 1x10-9M to 1x10-5M and from 1x10-4M to 1x10-2M.
[000170] In certain embodiments, an anti-alpha toxin and fragment antibody is a high affinity antibody. By "high affinity antibody" is meant an antibody that binds an alpha toxin epitope with an affinity of less than 10-8M (e.g., 10-9M, 10-10 M, and the like).
[000171] In certain embodiments, an anti-alpha toxin antibody or fragment is described as having a binding affinity for a specific molarity or better. "Or better" when used here means a stronger bond, represented by a lower numerical value Kd. For example, an antibody that has an affinity for an antigen of "0.6 nM or better", the antibody's affinity for the antigen is <0.6 nM, that is, 0.59 nM, 0.58 nM, 0 , 57 nM and the like, or any value less than 0.6 nM.
[000172] In some embodiments, the affinity of an anti-alpha toxin antibody or fragment is described in terms of the association constant (Ka), which is calculated as the kon / koff ratio. In this case, the present anti-toxin antibodies and fragments have binding affinities with an alpha-toxin epitope that include an association constant (Ka) of 1x102M-1or more, 1x103M-1or more, 1x10M-1or more, 1xi05M- 1or more, 1xio6 M-1 or more, 1xi07M-1or more, 1xio8 M-1or more, 1x109 M-1 or more, 1x1010M-1 or more 1x1011 M-1 or more 1x1012M-1or more, 1x1013M-1or more, 1x1014M -1or more or 1x1015 M-1or more. For example, the Ka can be 1x102M-1a 1x107M-1, 1x107M-1a 1x1010M-1, and 1x1010M-1a 1x1015M-1.
[000173] In certain embodiments, the rate at which an anti-alpha toxin antibody or fragment dissociates from an alpha-toxin epitope may be relevant. In some embodiments, an anti-alpha toxin antibody or fragment may bind to the alpha toxin with a koff of less than 10-3s-1, less than 5x10-3s-1, less than 10-4s-1, less than 5x10 -4s-1, or less than 10-5s-1. In some embodiments, the rate at which anti-alpha toxin antibodies and fragments are associated with an alpha-toxin epitope may be more relevant than the Kd or Ka value. In that case, the present anti-toxin antibodies and fragments bind to the alpha toxin with a kon rate of at least 10-4M-1s-1, at least 5 x 10-4M-1s-1, at least 105M -1s-1, at least 5 x105M-1s-1, at least 106M-1s-1, at least 5 x 106M-1s-1, at least 107M-1s-1.
[000174] The determination of binding affinity can be measured using the specific techniques described below in the examples section, see example 1 and methods known in the art. An example of such a method includes the measurement of the dissociation constant "Kd" by a radiolabeled antigen (RIA) binding assay performed with the Fab version of an antibody of interest and its antigen as described by the following test that measures the binding affinity of the Fabs solution with antigen balancing the Fab with a minimal concentration of labeled antigen (125I) in the presence of a series of unlabeled antigen titration, then capturing antigen bound with an anti-Fab plate coated with antibodies. To establish the conditions for the assay, microtiter plates (Dynex) were coated overnight with 5 μg / ml of an anti-capture Fab antibody (Cappel Labs) in 50 mM sodium carbonate (H 9, 6), and subsequently blocked with 2% (w / v) bovine serum albumin PBS for two to five hours at room temperature (approximately 23 degrees Celsius). In a non-absorbent plate (Nunc # 269620), 100 pM or 26 pM [125 I] of antigen were mixed with serial dilutions of a Fab of interest. The Fab of interest is then incubated overnight; however, the incubation can continue for a longer period (for example 65 hours) to ensure that equilibrium is achieved. Then, the mixtures are transferred to the capture plate for incubation at room temperature (for example, for one hour). The solution is then removed and the plate washed eight times with 0.1% Tween-20 in PBS. When the plates are dried, 150 μl / well of scintillant (MicroScint-20; Packard) is added, and the plates are counted in a Topcount gamma counter (Packard) for 10 minutes. The concentrations of each Fab that give less than or equal to 20% of the maximum binding are chosen for use in competitive binding assays.
[000175] In another case, the Kd value can be measured using surface plasmon resonance assays that can be performed for example using a BIAcore ™ -2000 or a BIAcore ™ -3000 (BIAcore, Inc., Piscataway, New Jersey) a 25 degrees Celsius with CM5 platelets of immobilized antigen at “10 response units (UK). Briefly, in an example of such a method, carboxymethylated dextran biosensory platelets (CM5, BIAcore Inc.) are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) hydrochloride (NHS) according to the manufacturer's instructions. The antigen is diluted with 110 mM sodium acetate, pH 4.8, at 5 μg / microliter (“0.2 µM) before being injected at a flow rate of 5 μl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the antigen injection, IM ethanolamine is injected to block unreacted groups. For kinetic measurements, two Fab series dilutions (0.78 nM to 500 nM) are injected into PBS with 0.05% Tween 20 (PBST) at 25 degrees Celsius at a flow rate of approximately 25 μl / min. Association (kon) and dissociation (koff) rates are used using a custom Langmuir single-link model (BIAcore Evaluation Software version 3.2) while simultaneously adjusting the association and dissociation sensorgram.
[000176] If the on-rate exceeds 106M-1 s-1 by the surface plasmon resonance assay, then the on-rate can be determined using a fluorescent suppression technique, for example, which measures the increase or decrease in the intensity of the fluorescent emission (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) at 25 degrees Celsius of a 20 nM antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a spectrophotometer equipped with flow interruption (Aviv Instruments) or a spectrophotometer of the 8000 SLM-Aminco series (ThermoSpectronic) with an agitated tank. A "rate-on" or "rate of association" or "rate of association" or "kon" according to this technology can also be determined with the same surface plasmon resonance technique described above using a BIAcore ™ -2000 or a BIAcore ™ -3000 (BIAcore, Inc., Piscataway, New Jersey) as described above.
[000177] Suitable methods and reagents for determining binding characteristics of an isolated antibody or its binding fragment to displayed antigens, or an altered / mutated derivative (discussed below) are known in the art and / or are available commercially. Equipment and software designed for such kinetic analysis is commercially available (for example, Biacore® A100, and Biacore® 2000 instruments; Biacore International AB, Uppsala, Sweden).
[000178] In some embodiments, a connection test can be performed as direct connection tests or as competition connection tests. The binding can be detected using standard ELISA assays or standard Flow Cytometry. In a direct binding assay, a candidate antibody is tested for binding to alpha toxin antigen. The competition binding assay, on the other hand, assesses the ability of a candidate antibody to compete with an anti-alpha toxin antibody or known fragment or other compound that binds alpha toxin (e.g., receptor, inhibitor). In general, any method that allows an antibody to bind to the alpha toxin that can be detected is within the scope of the present technology to detect and measure the binding characteristics of the antibodies. These methods can also be used to screen a panel of antibodies for those that provide the desired trait.
[000179] In certain embodiments, an isolated antibody or its antigen-binding fragment binds immunospecifically to an alpha-toxin and has one or more of the characteristics selected from the group consisting of: (a) affinity constant (KD) for alpha toxin of about 13 nM or less; (b) binds to alpha-toxin monomers, but does not inhibit the binding of the alpha-toxin to the alpha-toxin receptor; . (c) inhibits the formation of alpha-toxin oligomers by at least 50%, 60%, 70%, 80%, 90% or 95%; (d) reduces the alpha-toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (for example, as determined by hemolysis cell lysis assays); (e) reduces cell infiltration and pro-inflammatory cytokine release (for example, in animal pneumonia models).
[000180] In some embodiments, an isolated antibody or its antigen-binding fragment binds an antigen (for example, alpha-toxin) with an affinity characterized by a dissociation constant (KD) in the range of 0.01 nM to about 50 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30nM, or 40 nM. Functional characteristics
[000181] In certain embodiments, an anti-alpha toxin antibody or fragment alters the biological properties of the alpha toxin and / or cells that express the alpha toxin. In some embodiments, an anti-alpha toxin antibody or fragment neutralizes the biological activity of the alpha toxin by binding the polypeptide and inhibiting the assembly of alpha toxin monomers in a transmembrane pore (for example, alpha toxin heptamer). Neutralization tests can be performed using methods known in the art, in some circumstances, commercially available reagents. Alpha-toxin neutralization is often measured with an IC50 of ixio-6M or less, 1x10-7M or less, ixio-8M or less, ixio — 9 M or less, ixio-10M or less and ixio-11M or less . In certain embodiments, neutralization occurs when the antibody or antigen binding fragment that immunospecifically binds to the alpha a toxin S. aureus or less is at a concentration as described in examples 3-6. In certain embodiments, an anti-alpha toxin antibody or fragment neutralizes the alpha toxin's ability to oligomerize and form a transmembrane pore. The term "50% inhibitory concentration" (abbreviated as "IC50") represents the concentration of an inhibitor (for example, an anti-toxin antibody or fragment provided herein) that is required for 50% inhibition of a given activity of molecule that the inhibitor targets (for example, the oligomerization of the alpha toxin to form a heptamer complex with a transmembrane pore). A value below IC 50 generally corresponds to a more potent inhibitor.
[000182] In certain embodiments, an anti-alpha toxin antibody or fragment inhibits one or more biological activities of the alpha toxin. The term "inhibition" is used here to refer to any statistically significant decrease in biological activity, including total blocking of activity. For example, "inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of biological activity. In certain embodiments, an anti-alpha toxin antibody or fragment inhibits one or more biological activities of the alpha toxin by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 %, or 100%.
[000183] In some embodiments, an anti-alpha toxin antibody or fragment may deplete the alpha toxin secreted by the pathogenic S. aureus. In some embodiments, an anti-toxin antibody or fragment can reach at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% depletion of the alpha toxin secreted by S. aureus. In particular modalities, practically all the detectable secreted alpha-toxin is depleted of cells infected with S. aureus.
[000184] In certain embodiments, an anti-alpha toxin antibody or fragment may inhibit in vitro stimulated alpha-toxin activity (e.g., receptor binding, oligomerization) and / or cell proliferation expressing or secreting alpha-toxin. An anti-alpha toxin antibody or fragment sometimes inhibits the activity of the alpha toxin in vitro, pathogenicity S. aureus by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% or at least about 75%. Methods for measuring cell proliferation, pathogenicity and hemolysin alpha activity are known in the art.
[000185] In certain embodiments, an anti-alpha toxin antibody or fragment may inhibit the expression of one or more inducible genes that respond directly or indirectly to the environment created by S. aureus infection and / or expression and function of the alpha toxin. In specific embodiments, an anti-alpha toxin antibody or fragment can inhibit the expression of one or more inducible genes that respond directly or indirectly to the environment created by S. aureus infection and / or alpha-toxin expression and function in at least 20 %, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, or at least 200%. Production of anti-toxin antibodies and fragments
[000186] The following describes exemplary techniques for the production of antibodies. An alpha-toxin antigen used for the production of antibodies can be a peptide fragment that, for example, includes a zone of the alpha-toxin peptide sequence involved in oligomerization. Antibodies against alpha toxin can be generated using native S. aureus alpha toxin comprising SEQ ID NO: 39, or mutant alpha toxin comprising SEQ ID NO: 40, a variant, or an antigenic fragment thereof. S. aureus cells expressing and secreting alpha toxin can also be used to generate antibodies. Examples of nucleotide sequences and alpha-toxin amino acid sequences are available as provided in table 10, for example. Alpha-toxin can be produced recombinantly in an isolated form of bacterial or eukaryotic cells using standard recombinant DNA methodology. Alpha-toxin can be expressed as labeled (eg, epitope tag) or other fusion protein (eg, GST fusion) to facilitate isolation, as well as for identification in various assays. Antibodies or binding proteins that bind to various markers and fusion sequences are available as indicated below. Other forms of alpha-toxin useful for generating antibodies can be used.
[000187] Various marker polypeptides and their respective antibodies are known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; flu HA polypeptide tag and its 12CA5 antibody; c-myc marker and antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 for that; and Herpes Simplex virus (gD) glycoprotein D marker and its antibody. The FLAG peptide is recognized by an anti-FLAG M2 monoclonal antibody. Purification of a protein containing the FLAG peptide can be performed by immunoaffinity chromatography using an affinity matrix comprising the anti-FLAG M2 monoclonal antibody covalently linked to agarose. Other marker polypeptides include the KT3 epitope peptide; an α-tubulin epitope peptide; and peptide marker of 10 T7 gene proteins.
[000188] Polyclonal antibodies to an antigen of interest can be produced by various procedures known in the art. For example, an alpha toxin polypeptide or its immunogenic fragment can be administered to various host animals including, but not limited to, rabbits, mice, and the like to induce the production of polyclonal antibodies containing antigen-specific sera. Various adjuvants can be used to increase the immune response, depending on the host species, and include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyions , peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol and potentially useful human adjuvants such as BCG (bacillus Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are known in the art.
[000189] Polyclonal antibodies can be created in animals through various subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) with a protein that is immunogenic to the species to be immunized. For example, the antigen can be conjugated to Californian keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, using a bifunctional or derivatizing agent (reactive group), for example, activated ester (conjugation via cysteine or lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N = C = NR, where R and R1 are different alkyl groups. Conjugates can also be made in recombinant cell culture as fusion proteins.
[000190] Normally, animals are immunized against the antigen, immunogenic conjugates or derivatives by combining an appropriate concentration of antigen or conjugated with adjuvant and injecting the solution in several locations. Immunizations can also be performed as described in example 1 (immunization / hybridoma generation).
[000191] Monoclonal antibodies can be prepared using various techniques known in the art, including the use of hybridoma, recombinant and phage display technology, or a combination thereof. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous or isolated antibodies, for example, the individual antibodies comprising the population are identical except for any natural mutations that may occur that may be present in quantities minors. Monoclonal antibodies are highly specific, being directed against a single antigenic site or multiple antigenic sites in the case of modified multispecific antibodies. In addition, in contrast to polyclonal antibody preparations that include different antibodies targeting different determinants (epitopes), each monoclonal antibody is directed against the same determinant in the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they can be synthesized without being contaminated by other antibodies. The "monoclonal" modifier is not to be understood as requiring production of the antibody by any particular method. The following is a description of representative methods of producing monoclonal antibodies that is not intended to be limiting and can be used to produce, for example, mammalian monoclonal, chimeric, humanized, human, domain, diabody, vaccine, linear and multispecific antibodies.
[000192] Methods for producing and screening specific antibodies using hybridoma technology are routine and known in the art. In the hybridoma method, mice or other suitable host animals, such as hamsters, are immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the antigen used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusion agent or fusion partner, such as polyethylene glycol to form a hybridoma cell. In certain embodiments, the selected myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a selective medium that selects against unfused parental cells. In one aspect, myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available at the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 and derivatives , for example X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.
[000193] Once the hybridoma cells that produce the antibodies of the desired specificity are identified, affinity and / or activity are identified, the clones can be subcloned limiting the dilution procedures and developed by standard methods. Suitable culture media for this purpose include, for example, D-MEM medium or RPMI-1640 medium. In addition, hybridoma cells can be developed in vivo as ascites tumors in an animal, for example by intraperitoneal injection of cells into mice.
[000194] Monoclonal antibodies secreted by the subclones are appropriately separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (for example, using protein A or protein G -Sepharose) or ion exchange chromatography, affinity markers, hydroxylapatite chromatography, gel electrophoresis, dialysis and the like. Exemplary purification methods are described in more detail below. Recombinant DNA techniques
[000195] Methods for producing and screening specific antibodies using recombinant DNA technology are routine and known in the art. Monoclonal antibodies encoding DNA can be readily isolated and / or sequenced using conventional procedures (for example, using oligonucleotide probes that are able to specifically bind to genes encoding murine antibody heavy and light chains). Once isolated, DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that would not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. As described below for antibodies generated by phage display and humanization of antibodies, the DNA or genetic material for recombinant antibodies can be obtained from source (s) other than hybridoma to generate an anti-alpha toxin antibody or fragment.
[000196] Recombinant expression of an antibody or its variant often requires the construction of an expression vector containing a polynucleotide that encodes the antibody. Replicable vectors are provided herein comprising a nucleotide sequence encoding an antibody molecule, an antibody heavy or light chain, an heavy or light chain variable domain of an antibody or its portion, or an operably linked heavy or light chain CDR to a prosecutor. Such vectors can include the nucleotide sequence encoding the constant zone of the antibody molecule and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both heavy and light chains.
[000197] Once an expression vector has been transferred to a host cell using conventional techniques, the transfected cells are then cultured by conventional techniques to produce an antibody. Thus, host cells containing a polynucleotide encoding an isolated antibody or its antigen-binding fragment or fragments thereof, or its heavy or light chain, or its portion, or a single chain antibody, operably linked to a heterologous promoter are provided herein. In certain embodiments, for the expression of double chain antibodies, vectors encoding the heavy and light chains can be coexpressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
[000198] Mammalian cell lines available as hosts for expression of recombinant antibodies are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells ), HeLa cells, baby hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney cells 293, and several other cell lines. Different host cells have specific characteristics and mechanisms for post-translational processing and modification of proteins and genetic products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the antibody or its expressed portion. For this purpose, eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the genetic product can be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 cells (a murine myeloma cell line that does not produce endogenously any functional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst. In certain embodiments, human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies. In some embodiments, the human cell line PER.C6. (Crucell, Netherlands) can be used to recombinantly produce monoclonal antibodies.
[000199] Additional cell lines that can be used as hosts for expression of recombinant antibodies include but are not limited to (eg, Sf21 / Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (eg, S. cerevisiae , Pichia, US7326681; etc), plant cells (US20080066200); and chicken cells.
[000200] In certain embodiments, the antibodies presented here are expressed in a cell line with stable antibody expression. Stable expression can be used for long-term production of high yield of recombinant proteins. For example, cell lines can be generated that express the antibody molecule stably. Host cells can be transformed with an appropriately modified vector comprising expression control elements (e.g., promoter, enhancer, transcription terminators, polyadenylation sites and the like) and a selectable marker gene. Following the introduction of the foreign DNA, cells can grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection and allows cells that have stably integrated the plasmid into their chromosomes to develop and form foci that in turn can be cloned and expanded into cell lines. Methods for producing stable cell lines in high yield are known in the art and reagents are generally commercially available.
[000201] In certain embodiments, the antibodies presented here are expressed in a cell line with transient expression of the antibody. Transient transfection is a process in which the nucleic acid introduced into a cell does not integrate into the genome or chromosomal DNA of that cell. Often nucleic acid is maintained as an extrachromosomal element, for example as an episome. The episome's nucleic acid transcription processes are unaffected and a protein encoded by the episome's nucleic acid is produced.
[000202] The cell line, which can be transfected stably or transiently, is maintained in a cell culture medium and under conditions known in the art, resulting in expression and production of monoclonal antibodies. In certain embodiments, the mammalian cell culture media are based on commercially available medium formulations, including, for example, DMEM or Ham's F12. In some embodiments, cell culture media are modified to support increases in cell development and biological protein expression. As used herein, the terms "cell culture medium", "culture medium" and "medium formulation" refer to a nutritive solution for the maintenance, development, propagation or expansion of cells in an artificial in vitro environment outside a multicellular organism or tissue. The cell culture medium can be optimized for the use of a specific cell culture, including for example a cell culture development medium that is formulated to promote cell development, or a cell culture production medium that is formulated to promote production of recombinant protein. The terms nutrient, ingredient and component are used interchangeably here to refer to the constituents that make up a cell culture medium.
[000203] In several modalities, cell lines are maintained using a batch method. As used herein, "batch method" refers to a method by which a batch cell culture is fed with additional nutrients after being first incubated with a basal medium. For example, a batch method may comprise adding supplementary means according to a given feeding schedule within a given period of time. Thus, a "batch cell culture" refers to a cell culture where cells, typically mammals, and culture medium are fed with the culture vessel and additional culture nutrients are fed, either continuously or in discrete increments, to culture during cultivation, with or without periodic cell and / or product harvest before the end of culture.
[000204] The cell culture medium used and the nutrients contained therein are known in the prior art. In some embodiments, the cell culture medium comprises a basal medium and at least one hydrolyzate, for example, a soy-based hydrolyzate, a yeast-based hydrolyzate, or a combination of the two types of hydrolysates resulting in a modified basal medium. The additional nutrients can sometimes include only a basal medium, such as a concentrated basal medium, or it can include only concentrated hydrolysates or hydrolysates. Suitable basal media include but are not limited to Dulbecco's modified Eagle Essential Medium (DMEM), DME / F12, Medium Essential Medium (MEM), Medium Basal Eagle (BME), RPMI 1640, F-10, F-12, α -Minimum Essential Medium (α-MEM), Glasgow Minimal Essential Medium (G-MEM), PF CHO (see, for example Protein-Free CHO Medium (Sigma) or EX-CELL ™ 325 PF CHO Serum-Free Medium for CHO Cell Without Protein (SAFC Bioscience), and Iscove Modified Dulbecco Medium Other examples of basal media that can be used in the technology in question include BME Basal Medium; Dulbecco Modified Eagle Medium (DMEM, powder) (Gibco- Invitrogen (# 31600). In certain embodiments, the basal medium may lack serum, which means that the medium has no serum (for example, fetal bovine serum (FBS), horse serum, goat serum, or any other serum derived from animals known in the state of the art) or medium without animal protein or chemically defined medium.
[000205] The basal medium can be modified to remove certain non-nutritive components found in standard basal medium, such as various inorganic and organic buffers, surfactant (s) and sodium chloride. Removing such components from the basal cell environment allows for a greater concentration of the remaining nutrient components and can improve overall cell growth and protein expression. In addition, omitted components can be added back into the cell culture medium containing the basal cell medium modified according to the requirements of the cell culture conditions. In certain embodiments, the cell culture medium contains a modified basal cell medium and at least one of the following nutrients, a source of iron, a recombinant development factor; a buffer, a surfactant; an osmolarity regulator; a source of energy; and non-animal hydrolysates. In addition, the modified basal cell medium can optionally contain amino acids, vitamins or a combination of amino acids and vitamins. In some embodiments, the modified basal cell medium also contains glutamine, for example L-glutamine and / or methotrexate.
[000206] In some embodiments, the production of antibodies is conducted in large quantities by a bioreactor process using batch, batch, perfusion or continuous feed bioreactor methods known in the art. Large-scale bioreactors have at least 1000 liters of capacity, sometimes around 1000 to 100,000 liters of capacity. These bioreactors can use impellers to distribute oxygen and nutrients. Small-scale bioreactors generally refer to cell culture at a maximum of 100 liters of volumetric capacity and can range from about 1 liter to about 100 liters. Alternatively, bioreactors (SUB) can be used for large or small scale culture.
[000207] Temperature, pH, agitation, aeration and density of inoculation may vary depending on the host cells used and the recombinant protein to be expressed. For example, a recombinant protein cell culture can be maintained at a temperature between 30 to 45 degrees Celsius. The pH of the culture medium can be monitored during the culture process so that the pH remains at an optimal level, which can be for certain host cells within a pH between 6.0 and 8.0. An impeller-driven mixture can be used by such culture methods for stirring. The rotational speed of the impeller can be approximately 50 to 200 cm / sec. speed, but other elevation or mixing / aeration systems known in the art can be used, depending on the type of host cell to be cultured. Sufficient aeration is provided to maintain a dissolved oxygen concentration of approximately 20% to 80% air saturation in the culture, again depending on the selected host cell to be cultured. Alternatively, a bioreactor can spray air or oxygen directly into the culture medium. There are other methods of oxygen supply, including bubble-free aeration systems employing hollow fiber membrane aerators. Phage display techniques
[000208] In some embodiments, monoclonal antibodies or antibody fragments can be isolated from phage antibody libraries generated using techniques known in the art. In such methods, an anti-alpha toxin antibody or fragment can be isolated by screening a recombinant combinatorial antibody library, sometimes a scFv phage display library, prepared using VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. Antibody purification and isolation
[000209] Once the antibody molecule has been produced by recombinant expression or hybridoma, it can be purified by any method known in the art to purify an immunoglobulin molecule, for example by chromatography (for example ion exchange, affinity, particularly by affinity by specific antigens Protein A or Protein G, and design of column chromatography), centrifugation, differential solubility or any other standard technique for protein purification. In addition, the antibodies of the present technology or fragments thereof can be fused to heterologous polypeptide sequences (referred to herein as "markers") described above or also known in the art to facilitate purification. Humanized antibodies
[000210] In certain embodiments, antibodies of the present technology are humanized antibodies that are generated using methods known in the art. Humanized antibodies can be chimeric antibodies. Chimeric antibodies are antibodies in which a portion of the heavy and / or light chain is identical to or homologous with corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while another portion of the chain (s) ( s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity. Chimeric antibodies of interest include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., Old World monkey, such as baboons, rhesus monkey or cinomolg) and human constant zone sequences. Human antibodies
[000211] As an alternative to humanization, human antibodies can be generated using methods known in the art. Human antibodies avoid some of the problems associated with antibodies that have variable and / or constant murine or rat areas. The presence of such murine or rat-derived proteins can lead to rapid removal of antibodies or can lead to the generation of an immune response against a patient's antibody. In order to avoid the use of murine or rat derived antibodies, fully human antibodies can be generated by introducing a locus of functional human antibodies into a rodent, mammal or animal so that the rodent, other mammal or animal produces antibodies fully humans.
[000212] For example, it is now possible to produce transgenic animals (for example, mice) that are capable, after immunization, of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been reported that homozygous deletion of the antibody heavy chain (JH) binding zone gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The transfer of the human germline immunoglobulin gene set to such germline mutant mice results in the production of human antibodies after the antigen challenge. In practice, the use of XenoMouse® mouse strains that have been modified to contain configured germline fragments with not less than 1000 kb of the heavy chain locus and hood light chain locus. XenoMouse® strains are marketed by Amgen, Inc. (Fremont, California).
[000213] The production of the XenoMouse® strains of mice and antibodies produced in those mice is known in the prior art. Essentially, XenoMouse® lines are immunized with an antigen of interest (for example, alpha toxin), lymphatic cells (such as B cells) are recovered from hyperimmunized mice and the recovered lymphocytes are fused with a myeloid cell line to prepare cell lines. immortal hybridoma using techniques described above and known in the art. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest.
[000214] In an alternative approach, the miniloccus approach, an exogenous Ig locus is imitated by including parts (individual genes) of the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a constant mu zone, and usually a second constant zone (for example, a constant gamma zone) are formed in a construct for insertion into an animal .
[000215] The generation of human antibodies from mice in which large pieces of chromosomes, or whole chromosomes, have been introduced, through microcellular fusion, is known in the prior art. In addition, KM ™ mice were generated, which are the result of miscegenation of Kirin's Tc mice with Medarex miniloccus mice (Humab). These mice have the human IgG transromosome of the Kirin mice and the cover chain transgene of the Genpharm mice.
[000216] Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display (MedImmune (formerly CAT), Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display (MedImmune (formerly CAT)), display of yeast, and the like. Phage display technology can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin repertoires of the variable domain (V) gene from unimmunized donors. In accordance with this technique, antibody V domain genes are cloned into the structure of a larger or smaller coat protein gene from a filamentous bacterophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a copy of single-stranded DNA from the phage genome, selections based on the antibody's functional properties also result in the selection of the gene encoding the antibody displaying those properties. Thus, the phage mimics the properties of the B cell. The phage display can be performed in several formats. Various sources of V gene segments can be used for phage display. A diverse set of anti-oxazolone antibodies was isolated from a small random combinatorial library of V genes from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse set of antigens (including autoantigens) can be isolated essentially following techniques known in the art. As discussed above, human antibodies can also be generated in B cells activated in vitro.
[000217] The immunoglobulin genes undergo several changes during the maturation of the immune response, including recombination between segments of genes V, D and J isotypes exchange, and hypermutation in the variable zones. Somatic recombination and hypermutation are the foundation for the generation of antibody diversity and affinity maturation, but they can also generate sequence susceptibilities that can hinder the commercial production of such immunoglobulins as therapeutic agents or increase the risk of antibody immunogenicity. In general, mutations in the CDR zones can likely contribute to improved affinity and function, while mutations in the structure zones can increase the risk of immunogenicity. The risk can be reduced by reversing structure mutations to the germline while ensuring that antibody activity is not adversely affected. Diversification processes can also generate some susceptibilities or these susceptibilities can exist within the germline sequences, contributing to the variable domains of heavy and light chains. Regardless of the source, it may be desirable to remove any structural susceptibilities that may result in instability, aggregation, heterogeneity of the product or increased immunogenicity. Examples of undesirable susceptibilities include cysteines (which can lead to disulfide bond shuffling, variable sulfhydryl adduct formation), N-linked glycosylation sites (resulting in heterogeneity of structure and activity), as well as diamidation sites (for example, NG , NS), isomerization (DG), oxidation (exposed methionine) and hydrolysis (DP).
[000218] Accordingly, to reduce the risk of immunogenicity and improve the pharmaceutical properties of the antibodies disclosed in example 11, tables 1-8, it may be desirable to revert a germline structure sequence, revert a germline and / or a CDR remove a structural susceptibility. Thus, in some embodiments, when a particular antibody is different from its germline sequence at the amino acid level, the antibody sequence can be reassigned to the germline sequence. Such corrective mutations can occur in one, two, three or more positions or a combination of any of the mutated positions, using standard molecular biological techniques.
[000219] Other approaches include VelocImmune® technology (Regeneron Pharmaceuticals). Velocimmune® technology can be used to generate fully human monoclonal antibodies for targets of therapeutic interest and involves the generation of a transgenic mouse with a genome comprising variable human zones of heavy and light chain operationally linked to endogenous loci of constant mouse zones. that the mouse produces an antibody comprising a variable human zone and a constant mouse zone in response to the antigenic stimulus. The DNA encoded from the variable zones of the heavy and light chains of the antibody is isolated and operationally linked to the DNA encoded from the human zones of the heavy and light chain. The DNA is then expressed in a cell capable of expressing the fully human antibody. See, for example, U.S. Patent No. 6,596,541. Antibody fragments
[000220] In certain embodiments, the antibodies present are antibody fragments or antibodies comprising those fragments. The antibody fragment comprises a portion of the integral antibody, which is usually its antigen binding or variable zone. Examples of antibody fragments include Fab, Fab ', F (ab') 2, Fd and Fv fragments. Diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies are antibodies formed from these antibody fragments.
[000221] Traditionally, these fragments were derived by means of proteolytic digestion of intact antibodies using techniques known in the art. However, these fragments can now be produced directly by recombinant host cells. Fragments of Fab, Fv and scFv antibodies can all be expressed and secreted from E. coli, thus allowing easy production of large quantities of these fragments. In some embodiments, antibody fragments can be isolated from the phage antibody libraries mentioned above. Alternatively, Fab'-SH fragments can also be directly recovered from E. coli and chemically coupled to form F (ab ') 2 fragments. According to another approach, F (ab ') 2 fragments can be isolated directly from a culture of recombinant host cells. Other techniques for producing antibody fragments are known. In some embodiments, the antibody chosen is a single chain Fv fragment (scFv). In certain embodiments, the antibody is not a Fab fragment. Fv and scFv are the only species with intact combining sites that do not have constant zones; thus, reduced nonspecific binding during in vivo use is suitable. ScFv fusion proteins can be constructed to fuse an effector protein at the amino or carboxy terminus of an scFv.
[000222] In certain embodiments, the antibodies present are domain antibodies, for example antibodies containing small functional antibody binding units, corresponding to the variable zones of the heavy (VH) or light (VL) chains of human antibodies. Examples of domain antibodies include, but are not limited to, those marketed by Domantis, which are specific for therapeutic purposes. Commercially available domain antibody libraries can be used to identify anti-alpha toxin domain antibodies. In certain embodiments, anti-toxin antibodies and fragments comprise an alpha-toxin functional binding unit and a functional gamma Fc receptor binding unit.
[000223] In certain embodiments here, the antibodies present are linear antibodies. Linear antibodies comprise a pair of Fd segments in series (VH-CH1-VH-CH1) that form a pair of antigen binding zones. Linear antibodies can be bispecific or monospecific. Certain changes in the amino acid sequence
[000224] In addition to human, humanized and / or chimeric antibodies, the present technology also includes other modifications, and their variants and fragments, of an anti-alpha toxin antibody or fragment comprising one or more of the following: amino acid residues and / or polypeptide substitution, addition and / or elimination in the variable light domain (VL) and / or heavy variable domain (VH) and / or Fc zone and a post-translational modification. Included in these modifications are antibody conjugates in which an antibody has been covalently linked to a fraction. Suitable fractions for binding to antibodies include, but are not limited to, proteins, peptides, drugs, labels and cytotoxins. Such changes to antibodies can be made to alter or improve characteristics (eg, biochemical, binding and / or functional) of antibodies as appropriate for treatment and / or diagnosis of diseases associated with S. aureus and / or mediated by alpha toxin . Methods for forming conjugates, making changes to amino acids and / or polypeptides and post-translational modifications are known in the art, some of which are detailed below. Any combination of elimination, insertion and replacement to arrive at a final construct, as long as the final construct has the desired characteristics.
[000225] Amino acid changes to antibodies result in sequences that are less than 100% identical to an antibody sequence or main antibody sequence described herein. In certain embodiments, in that context, antibodies may have about 25% to about 95% sequence identity to the amino acid sequence of the heavy or light chain variable domain of an anti-alpha toxin antibody or fragment as described herein. . Thus, in certain embodiments, a modified antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% identity of the amino acid sequence or similarity to the amino acid sequence of the heavy or light chain variable domain of an anti-alpha toxin antibody or fragment as described herein. In certain embodiments, an altered antibody can have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% sequence identity amino acid or similarity to the amino acid sequence of the heavy or light chain variable domain CDR1, CDR2, or CDR3 of an anti-alpha toxin antibody or fragment as described herein. An altered antibody can sometimes have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity to the amino acid sequence of the heavy or light chain variable domain FR1, FR2, FR3 or FR4 of an anti-alpha toxin antibody or fragment as described herein.
[000226] In certain embodiments, altered antibodies are generated by one or more amino acid changes (for example substitutions, deletion and / or additions) introduced in one or more variable zones of the antibody. In various modalities, amino acid changes are introduced in the structure zones. One or more changes in the residues of the framework zone may result in an improvement in the binding affinity of the antibody to the antigen. This can be especially true when these changes are made to humanized antibodies when the structure zone may be of a different species than the CDR zones. Examples of structural zone residues to be modified include those that non-covalently bind antigen directly, interact with / effect the formation of a CDR and / or participate in the VL-VH interface. In some embodiments, between one and about five structure residues can be changed. This can sometimes be sufficient to make a mutant antibody suitable for use in pre-clinical trials, even when none of the residues in the hypervariable zone has been altered. Typically, however, an altered antibody will comprise one or more additional hypervariable zone changes. In certain embodiments, the hypervariable zone residues can be changed randomly, especially when the onset binding affinity of an anti-toxin antibody or fragment to the antigen of the second mammalian species is such that such randomly produced antibodies can be screened readily.
[000227] A useful procedure for generating altered antibodies is called "alanine scan mutagenesis". In this method, one or more hypervariable zone residues are replaced by one or more alanine or polyalanine residues to alter the interaction of amino acids with alpha toxin. This or these hypervariable zone residues demonstrating functional sensitivity to substitutions are then refined by introducing more or other mutations at or to the substitution sites. Thus, while the location for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se does not need to be predetermined. The Ala-mutants thus produced are screened for their biological activity as described herein.
[000228] In certain embodiments, the substitutional variant involves replacing one or more residues of the hypervariable zone of a main antibody (for example a humanized or human antibody). Generally, the resulting variant (s) selected for further development will have improved biological properties over the main antibody from which they were generated. A convenient way to generate such substitutional variants involves affinity maturation using phages. Soon, several hypervariable zone sites (for example 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus generated are displayed monovalently from filamentous phage particles as fusions for the M13 gene III product packaged with each particle. Phage display mutants are then screened for their biological activity (e.g., binding affinity) as disclosed herein.
[000229] Mutations in antibody sequences can include substitutions, deletions, including internal deletions, additions, including additions giving fusion proteins, or conservative substitutions of amino acid residues within and / or adjacent to the amino acid sequence, but this results in a change "silent" because the change produces a functionally equivalent anti-alpha toxin antibody or fragment. Conservative amino acid substitutions can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues involved. For example, non-polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; neutral polarity amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged amino acids (acids) include aspartic acid and glutamic acid. In addition, glycine and proline are residues that can influence the orientation of the chain. Non-conservative substitutions will imply exchanging a member of one of these classes for a member of another class. In addition, if desired, non-classical amino acids or chemical chemical amino acids can be introduced as a substitution or addition to the antibody sequence. Non-classic amino acids include, but are not limited to, D isomers of common amino acids, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cystic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro amino acids, amino acids such as beta-methyl amino acids, C-alpha-methyl amino acids, N-alpha-methyl amino acids, and analogous amino acids in general.
[000230] In certain embodiments, any cysteine residue not involved in maintaining the proper conformation of an anti-alpha toxin antibody or fragment can also be replaced, usually with serine, to improve the oxidative stability of the molecule and avoid aberrant crosslinking. Conversely, one or more cysteine bonds can be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).
[000231] In some embodiments, an antibody can be modified to produce fusion proteins; that is, the antibody, or a fragment thereof, fused to a heterologous protein, polypeptide or peptide. In several embodiments, the protein fused to the antibody portion is an enzyme component of Antibody-Directed Therapy-Enzyme-Pro-Drug (ADEPT). Examples of other proteins or polypeptides that can be modified as an antibody fusion protein include, but are not limited to toxins such as ricin, abrin, ribonuclease, DNase I, enterotoxin-A staphylococcus, anti-viral protein Phytolacca, gelonin, toxin diphtheria, exotoxin Pseudomonas, and endotoxin Pseudomonas. Enzymatically active toxins and fragments thereof which may include diphtheria A chain, active fragments of non-binding diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha -sarcina, Aleurites fordii proteins, diantina proteins, American Phytolaca proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcine, croton, saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecenes .
[000232] Additional fusion proteins can be generated through known techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA scrambling can be used to alter the characteristics of the antibody or its fragments (for example, an antibody or fragment with higher affinities and lower dissociation rates). An antibody can further be a binding domain immunoglobulin fusion protein, as known in the art. Fc variant zones
[000233] The present invention also includes binding members of the invention, and in particular the antibodies of the invention having modified IgG constant domains. Antibodies of the human class lgG that have functional characteristics such as long serum half-life and the ability to mediate various effector functions are used in certain modalities of the invention (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., chapter 1 (1995) ). The human class antibody lgG is further classified into the following 4 classes: IgG1, IgG2, IgG3 and IgG4. A large number of studies have so far been conducted for ADCC and CDC as effector functions in the lgG class antibody and it has been reported that among antibodies of the human class lgG, the subclass lgG1 has the highest ADCC and CDC activity in humans ( Chemical Immunology, 65, 88 (1997)).
[000234] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (eg, Natural Killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and subsequently cause lysis of the target cell. In one embodiment, such cells are human cells. Despite not wanting to limit it to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs). Primary cells to mediate ADCC, NK cells, express FCYRIII, while monocytes express FCYRI, FCYRII, FCYRIII and / or FCYRIV. FcR expression in hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9: 457-92 (1991). To assess the ADCC activity of a molecule, an in vitro ADCC assay such as that described in U.S. Patent No. 5,500,362 or 5,821,337 can be performed. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer cells (NK). Alternatively, or in addition, the ADCC activity of the molecules of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95: 652-656 (1998).
[000235] "Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to initiate complementary activation and lysis to a target in the presence of a complement. The complement activation pathway is initiated by binding the first component of the component system (C1q) to a molecule (for example, an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed, for example as described by Gazzano-Santaro et al., J. Immunol. Methods, 202: 163 (1996).
[000236] Expression of ADCC activity and CDC activity of antibodies of the human subclass lgG1 generally involves binding the antibody's Fc zone to a receptor for an antibody (hereinafter referred to as "FCYR") that exists on the surface of effector cells such as cells killer cells, natural killer cells or activated macrophages. Several add-on components can be connected. Regarding binding, it has been suggested that several amino acid residues in the hinge zone and the second domain of zone C (hereinafter referred to as "CY2 domain") of the antibody are important (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319 (1995), Chemical Immunology, 65, 88 (1997)) and that a sugar chain in the Cy2 domain (Chemical Immunology, 65, 88 (1997)) is also important.
[000237] "Effector cells" are leukocytes that express one or more FcRs and perform effector functions. The cells express at least FCYRI, FCyRII, FcyRIII and / or FCYRIV and play an ADCC effector role. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils.
[000238] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to an Fc zone of an antibody. In one embodiment, Fcr is a native human FcR sequence. In addition, in certain embodiments, the FcR binds an IgG antibody (a gamma receptor) and includes receptors from the subclasses FCYRI, FcyRII, FCYRIII, and FcyRIV, including allelic variants and alternatively joined forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ essentially in their cytoplasmic domains. The FCYRIIA activation receptor contains an immunoreceptor tyrosine-based activation (ITAM) motif in its cytoplasmic domain. The FCYRIIB inhibitor receptor contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, Daêron, Annu. Rev. Immunol., 15: 203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9: 457-92 (1991); Capel et al., Immunomethods, 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are covered by the term "FcR" here. The term also includes the neonatal FcRn receptor, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., Immunol., 117: 587 (1976) and Kim et al., J. Immunol., 24: 249 (1994 )).
[000239] In certain embodiments, an anti-alpha toxin antibody or fragment comprises an altered Fc zone (also referred to herein as "variant Fc zone") in which one or more changes have been made to the Fc zone in order to alter the functional and / or pharmacokinetics of antibodies. Such changes may result in a decrease or increase in Clq binding and complement-dependent cytotoxicity (CDC) or FcgammaR binding, for IgG. The present technology encompasses the antibodies described here with variant Fc zones where changes have been made to alter the effector function, providing a desired effect. Accordingly, in some embodiments, an anti-alpha toxin antibody or fragment comprises a variant Fc zone (i.e., Fc zones that have been altered as noted below). Anti-alpha toxin antibodies and fragments referred to herein comprising a variant Fc zone are also referred to herein as "Fc variant antibodies". As used herein, native refers to the unmodified main sequence and the antibody comprising a native Fc zone is referred to herein as a "native Fc antibody". In some modalities, the variant Fc zone exhibits a similar level of inducing effector function when compared to the native Fc zone. In certain modalities, the variant Fc zone exhibits a greater induction of the effector function when compared to the native Fc zone. In certain modalities, the variant Fc zone exhibits less induction of the effector function when compared to the native Fc zone. Some specific types of variant Fc zones are detailed here. Methods of measuring effector function are known in the art.
[000240] The effector function of an antibody can be modified through changes in the Fc zone, including but not limited to amino acid substitutions, amino acid additions, amino acid deletion and changes in post-translational modifications to Fc amino acids (for example, glycosylation). The methods described below can be used to alter the effector function of an isolated antibody or antigen binding fragment as described herein, resulting in an antibody or antigen binding fragment having certain advantageous properties for the prophylaxis or treatment of a particular disease or condition. associated with Staphylococcal aureus.
[000241] In some embodiments, a variant Fc antibody is prepared that has altered binding properties for an Fc ligand (e.g., an Fc, C1q receptor) relative to a native Fc antibody. Examples of binding properties include, but are not limited to, binding specificity, equilibrium dissociation constant (Kd), dissociation and association rates (koff and kon, respectively), binding affinity and / or avidity. It is known in the prior art that the equilibrium dissociation constant (Kd) is defined as koff / kon. In certain aspects, an antibody comprising a variant Fc zone with a low Kd may be more desirable than an antibody with a high Kd. However, in some cases, the kon or koff value may be more relevant than the Kd value. The most important kinetic parameter for a given antibody application can be determined.
[000242] In some embodiments, variant Fc antibodies exhibit altered binding affinity for one or more Fc receptors including, but not limited to, FcRn, FcgammaRI (CD64) including FcgammaRIA, FcgammaRIB, and FcgammaRIC isoforms; FcgammaRII (CD32 including FcgammaRIIA, FcgammaRIIB, and FcgammaRIIC isoforms); and FcgammaRIII (CD16, including FcgammaRIIIA and FcgammaRIIIB isoforms) when compared to a native Fc antibody.
[000243] In certain embodiments, a variant Fc antibody has improved binding to one or more Fc ligands over a native Fc antibody. In certain embodiments, the variant Fc antibody exhibits increased or decreased affinity for an Fc linker that is at least 2 times, or at least 3 times, or at least 5 times, or at least 7 times, or at least 10 times, or at least at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or is between 2 times and 10 times, or between 5 times and 50 times, or between 25 times and 100 times, or between 75 times and 200 times, or between 100 and 200 times, more or less than a native Fc antibody. In various embodiments, variant Fc antibodies exhibit affinities for an Fc linker that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than a native Fc antibody. In certain embodiments, a variant Fc antibody has an increased affinity for an Fc linker. A variant Fc antibody may sometimes have less affinity for an Fc linker.
[000244] In some embodiments, a variant Fc antibody has improved binding with the Fc FcgammaRIIIA receptor. In some embodiments, a variant Fc antibody has improved binding to the Fc FcgammaRIIIB receptor. In some embodiments, a variant Fc antibody has improved binding to both Fc FcgammaRIIIA and FcgammaRIIB receptors. In certain embodiments, variant Fc antibodies that have improved binding to FcgammaRIIIA do not have a concomitant increase in binding to the FcgammaRIIB receptor when compared to a native Fc antibody. In some embodiments, a variant Fc antibody has reduced binding to the Fc FcgammaRIIIA receptor. A variant Fc antibody may sometimes have less binding to the Fc FcgammaRIIB receptor. In various embodiments, a variant Fc antibody exhibiting altered affinity for FcgammaRIIIA and / or FcgammaRIIB has improved binding to the Fc FcRn receptor. In some embodiments, a variant Fc antibody exhibiting altered affinity for FcgammaRIIIA and / or FcgammaRIIB has altered binding with C1q relative to a native Fc antibody.
[000245] In certain embodiments, variant Fc antibodies exhibit affinities for the FcgammaRIIIA receptor that are at least 2 times, or at least 3 times, or at least 7 times, or at least 7 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or are between 2 times and 10 times, or between 5 times and 50 times, or between 25 times and 100 times, or between 75 times and 200 times, or between 100 and 200 times, more or less than than a native Fc antibody. In various embodiments, variant Fc antibodies exhibit affinities for FcgammaRIIIA that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than a native Fc antibody.
[000246] In certain embodiments, variant Fc antibodies exhibit affinities for the FcgammaRIIB receptor which are at least 2 times, or at least 3 times, or at least 5 times, or at least 7 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or are between 2 times and 10 times, or between 5 times and 50 times, or between 25 times and 100 times, or between 75 times and 200 times, or between 100 and 200 times, more or less than than a native Fc antibody. In certain embodiments, variant Fc antibodies exhibit affinities for FcgammaRIIB that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than a native Fc antibody.
[000247] In some embodiments, variant Fc antibodies exhibit greater or lesser affinities for C1q relative to a native Fc antibody. In some embodiments, variant Fc antibodies exhibit affinities for the C1q receptor that are at least 2 times, or at least 3 times, or at least 5 times, or at least 10 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or are between 2 times and 10 times, or between 5 times and 50 times, or between 25 times and 100 times, or between 75 times and 200 times, or between 100 and 200 times, more or less than an antibody Native FC. In certain embodiments, variant Fc antibodies exhibit affinities for C1q that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than a native Fc antibody. In several embodiments, a variant Fc antibody exhibiting altered affinity for C1q has improved binding to the Fc FcRn receptor. In yet another specific embodiment, a variant Fc antibody exhibiting altered affinity for C1q has altered binding to FcgammaRIIIA and / or FcgammaRIIB relative to a native Fc antibody.
[000248] It is contemplated that variant Fc antibodies are characterized by functional in vitro assays to determine one or more effector cell functions mediated by FcgammaR. In certain embodiments, variant Fc antibodies have similar binding properties and effector cell functions in in vivo models (such as those described and disclosed here) as those based on in vitro assays. The present technology does not exclude variant Fc antibodies that do not exhibit the desired phenotype in in vitro based assays, but do not exhibit the desired phenotype in vivo.
[000249] The half-life of proteins comprising Fc zones can be increased by increasing the binding affinity of the Fc zone to FcRn. The term "antibody half-life" as used herein means a pharmacokinetic property of an antibody that is a measure of the average lifetime of the antibody molecules following their administration. The half-life of the antibodies can be expressed as the time needed to eliminate 50 percent of a known amount of immunoglobulin from the patient's body (or another mammal) or a specific compartment, for example, as measured in the serum, that is, circulating half-life, or in other tissues. The half-life can vary from one immunoglobulin or class of immunoglobulin to another. In general, an increase in antibody half-life results in an increase in mean residence time (MRT) in the circulation for the administered antibody.
[000250] An increase in half-life allows for a reduction in the amount of drug given to a patient, as well as a reduction in the frequency of administration. An increase in half-life can also be beneficial, for example, to avoid a disease or condition associated with Staphylococcal aureus and also to prevent the recurrence of infection that can occur after the patient is discharged from the hospital. To increase the serum half-life of the antibody, a recovery receptor binding epitope can be incorporated into the antibody (especially an antibody fragment) as known in the art. As used herein, the term "recovery receptor binding epitope" refers to an Fc zone epitope of a lgG molecule (eg, IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the half-life of the in vivo serum of the lgG molecule. Antibodies with an increased half-life can also be generated by modifying the amino acid residues identified as being involved in the interaction between the Fc and the FcRn receptor. In addition, the half-life of an anti-alpha-toxin antibody or fragment can be increased by conjugation with PEG or Albumin using techniques widely used in the prior art. In some embodiments antibodies comprising zones of Fc variant of an anti-alpha toxin antibody have an increased half-life of about 5%, about 10%, about 15%, about 20%, about 25%, about about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150% or more when compared to an antibody comprising a native Fc zone. In some embodiments antibodies comprising variant Fc zones have an increased half-life of about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 20 times, about 50 times or more, it is between 2 times and 10 times, or between 5 times and 25 times, or between 15 times and 50 times, when compared to an antibody comprising a native Fc zone.
[000251] In some embodiments, the technology presented here provides Fc variants where the Fc zone comprises a modification (for example, amino acid substitutions, amino acid insertions, amino acid deletions) in one or more selected positions from the group consisting of 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443 as numbered by the EU index proposed by Kabat. Optionally, the Fc zone can comprise an amino acid residue not naturally occurring at additional positions and / or alternatives known in the art.
[000252] In certain embodiments, an Fc variant is provided here in which the Fc zone comprises at least one substitution selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241 R. 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T , 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265, 265I , 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269 , 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T , 325V, 325H, 327G, 327W , 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I , 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W , 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EU index proposed by Kabat. Optionally, the Fc zone can comprise additional and / or alternative non-naturally occurring amino acid residues known in the art.
[000253] In various embodiments, a variant Fc antibody is provided here in which the Fc zone comprises at least one modification (for example, amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from the group consisting of 234, 235 and 331. In some embodiments, non-naturally occurring amino acids are selected from the group consisting of 234F, 235F, 235Y, and 331S. Here, a variant Fc is provided in which the Fc zone comprises at least one non-naturally occurring amino acid in one or more positions selected from the group consisting of 239, 330 and 332. In some embodiments, the non-naturally occurring amino acids are selected from the group consisting of 239D, 330L and 332E.
[000254] In some embodiments, a variant Fc antibody is provided here in which the Fc zone comprises at least one amino acid not naturally occurring in one or more positions selected from the group consisting of 252, 254 and 256. In certain embodiments, the amino acids do not naturally occurring are selected from the group consisting of 252Y, 254T and 256E, described in US Patent No. 7,083,784, the content of which is incorporated herein by reference in its entirety.
[000255] In certain embodiments, an alpha-toxin anti-staphylococcal antibody with a variant Fc zone is provided here that increases the serum half-life of the antibody, where the antibody has the following heavy and light chain sequences: Heavy chain: LC10-IgG1-YTE
[000256] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHDMHWVR QATGKGLEWVSGIGTAGDTYYPDSVKGRFTISRENAKNSLYLQMNSL RAGDTAVYYCARDRYSPTGHYYGMDVWGQGTTVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: XX). Light chain: LC10-Capa
[000257] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQK PGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYY CKQYADYWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: YY)
[000258] In certain embodiments, the effector functions elicited by IgG antibodies depend largely on the carbohydrate fraction linked to the Fc zone of the protein. Thus, the glycosylation of the Fc zone can be modified to increase or decrease the effector function. Accordingly, in some embodiments, the Fc zones of anti-alpha-toxin antibodies and fragments provided herein comprise the altered glycosylation of amino acid residues. In certain embodiments, altered glycosylation of amino acid residues results in reduced effector function. In certain embodiments, altered glycosylation of amino acid residues results in increased effector function. In some embodiments, the Fc zone has reduced fucosylation. In certain embodiments, the Fc zone is afucosylated.
[000259] In some embodiments, the Fc variants contained herein can be combined with other Fc variants as known in the prior art. Other modifications and / or substitutions and / or additions and / or deletions from the Fc domain may be introduced. Glycosylation
[000260] In addition to the ability of glycosylation to alter the effector function of antibodies, modified glycosylation in the variable zone can change the affinity of the antibody to a target antigen. In some embodiments, the pattern of glycosylation in the variable zone of the antibodies present is modified. For example, an aglycosylated antibody can be made (that is, the antibody has no glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody to a target antigen. Such carbohydrate modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence. For example, a substitution of one or more amino acids can be made which results in the elimination of one or more glycosylation sites from variable zone framing to thereby eliminate glycosylation at that location. Such aglycosylation can increase the affinity of the antibody for the antigen. One or more amino acid substitutions can also be made that result in the elimination of a glycosylation site present in the Fc zone (for example, IgG Asparagine 297). In addition, aglycosylated antibodies can be produced in bacterial cells that do not have the necessary glycosylation machinery. Antibody conjugates
[000261] In certain embodiments, the antibodies presented herein are conjugated or covalently linked to a substance using methods known in the art. In some embodiments, the bound substance is a detectable marker (also referred to herein as a reporter molecule) or a solid support. Suitable substances for antibody binding include, but are not limited to, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a hormone, a lipid , a lipid pool, a synthetic polymer, a polymeric microparticle, a biological cell, a virus, a fluorophore, a chromophore, a dye, a toxin, a hapten, an enzyme, an antibody, an antibody fragment, a radioisotope, matrices solid, semi-solid matrices and their combinations. Methods of conjugating or covalently binding another substance to an antibody are known in the art. Diagnostic use methods
[000262] Alpha-toxin is a virulence factor normally found only in pathogenic strains of S. aureus. Alpha-toxin sometimes binds to a cell surface receptor as part of the formation of active complexes. However, oligomerization and formation of a transmembrane pore (e.g., alpha-toxin heptamer) can occur in the absence of binding to a specific receptor. The action of the oligomerized alpha-toxin on cell dysfunction and lysis (for example, formation of a transmembrane pore) facilitates the colonization of invasive S. aureus bacteria. The antibodies described here inhibit the junction or oligomerization of alpha-toxin monomers to a transmembrane pore by recognizing the zone in the alpha-toxin molecule directly or indirectly involved with the monomer-monomer alpha-toxin interaction.
[000263] In certain embodiments, anti-alpha-toxin antibodies and fragments and compositions presented herein can be used in vivo and / or in vitro to diagnose S. aureus strains that produce alpha-toxin and / or diseases and conditions associated with alpha-toxin toxin. This can be achieved, for example, by contacting a sample to be tested, optionally together with a control sample, with the antibody under conditions that allow the formation of a complex between the antibody and the alpha toxin. Complex formation is then detected (for example, using an ELISA). When using a control sample together with the test sample, the complex is detected in both samples and any statistically significant difference in complex formation between the samples is indicative of the presence of S. aureus, alpha-toxin or S. aureus alfa -toxin in the test sample.
[000264] In some embodiments, the technology presented here provides a method of determining the presence of S. aureus strains that produce alpha-toxin and / or alpha-toxin in a sample suspected to contain S. aureus and / or other bacteria producing homologous alpha-toxin, the method comprising exposing the sample to an anti-alpha-toxin antibody or fragment and determining the binding of the antibody to the alpha-toxin in the sample where the binding of the antibody to the alpha-toxin in the sample is indicative of the presence of S. aureus and / or alpha-toxin in the sample. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is from a mammal that has or is suspected of having a disease or disorder associated with S. aureus.
[000265] In certain embodiments, an anti-alpha-toxin antibody or fragment can be used to detect the development of S. aureus and / or alpha-toxin overexpression using an in vivo diagnostic assay. In some embodiments, the anti-alpha toxin antibody or fragment is added to a sample in which the antibody binds to the alpha toxin to be detected and is marked with a detectable marker (for example, a radioactive isotope or a fluorescent marker) and an external screening of the patient is done to locate the marker.
[000266] FISH assays like INFORM ™ (marketed by Ventana, Arizona) or PATHVISION ™ (Vysis, Illinois) can be performed on formalin-spun and paraffin-embedded tissue to determine the extent (if any) of alpha-toxin overexpression in the tissue sample.
[000267] In certain embodiments, anti-alpha-toxin antibodies and fragments can be used in a method of detecting alpha-toxin in a tissue, blood or serum sample (eg, soluble alpha-toxin). In some embodiments, the method comprises contacting a tissue, blood or serum test sample from a mammal suspected of having an alpha-toxin-mediated disorder of S. aureus with an anti-alpha-toxin antibody or fragment shown here and detecting a increase in alpha-toxin in the test sample compared to a control sample of blood or serum from a normal mammal. In some embodiments, the detection method is useful as a method of diagnosing a disorder mediated by S. aureus and / or alpha toxin associated with an increase in alpha toxin in the tissue, blood or serum of a mammal. Therapeutic use methods
[000268] In certain embodiments, an anti-alpha-toxin antibody or fragment can be administered to prevent and / or treat an alpha-toxin-mediated condition. Methods of preventing, treating, maintaining, ameliorating and / or inhibiting an alpha toxin-mediated disorder or disease are presented herein, wherein the methods comprise administering anti-alpha toxin antibodies and fragments provided herein. Alpha toxin-mediated disorders of pathogenic Staphylococcus aureus
[000269] In certain embodiments, methods of administering and using compositions and antibodies as provided herein are provided to treat and prevent a wide range of diseases / conditions mediated by aureuspathogenic Staphylococcus, including chronic conditions and acute conditions, such as, but not limited to , bacteremia, burns, cellulite, dermonecrosis, eyelid infections, food poisoning, joint infections, neonatal conjunctivitis, osteomyelitis, pneumonia, skin infections, surgical wound infection, scalded skin syndrome, endocarditis, meningitis, abscess formation and syndrome of toxic shock.
[000270] Both CA-MRSA and HA-MRSA are resistant to traditional anti-staphylococcal antibiotics, such as cephalexin. CA-MRSA has a broader spectrum of antimicrobial susceptibility, including sulfa drugs (such as co-trimoxazole / trimethoprim-sulfamethoxazole), tetracyclines (such as doxycycline and minocycline) and clindamycin, but the drug of choice for treating CA-MRSA is currently thought to be vancomycin, according to a study by Henry Ford hospital. HA-MRSA is resistant even to these antibiotics and is often only susceptible to vancomycin. New drugs, such as linezolid (belonging to the new class of oxazolidinones) and daptomycin, are effective against CA-MRSA and HA-MRSA.
[000271] In certain embodiments, the antibody or an antigen-binding fragment thereof can be used in combination with an antibiotic that corresponds, for example, to beta-lactama antibiotics (such as cephalexin), sulfa drugs (such as co-trimoxazole / trimethoprim-sulfamethoxazole), tetracyclines (such as doxycycline and minocycline), clindamycin, vancomycin, linezolid, daptomycin, teicoplanin, quinupristin / dalfopristin (sinercide), or tigecycline. Pharmaceutical formulations
[000272] In certain embodiments, an anti-alpha-toxin antibody or fragment provided herein can be formulated with a pharmaceutically acceptable carrier as pharmaceutical (therapeutic) compositions and can be administered by various methods known in the art. The route and / or mode of administration may vary depending on the desired results. As used herein, pharmaceutical formulations comprising an anti-alpha toxin antibody or fragment are referred to as formulations of the technology. The term "pharmaceutically acceptable carrier" means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations can routinely contain salts, buffering agents, preservatives, compatible carriers and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate application. The components of the pharmaceutical compositions are also capable of being mixed with the antibodies of the present technology and with each other, so that there is no interaction that substantially impairs the desired pharmaceutical efficacy.
[000273] The therapeutic compositions of the present technology can be formulated for a particular dosage. Dosage regimens can be adjusted to provide the optimal response desired (for example, a therapeutic response). For example, a single bolus can be administered, multiple doses divided over time can be administered or the dose can be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form that facilitate administration and uniformity of dosage. The unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the patients to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for unit dosage forms are dictated and directly dependent on (a) the unique characteristics of the anti-alpha-toxin antibody or fragment and the particular therapeutic effect to be achieved and (b) the inherent limitations of the compound area as an antibody anti-alpha-toxin or fragment for the treatment of sensitivity in individuals.
[000274] The therapeutic compositions of the present technology can be formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and / or parenteral. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods known in the field of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the patient being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Therapeutically effective dosages
[000275] An antibody formulation described herein can be administered in an appropriate dosage and dosage regimen, and such dosage and dosage regimen may depend on the disease or condition to be treated. A therapeutically effective dosage can be identified by determining whether the dosage or dosing regimen gives rise to a therapeutic effect or therapeutic purpose. Production items and kits
[000276] Here is provided a pharmaceutical package or kit comprising one or more containers filled with a liquid formulation or lyophilized formulation contained herein. In some embodiments, a container filled with a liquid formulation contained herein is a pre-filled syringe. In specific embodiments, the formulations presented herein comprise anti-alpha toxin antibodies and recombinantly fused or chemically conjugated fragments with another fraction, including, but not limited to, a heterologous protein, a heterologous polypeptide, a heterologous peptide, a large molecule, a small molecule, a marker sequence, a diagnostic agent or detectable agent, a therapeutic fraction, a drug fraction, a radioactive metal ion, a second antibody and a solid support. In certain embodiments, the formulations contained herein are formulated in unit dose vials as a sterile liquid. A formulation contained herein is sometimes supplied in a pre-filled syringe.
[000277] In certain embodiments, kits are also provided comprising anti-alpha-toxin antibodies and fragments that are useful for various purposes, for example research and diagnosis including for purification or immunoprecipitation of cell alpha-toxin, detection of alpha-toxin and similar. For isolation and purification of the alpha-toxin, a kit may contain an anti-alpha-toxin antibody or fragment attached to strands (for example, Sepharose strands). A kit may be provided that contains the antibodies for detection and quantification of the alpha toxin in vitro, for example in an ELISA or Western blot. As with the production item, the kit comprises a container and a label or packaging insert in or associated with the container. The container has a composition comprising at least one anti-alpha-toxin antibody or fragment as disclosed herein. Additional containers can be included that contain, for example, diluents and buffers, control antibodies. The label or packaging insert can provide a description of the composition, as well as instructions for the intended diagnostic or in vitro use.
[000278] The present technology also covers a final packaged and labeled pharmaceutical product. This production article includes the appropriate unit dosage form in a suitable container or container such as a glass bottle, pre-filled syringe or other hermetically sealed container. In some embodiments, the unit dosage form is provided as a sterile, non-particulate solution comprising an anti-alpha toxin antibody or fragment that is suitable for parenteral administration. In certain embodiments, the unit dosage form is provided as a sterile lyophilized powder comprising an anti-alpha-toxin antibody or fragment that is suitable for reconstitution.
[000279] In some embodiments, the unit dosage form is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous administration. Thus, the technology includes sterile solutions for each route of administration. The technology also includes sterile lyophilized powders that are suitable for reconstitution.
[000280] As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and shipping. In addition, the products referred to here include instructions for use or other information material that advise the doctor, technician or patient on how to prevent or properly treat the disease or disorder in question. In other words, the production article includes means of instruction indicating or suggesting a dosage regimen including, but not limited to, actual doses, monitoring procedures, and other monitoring information.
[000281] Specifically, the technology provides a production item comprising packaging material, such as a box, bottle, tube, vial, container, pre-filled syringe, spray, insufflator, intravenous bag (i.v.), envelope and the like; and at least one unit dosage form of a pharmaceutical agent contained within the packaging material, wherein the pharmaceutical agent comprises a liquid formulation containing the antibody. The packaging material includes means of instruction that indicate how the antibody can be used to prevent, treat and / or manage one or more symptoms associated with a disease or disorder. Examples
[000282] The examples presented here illustrate certain modalities and do not limit the technology. Example 1: Materials and methods
[000283] Materials and methods used for examples 2 to 9 and example 11 to 13 are now provided. Cloning and expression of wild-type and non-hemolytic H35L mutant S. aureusAT
[000284] Genomic DNA from the strain Staphylococcus aureus ATCC BAA1556 was used to amplify the wild-type alpha-toxin (AT) gene by PCR. The reaction contained a direct primer atatatgagctcgcagattctgatattaatattaaaacc (SEQ ID NO: 41), indirect primer, atatataagcttaatttgtcatttcttctttttccc (SEQ ID NO: 42), and approximately 10 ng of genomic DNA in a 50 μl reaction using a polymerase. The resulting fragment was digested with Sac I and Hind III and ligated into the pCold II DNA vector (TaKaRa), framed with a 6 X His label N-terminus. The H35L mutant was generated by mutagenesis targeting the wild type gene site using a mutagenesis kit targeting the QuikChange II XL site (Stratagene), according to the manufacturer's instructions. The mutagenic initiators used for mutagenesis were gataaagaaaatggcatgctcaaaaaagtattttatagttttatc (SEQ ID NO: 43) and gataaaactataaaatacttttttgagcatgccattttctttatc (SEQ ID NO: 44).
[000285] The sequences of the wild-type mutant AT and ATH35L were confirmed by automatic DNA sequencing. Mutant alpha-toxin and wild-type H35L were expressed in strain E. coli BL21. A 50 ml culture overnight grown on carbenicillin LB plus was diluted 1:10 in a 500 ml culture and grown at 37 ° C to an A600 of about 0.5. The culture was changed to 15 ° C for 30 minutes and then 1 M IPTG was added to reach the final concentration of about 100 mM. The culture was incubated for an additional 24 hours at 15 ° C. The cells were harvested by centrifugation. Purification of recombinant, his marker alpha-toxin (rAT-his)
[000286] Pellets of bacterial cells were thawed on ice and resuspended in Ni-NTA buffer A (20 mM Sodium Phosphate, pH 7.2, 300 mM NaCl). The cells were lysed by microfluidization (Microfluidics Model M-110P) at 20,000 psi and the crude lysate was clarified by centrifugation at 27,000 x g for 10 min. at 4 ° C. Following 0.2 μm filtration, the supernatant was loaded onto a 5 ml Ni-NTA Superflow (Qiagen) column equilibrated with Ni-NTA buffer A. rAT-his was eluted with a 300 mM and 500 mM imidazole step gradient , fractions were collected in tubes containing EDTA at a final concentration of 1 mM, and dialyzed in SP buffer A (50 mM Sodium Phosphate, pH 7.0, 25 mM NaCl, 1 mM EDTA). Dialysates were loaded onto a 5 ml HiTrap SP Sepharose FF column (GE Healthcare) in SP buffer A and rAT-his was eluted with a step gradient to 1 M NaCl. Fractions containing rAT-his were dialyzed to 1X PBS, pH 7.2 with 1 mM EDTA and aliquots were frozen at -80 ° C. Purification of S. aureus native alpha-toxin
[000287] Native alpha-toxin (nAT) from S. aureus Wood strain was purified. S. aureus Wood was grown overnight in tryptic soy broth (TSB) at 37 ° C, with stirring (for example, about 250 RPM). The supernatant culture was harvested by centrifugation and then brought to 75% saturation with solid ammonium sulfate. After stirring for 3 hours at 4 ° C, the precipitate was captured by centrifugation at 12,000 xg for 45 min., Again suspended in SP buffer A (25 mM sodium acetate, pH 5.2, 20 mM NaCl, 1 mM EDTA) and dialyzed against SP buffer A overnight at 4 ° C with an exchange. Insoluble material was removed by centrifugation at 27,000 x g for 30 min. at 4 ° C. The soluble dialysate was filtered (0.2 μm) and loaded onto a 10 ml SP Sepharose FF column (GE Healthcare) equilibrated with SP buffer A. nAT bound was eluted with a linear gradient to 300 mM NaCl, followed by steps at 0 , 5 and 1 M NaCl. Fractions containing nAT were pooled and dialyzed overnight in PBS, pH 7.2 containing 1 mM EDTA. For final processing, the dialysate was loaded onto a HiPrep Sephacryl S-200 High Resolution column (GE Healthcare) at a flow rate of 1.3 ml / min in 1X PBS, pH 7.2 with 1 mM EDTA. Fractions containing nAT were pooled, aliquoted and frozen at -80 ° C. Immunization / hybridoma generation
[000288] VelocImmune mice at eight weeks received 5 subcutaneous injections of rATH35L at various sites following the RIMMS immunization regimen proposed by Kilpatrick et al (1997). The mice were immunized over 13 days, at intervals of 2-3 days. In each round of immunization, the mice were first anesthetized with isoflurane. The immunogen was emulsified in complete or incomplete Freund's adjuvant and TiterMax Gold adjuvant and injected bilaterally in the neck, armpit, calf and groin. Blood was collected for testing on the 13th day and tested on an ELISA antigen. The mice received a pre-fusion boost intraperitoneally and sacrificed on day 17. Lymph nodes and splenocytes were fused into a myeloma partner to generate stable hybridomas. Neutralization of hemolytic activity
[000289] 50 microliters of each B cell hybridoma culture supernatant was mixed with recombinant alpha-toxin-His (rAT-his, 0.1 μg / ml final concentration) in 96-well plates, followed by the addition of 50 μl of 5% rabbit red blood cells (RBC) in PBS. Control wells contained RBC and culture media isolated with or without TA. The plates were incubated for 1 h at 37 ° C, and the intact cells were pelleted by centrifugation. 50 μl of supernatants were transferred to a new 96-well plate and A490 measured on a spectrophotometer. The neutralizing activity was calculated in relation to lysis with RBC and isolated rAT-his and calculated:% inhibition = 100 x [100- (A490 nAT + Ab) / (A490 nAT in Ab)].
[000290] Inhibition with purified mAbs was also tested. Anti-AT mAbs was added to a 96-well plate at about 80 μg / mL in PBS and the samples serially diluted (twice) in PBS to a final volume of 50 μL. A non-specific IgG1 (R347) was included as a control isotype. 25 microliters of mAb dilutions were mixed with 25 μL of nAT (native alpha-toxin) at about 0.1 μg / mL in 96-well round bottom plates, followed by the addition of 50 μL 5% RBC. The inhibition of hemolytic activity was calculated as above. Expression and purification of anti-AT mAbs
[000291] Clarified murine anti-AT supernatants (approximately 5L @ 30-50 mg / L) were concentrated by tangential flow filtration. The concentrated supernatants were then passed through 5 columns of 5 ml Protein G HiTrap HP sequentially and the bound IgG was eluted with 50mM sodium carbonate pH 11.0 and neutralized to approximately pH 7.0 with 1M phosphoric acid. The neutralized material was sequentially loaded onto two 1 mL HiTrap Q FF columns (GE Healthcare). The flow-through containing IgG was collected and dialyzed in PBS pH 7.2. A549 lysis neutralization
[000292] A549 cells were kept in a 5% CO2 incubator at 37 ° C in RMPI supplemented with non-essential acid, glutamine and 10% fetal bovine serum. The cells were washed once with Hank's balanced medium and coated at 104 / well under 50 μl in RPMI, 5% FBS, and incubated at 37 ° C with 5% CO2 for 20 hr. Anti-AT mAbs was added to a 96-well plate at 80 μg / mL in RPMI and the samples were serially diluted (twice) in RPMI. An irrelevant IgG1 (R347) was included as a control isotype. In a separate 96-well plate, 30 μl of the diluted antibodies were mixed with 30 μl of nAT (final concentration, 5 μg / ml). 50 microliters were transferred from each well to the plate containing adherent A549 cells. Control wells for A549 cells with or without nAT were included. The plates were incubated at 37 ° C with 5% CO2 for 3 h, centrifuged and 50 μl of supernatant were transferred to a new 96-well plate. Cell lysis was measured as the release of lactate dehydrogenase (LDH) using a non-radioactive Cytotox 96 assay kit (Promega) following the manufacturer's protocol. Bottom LDH was subtracted from each well and the inhibition of LDH release was calculated:% inhibition = 100 x [100- (A590 nAT + Ab) / (A590 nAT in Ab)]. Neutralization of THP-1 lysis
[000293] THP-1 cells were kept in a 5% CO2 incubator at 37 ° C in RMPI medium (Invitrogen) supplemented with non-essential amino acids (Invitrogen), 2mM glutamine (Invitrogen) and 10% fetal bovine serum (Invitrogen) . Anti-AT mAbs was added to a 96-well plate at 80 μg / mL in RPMI and the samples serially diluted (twice) in RPMI to a final volume of 50 μL. An irrelevant IgG1 (R347) was included as a control isotype. 25 microliters of the mAb dilutions were mixed with 25 μl of native alpha-toxin (nAT) at 1.5 μg / ml final, followed by the addition of 50 μl of THP-1 cells washed in RMPI (106 cells / ml in RPMI with 10% FBS) in a 96-well plate. Control wells consisted of THP-1 cells with or without nAT. The plates were incubated in an incubator and 5% CO2 37 ° C for 3h, centrifuged and 50 μl of the supernatant were transferred to a new 96-well plate. Cell lysis was measured as the release of lactate dehydrogenase (LDH) using the non-radioactive Cytotox 96 assay kit (Promega) following the manufacturer's instructions. The inhibition of LDH release was calculated as described above. Cloning of anti-AT IgG mAbs and expression as fully human mAbs
[000294] The mRNA of five hybridoma clones 2A3, 10A7, 12B8, 25E9 and 28F6 was isolated using the Dynabeads mRNA Direct kit (Invitrogen). The first cDNA strand was synthesized using SuperScript III reverse transcriptase (Invitrogen) and random heptomer primers. Human Ig VL (cap) and VH were amplified by PCR using an Ig primer set (Novagen, Catalog # 69830). The PCL amplified VL and VH products were cloned into TOPO TA vector (Invitrogen pCR2.1-TOPO) and sequenced. The VH and VL (cap) of each hybridoma were reamplified by PCR adding enzyme restriction sites for cloning into human vector IgG.capa.pOE, where VL was cloned into the BssHII / BsiWI site fused with human c-cap, and VH was cloned at the BsrGI / SalI site fused to a human IgG-1 heavy chain constant zone. The resulting pOE plasmids were verified by DNA sequencing. Expression and purification of alpha-toxin mAb
[000295] Plasmid DNA from the pOE constructs was prepared using the Endofree Plasmid Maxi kit (Qiagen). The pOE plasmids were transfected in 293F suspension using fectin 293 reagent (Invitrogen) in Freestyle 293 expression medium (GIBCO). On days 6 and 9 post-transfection, the culture medium was harvested and the IgG purified using a protein A-sepharose column (GE Healthcare). The peaks containing IgG were pooled, dialyzed in PBS, pH 7.4 and stored at -70 ° C. The purity of the IgG proteins was verified by SDS-PAGE. Murine pneumonia model
[000296] Twenty-four hours before infection, groups of 10 7-9 week old C57BL / 6J mice (Harlan) received 0.5 ml of mAb at the concentrations indicated via i.p. The animals were then anesthetized with isofluorane, kept upright and 0.05 ml of S. aureus bacterial suspension (1x108 CFU to 3x108CFU) were inoculated in sterile PBS in the left and right nostrils. The animals were placed in a supine cage for recovery and were observed twice a day throughout the study. The animals' survival was monitored and reached a maximum of 6 days.
[000297] Alternatively, the animals were euthanized with CO2 inhalation 48h after the bacterial infection. A lung and kidney were removed for sterile PBS, homogenized, diluted and coated for bacterial enumeration. The statistical significance of mortality studies was determined using the Mantel-Cox test. The significance of organ bacteria recovery was calculated using analysis of variance and Dunnett's post-test. Murine model of necrosis
[000298] Hair was removed from the backs of groups of five 6-8 week old female BALB / c mice (Harlan) and given an intraperitoneal injection of 0.5 ml IgG at the concentration shown in the graph. Twenty-four hours later, the mice were infected by subcutaneous infection of 50 μL of a bacterial suspension (1 * 108 S. aureus). The animals were monitored twice a day for signs of infection and the size of the abscess was measured at the same time each day. The lesion area was calculated using the formula A = L x W. Statistical significance was determined using analysis of variance and Dunnett's post-test. Receptor binding assay
[000299] Red blood cell ghosts were prepared by incubating 5 ml washed and packaged rabbit red blood cells (RBC) in 500 ml of lysis buffer (5 mM phosphate, 1 mM EDTA, pH 7.4) o / na 4 ° C with constant agitation . The ghosts were then removed by centrifugation at 15,000 x g and washed 3 times with lysis buffer. They were then washed in PBS and resuspended in a final volume of 3 ml.
[000300] To assess the binding of nAT to RBC cell membranes, phantoms were diluted to OD600 approximately 0.2 in PBS and 50 μL were coated on 96-well plates (Costar) and incubated overnight at 4 ° Ç. The liquid was then removed from the plates and the wells were blocked with 100 μL of 1% BSA in PBS, pH 7.4 for 2 hr at 4 ° C and washed 3 times with PBS. An excess of 20 mol IgG was mixed with 3 μg / mL nAT and 50 μL was added to the blocked plates. The plates were incubated at 4 ° C for 2 h and washed 3 times with PBS. Biotin-labeled rabbit anti-AT IgG was added to the wells at 1 mg / mL and incubated at 4 ° C for 1 h, washed 3 times and incubated with streptavidin peroxidase conjugate (1: 30,000, Jackson Immunoresearch). The wells were washed 3 times and developed with Sure Blue Reserve (KPL, Inc.). A450 was read using a plate reader (Molecular Devices) and the calculated% AT binding. % AT binding = 100 x (A450 - AT + IgG / A450 - AT isolated) Oligomerization test
[000301] Liposomes were generated using a Liposofast Extruder (Avestin, Inc.) and a membrane with a pore size of 100 nm. A mixture (5: 1: 4, molar ratio) of egg yolk phosphatidylcholine (15 mg, Avanti Polar Lipids), egg yolk phosphatidylglycerol (2.9 mg, Avanti Polar Lipids) and cholesterol (5.8 mg, Avanti Polar Lipids) in chloroform was dried at 40 ° C under nitrogen flow. The dry lipid film was then rehydrated with 3 ml PBS, pH 7.4 (Invitrogen) and incubated at 37 ° C for 30 min. The sample was then vigorously vortexed to form a regular suspension and then subjected to 3 rounds of freeze-thaw using an isopropanol bath of dry ice and water at room temperature. The solution was then passed through the Liposofast extruder 21 times.
[000302] AT (0.5 μg) was mixed with purified IgG, 5 μL of RBC and PBS phantoms in a final volume of 22 μL and incubated at 37 ° C for 45 min. The samples were then solubilized in 5 μL of SDS-PAGE sample buffer for 5 min at 37 ° C and 10 μL submitted to SDS-PAGE in 4-12% ready-made polyacrylamide gel (Invitrogen). The separated proteins were then transferred to nitrocellulose, blocked for 10 min with Blocker Casein in PBS (Thermo Scientific) and probed with rabbit anti-AT IgG (2 μg / mL) for 2 h at room temperature with constant agitation. AT bands were detected after 1 h with an alkaline phosphatase-labeled goat anti-rabbit 2 and developed using a membrane phosphatase BCIP / NBT substrate system (KPL, Inc.). Measurement of Kinetic Rate and Binding Constants (KD)
[000303] The kinetic rate constants (kon, koff) for binding anti-AT IgG antibodies to purified nAT were measured using an IgG capture assay format on a BIAcore 3000 instrument (BIAcore, Inc). Soon, an anti-mouse rat IgG was immobilized on a CM5 sensor plate according to the manufacturer's instructions. The final surface density of the capture reagent on the sensor plate was approximately 2500 response units (RUs), as described herein. A reference flow cell surface was also prepared on this sensor plate using the identical immobilization protocol and omitting nAT. IgG anti-AT antibodies were prepared at 20 nM in instrument buffer (HBS-EP buffer containing 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA and 0.005% P-20) together with two serial dilutions of nAT. Serial nAT dilutions were performed in an amplitude of 0.78 nM to about 50 nM, in instrument buffer.
[000304] A sequential approach was used in kinetic measurements. Each anti-AT lgG was first injected into the capture and reference surfaces at a flow rate of 50 μL / min. After the binding of the captured IgG had stabilized, a single concentration of the nAT protein was injected on both surfaces, at a flow rate of 50 μL / min. The resulting binding response curves were used to determine the association phase data. Following the injection of nAT, the flow was then switched back to instrument buffer for 10 minutes to allow collection of dissociation phase data followed by 1 minute of 10mM glycine pulse, pH 1.5 to regenerate the surface of captures IgG on the platelet. Binding responses for duplicate injections of each concentration of nAT were recorded against all anti-AT IgG.
[000305] Additionally, several buffer injections were interspersed throughout the series of injections. Selected buffer injections were used together with the cellular reference responses to correct the raw data sets for artifact injection and / or non-specific binding interactions commonly referred to as "double referencing" (DG Myszka, Improving biosensor analysis. J. Mol. Recognit. 12 (1999), pp. 279-284). Completely corrected connection data was then globally inserted into a 1: 1 connection model (BIAevaluation 4.1 software, BIAcore, Inc, Uppsala, Sweden) that included a term to correct the transport-limited mass connection, if detected. These analyzes determined the kinetic rate constants (on, off), from which the apparent KD was then calculated as koff / kon. Measurement of cytokine levels in lungs affected by S. aureus
[000306] C57BL / 6J mice from seven to nine weeks of age were treated with 2A3.1hu (2A3.1 fully human) or R347 (45 mg / kg) by intraperitoneal injection 24h before intranasal infection with 1.5 x 108cfu USA300 (BAA-1556, ATCC). 4 and 24 hours after infection, the mice were euthanized and the lungs were flooded 3 times with 1 ml of PBS. Bronchoalveolar lavage fluid (BAL) was stored at -70 ° C. Pro-inflammatory cytokines were quantified using the mouse pro-inflammatory cytokine kit 7 (Mesoscale, Gaithersburg, MD) according to the manufacturer's instructions. Cytokine levels were expressed as pg / ml. Cloning and expression of GST fusion proteins
[000307] The genetic sequences encoding AT1-50 and AT51-293 were amplified by PCR from the pColdII AT clone described above. The reactions contained in 10 ng of AT-pColdII DNA and 0.1 mg of each direct and indirect primer (AT1-50-F, atattggatccgcagattctgatattaatattaaaac (SEQ ID NO: 45) and AT1-50-R, atacttctcgagttatttattatgatttttatcatcgataaaac (SEQ ID: 46); or AT51- 293-F catagggatccaaactgctagttattagaacgaaag (SEQ ID NO: 47) and AT51- 293-R, catagctcgagtcaatttgtcatttcttctttttcccaatc (SEQ ID NO: 48)), and polymerase ** PCR used, according to the instructions of the PCR, according to the instructions manufacturer. The resulting PCR fragment was digested with BamHI and XhoI and ligated into the vector pGex 6P DNA (Stratagene) framed with the N-terminal glutathione S transferase (GST) marker. The sequences of the clones were confirmed by automatic DNA sequencing.
[000308] Fragment expression was achieved with the E. coli BL21 (DE3) strain as host. Several colonies were chosen from a plate and inoculated in 100 mL of ampicillin LB + 100 μg / mL (Sigma Chemical Company) and cultured o / n at 37 ° C. Overnight cultures were diluted 1: 100 in 3 x 1L cultures of ampicillin LB + 100 μg / mL and cultured with agitation at approximately 250 RPM to an OD600 of approximately 0.8. Protein expression was then induced by adding 1 mM IPTG. The cultures continued to incubate for 2 hours at 37 ° C, with shaking. The bacterial cells were harvested by centrifugation and frozen at -20 ° C.
[000309] Cell pellets were resuspended in 100mL PBS, pH 7.4 (Invitrogen) and lysed by microfluidization (Microfluidics Model M-110P) at 20,000 psi and the crude lysate was clarified by centrifugation at 27,000 x g for 10 min. at 4 ° C. The resulting supernatant was loaded onto a GSTrap FF (GE Healthcare) and GST-AT1-50 column and the soluble fraction of GST-AT51-293 was purified following the manufacturer's instructions. The insoluble GST-AT51-293 fraction was purified from insoluble cell pellets. The insoluble material was solubilized for about an hour at room temperature, with gentle agitation, in 3 M guanidine-HCl in 25 mM sodium phosphate, pH 7.4. The solubilized material was diluted 7 times with refolded buffer A [25 mM Sodium Phosphate, pH 7.4 with 2 M Guanidine-HCl]. GST-AT51-293 was increased by gradual dialysis. An equal volume of refolded buffer B [25 mM Sodium Phosphate, pH 7.4] was added to the dialysis pipette after every 12 - 15 hours of dialysis at 4 ° C to a guanidine concentration of approximately 2, 1, then 0 , 5 M. GST-AT51-293 was dialyzed against refolded buffer B for 24 hours. The final dialysate was clarified by centrifugation and the soluble fraction purified on a GSTrap column as described above. Dot blot tests
[000310] Overlapping peptides spanning amino acids 40 to 293 have been chemically synthesized (New England Peptide). The synthesis of AT1-50 has been tried without success. Alpha-toxin (AT), AT peptides and AT fragments (1 μg) were marked on nitrocellulose and blocked 10 min with Blocker Casein in PBS. The blots were then probed with 2 μg / mL of individual IgG for 3 hr at room temperature. The stains were washed and incubated with an anti-mouse alkaline phosphatase conjugate or goat anti-rabbit IgG (1: 1000, Caltag Laboratories) for 1 h and developed using a BCIP / NBT membrane phosphatase substrate system (KPL, Inc). ELISA characterization of LC10 YTE binding to alpha-toxin and LukF-PV.
[000311] The bacterial lysate containing His-tagged alpha toxin or LukF-PV was coated on the surface of a 96-well plate overnight at 4 ° C. The plates were washed six times with PBS / 0.05% Tween 20 and blocked with 10% Superblock buffering buffer (Pierce, Rockford, IL) at 37 ° C for 1 h. LC10 YTE or anti-His mouse mAb at 2μg / ml (R&D Systems, Minneapolis, MN) was added to the wells and incubated for 1 h at room temperature. The plates were then washed six times with PBS / 0.05% Tween 20. LC10 bound YTE or anti-mouse mAb was detected using anti-human or anti-mouse IgG HRP conjugates (Jackson ImmunoResearch laboratories, Inc. West Grove, PA), respectively. Generation of chimeric variants between alpha-toxin and LukF-PV
[000312] Chimeric variants composed of portions of alpha-toxin and LukF-PV were generated to identify the LC10 YTE binding zone in the alpha-toxin. DNA constructs from six chimeric alpha-toxin variants encoding LukF-PV zones were generated by genetic synthesis at aa 1-51, aa 52-110, aa 111-147, aa 148-205, aa 204-241, or aa 248 -293. DNA constructs were created encoding the other chimeric variants by overlapping the PCR extension using an alpha-toxin encoding plasmid pET3d or LukF-PV (internal plasmids) as models. All DNA constructs were then cloned into the bacterial expression vector pET3d (EMD Chemicals Inc, Philadelphia, PA) and transformed into E. coli BL21 (DE3) strain (Invitrogen, Carlsbad, CA). The transformed BL21 (DE3) cells were cultured in E.coli MagicMedia expression medium (Invitrogen, Carlsbad, CA) to express the variant proteins using standard protocols. Characterization of the binding characteristics of LC10 YTE to chimeric variants using ProteOn
[000313] LC10 YTE binding characteristics to chimeric alpha-toxin / LukF-PV variants were studied using a ProteOn XPR36 instrument (BioRad, Hercules, CA). Standard amine coupling was used to immobilize a polyclonal anti-alpha-toxin antibody (internally generated antibody) in 10 mM sodium acetate [H 5.0] on the surface of a GLC biosensory platelet at about 5000 resonance units (UK) ) for each channel. The chimeric alpha-toxin / LukF-PV proteins in bacterial lysed supernatant were injected into the immobilized GLC surface in order to obtain a capture response of about 200RU. Untransformed bacterial lysate supernatant was also injected under the same conditions as a reference channel. LC10 YTE samples were prepared in phosphate buffered saline (PBS) (pH 7.4), 0.005% Tween-20, and injected at 90 μL / min for 150 or 180 sec. at concentrations typically between 50nM and 3.125nM. 600 or 800 seconds of dissociation time were used. The expression levels of chimeric variants were also monitored following the injection of LC10 YTE as follows: polyclonal anti-alpha-toxin antibodies were fluid at 90 μL / min for 150 or 180 sec. with concentrations typically between 50 nM and 3.125nM with 600 or 800 seconds of dissociation time. The surface was regenerated twice by injecting glycine (10mM, pH 1.5) at 100 μL / min for 30 sec. All sensorgram data were processed with ProteOn Manager 3.0.1 software Example 2: mAb generation of anti-alpha toxin
[000314] Anti-alpha-toxin (AT) monoclonal antibodies (mAbs) were generated in VelocImmune mice that were genetically modified to contain an antibody repertoire with entirely human variable zones fused with constant murine domain. The resulting antibodies are human: mouse chimeras easily converted to fully human IgG by genetic fusion of the variable human domain of chimeric mAb with constant zones of a cloned human IgG-1. Mice were immunized with non-hemolytic mutant AT (ATH35L), described herein, and hybridomas were generated using standard methods. Initially, supernatants with more than 1800 hybridomas were found to contain IgG that binds recombinant AT (rAT-his) by the ELISA antigen. Hybridoma supernatants that exhibited binding to rAT-his were then screened for activity through inhibition of rAT-his mediated lysis of rabbit red blood cells (RBC) in a hemolytic assay, so that the pool of functional mAbs was reduced to about 250. Hybridoma supernatants were then normalized to IgG levels and their inhibitory activities were compared. Thirteen of the most potent rAT-his inhibitors were selected for limited-dilution cloning and used for small-scale IgG expression and purification. Following the screening of these clones and subsequent biochemical and in vivo characterization, as described below, the VH and VL sequences were further optimized to generate additional antibodies, as listed below in table 7. Example 3: Inhibition of cytolytic activity
[000315] The inhibitory activities of the 13 purified anti-AT IgG were compared in a hemolytic assay. Purified anti-AT mAbs were titrated in a hemolytic assay in the presence of constant amounts of nAT and rabbit red blood cells. The mAbs were each titrated to about 20 μg / mL in the presence of a constant amount of native AT (nAT) and rabbit red blood cells (RBC). Hemolysis was measured by the release of hemoglobin in the supernatant. The percentage inhibition (%) of hemolysis was calculated as follows:% inhibition = 100 * [100- (A490 nAT + Ab) / (A490 nAT in Ab)]. Representative hemolytic assays demonstrating the 13 most potent rAT-his inhibitors are shown in figures 1A and 1B. A non-specific IgG control (R347) was included as a negative control.
[000316] Only 7 of the 13 purified mAbs (mAbs; 2A3.1, 10A7.5, 11D12.1, 12B8.19, 15B6.3, 25E9.1 and 28F6.1) inhibited nAT-mediated RBC lysis (see figures 1A and 1B). Three of the antibodies (2A3.1, 10A7.5 and 12B8.19) were potent inhibitors and exhibited about 80% inhibition of nAT-mediated RBC lysis at a 1: 1 ratio (mol IgG: mol AT). These results suggested that the generated mAbs can inhibit the formation of pores in rabbit RBCs.
[000317] Human erythrocytes do not have a large number of receptors for AT. Consequently, human RBC is not as sensitive as rabbit RBC to nAT-mediated lysis and is probably not a primary target for AT during infection. Other types of cells (eg, epithelials, lymphocytes, monocytes and macrophages) are more relevant targets for the effects of nAT during a staph infection. The activity of purified antibodies was examined in nAT-mediated lysis of human cell lines, A549 (epithelial alveolar cell line) and THP-1 (monocytic cell line). Monoclonal antibodies (mAbs) were titrated against a constant level of nAT in the presence of A549 or THP-1 cells. Cell lysis was quantified by lactate dehydrogenase (LDH) release and the% inhibition of LDH release determined, as described herein. The results are presented graphically in figures 2A and 2B. The mAbs that inhibited RBC lysis in rabbits also inhibited nAT-mediated lysis of A549 and THP-1 cells (see figures 2A and B, respectively) with the exception of 11D12.1 which inhibited the lysis of A549 cells and had no effect on nAT-mediated lysis of THP-1 cells. The potent anti-AT activity exhibited by these mAbs highlights the potential usefulness of these antibodies to inhibit AT activity during an infection, thus limiting disease progression and symptoms related to staphylococcus. Example 4: Passive Immunization with Anti-AT mAbs reduces Dermonecrotic Lesions
[000318] S. aureus is a prominent cause of skin and soft tissue infections (SSTI) both in the hospital and in the community, and is often characterized by inflammation, tissue damage and pus formation. TA can play a role in these infections, leading to a hyperinflammatory response and tissue damage. Inhibition of AT function would then limit the bacteria's ability to cause serious illness. To determine the usefulness of anti-AT mAbs in minimizing, reducing or eliminating the effects of infection by S. aureus, groups of 5 mice were injected intraperitoneally (IP) with each of the 7 inhibitory mAbs (for example, about 5 mg / kg ) and an IgG-1 (R347) isotype control 24 hours before subcutaneous infection by S. aureus Wood. The size of the dermonecrotic lesions was measured daily for 6 days and documented using photographs as shown in Figure 3A (day 6 shown in Figure 3A). The 5 most potent in vitro inhibitors of nAT function (2A3.1, 10A7.5, 12B8.19, 25E9.1 and 28F6.1), substantially reduced the size of the lesion compared to the R347 control while the less potent mAbs, in vitro (11D12.1, 15B6.3), did not have a substantial effect on the size of the lesion relative to the control, as shown in Figures 3A and 3B. Figure 3B graphically illustrates the reduction in the size of the lesion over time. 2A3.1, 10A7.5, 12B8.19, 25E9.1 and 28F6.1 are potent inhibitors of AT function in vitro and also exhibit a potent prophylactic effect in a murine model of SSTI. Additional antibodies, LC10, QD20, QD33, and QD37 were also tested in the dermonecrosis model. These monoclonal antibodies were injected intraperitoneally (IP) in five mice per group at 1 and 0.5 mg / kg 24 hours before the subcutaneous infection by S. aureus Wood, as described above. The results are shown in Figures 17 A and B. The p-values are calculated using Dunnett's post-test. For the 1 mg / kg experiments, the p-value for the R347 control when compared to the Abs test was p <0.0001. For experiments with 0.5 mg / kg, the p-value for the R347 control when compared to the Abs test was p <0.05. Example 5: Passive Immunization with Improved Survival mAbs Anti-AT in Pneumonia in a Murine
[000319] Prophylaxis was tested with the most potent anti-AT mAbs generated in a model of murine pneumonia. C57BL / 6J mice were passively immunized with about 5 mg / kg, about 15 mg / kg, and about 45 mg / kg of 2A3.1, 10A7.5, 12B8.19 or 28F6.1, 24 hours before infection intranasal by S. aureus USA300 (BAA-1556). Survival was then monitored for 6 days and compared with an isotype control (R347) at 45mg / kg, as shown in Figures 4-7. Statistical significance was calculated using the Mantel-Cox test. Figure 4 graphically illustrates the percentage of survival over S. aureus infection after passive immunization with various amounts of mAB 12B.19. Figure 5 graphically illustrates the percentage of survival over S. aureus infection after passive immunization with various amounts of mAB 2A3.1. Figure 6 graphically illustrates the percentage of survival over S. aureus infection after passive immunization with various amounts of 28F6.1 mAB. Figure 7 graphically illustrates the percentage of survival over S. aureus infection after passive immunization with various amounts of 10A7.5 mAB.
[000320] All anti-AT antibodies shown resulted in a significant improvement in survival over control, leading to at least 90% survival at the 45 mg / kg dose (see Figures 4-7). Alpha-toxin is considered to be a key determinant of virulence in staphylococcal pneumonia. The results presented here illustrate that passive administration of potent inhibitory mAbs is a valid approach to disease prevention. Taken together, the animal studies presented here support a role for AT in staphylococcal disease and provide support for the use of mAbs that inhibit AT function to limit the severity of the disease or even death associated with infections with S. aureus infection.
[000321] To further characterize the impact of an anti-AT mAb on bacterial numbers during an infection, a fully human version of mAb 2A3.1 (eg, 2A3hu) was administered to mice 24 hours before intranasal infection with approximately 1 , 3 x 108cfu of S. aureus USA300. 48 hours after infection, the mice were euthanized and their lungs and kidneys were collected and processed for bacterial enumeration (see Figures 8A and 8B). 4 and 24 hours after infection, the mice were euthanized and samples were taken to measure cytokine production (described below and see Figure 9) and for histopathological analysis (described below and see Figure 10). The p-values are calculated using the Dunnett post-test. Representative results of bacterial enumeration are shown in Figures 8A and 8B. The prophylactic administration of 2A3hu resulted in a significant reduction in bacterial values in both the lungs (see Figure 8A) and kidneys (see Figure 8B) compared to the R347 control, indicating that inhibition of AT function may limit disease progression, improve its removal and also limit the systemic spread of the invading organism.
[000322] Additional antibodies, LC10, QD20, QD33, and QD37 have also been tested in the pneumonia model. These monoclonal antibodies were injected intraperitoneally (IP) in 10 mice per group at 5 mg / kg, 24 hours before intranasal infection (IN) with approximately 2 x 108 cfu of S. aureus USA300. The results of these experiments are shown in Figure 18. The p-values are calculated using Dunnett's post-test. The p-value for 2A3 mAb when compared to QD37 was p = 0.0072; the p-value for 2A3 mAb when compared to LC10 was p = 0.0523; the p-value for 2A3 mAb when compared to QD33 was p = 0.0521. Example 6: Inhibition of AT in the Pneumonia Model Reduces Pro-inflammatory Cytokine Production
[000323] Pneumonic infection by S. aureus is typically accompanied by an overproduction of pro-inflammatory cytokines that were thought to lead to increased activation and infiltration of immune cells, eventually leading to greater tissue congestion and necrosis (Bubeck Wardenburg, J 2007). An AT S. aureus deletion mutant has been shown to exhibit reduced virulence over its wild type S. aureusisogenic in the murine pneumonia model. It has also been proven that active and passive immunization against AT reduced the expression of IL-1β, a known mediator of acute lung injuries and protected mice from severe pneumonia (Bubeck Wardenburg, J. 2007; Bubeck Wardenburg, J. 2008). These results suggest that inhibition of AT during infection by S. aureus may reduce the production of pro-inflammatory cytokines and thus limit excessive cell infiltration with the end result being less symptoms of pneumonia and better bacterial removal, as seen above.
[000324] To test this hypothesis, mice were passively immunized with 2A3hu 24 hours before intranasal infection with approximately 1.3 x 108 cfu of S. aureus USA300. Four and twenty-four hours after infection, the mice were euthanized and half of the lung was arranged and prepared for staining with hematoxylin and eosin and microscopic examination, while the bronchoalveolar lavage fluid was collected on the other hand and processed to determine the levels of cytokine. Representative results of cytokine production after passive immunization are shown in Figure 9. Figure 10 illustrates through photography the effectiveness of passive mAbs immunization described here.
[000325] Four hours after infection, cytokine levels were similar in mice treated with R347 and 2A3hu; however, 24 hours after infection, the levels of IL-6, TNF-α KC and IL-1β were all reduced in animals treated with 2A3hu, as shown in Figure 9 (see results marked with a 24-hour time point circle ), indicating that prophylactic administration of 2A3hu resulted in reduced cytokine levels compared to control. These data are supported by the results of the histopathological examination of the lung, in which the mice treated with R347 had evident signs of pulmonary inflammation, necrosis and alveolitis, together with the presence of bacterial colonies (see the images at the top left and bottom left at Figure 10). In contrast, animals treated with 2A3hu had limited pulmonary inflammation, with no visible necrosis, alveolitis or bacterial colonies (see the images at the top right and bottom right in Figure 10). The protective effect of anti-AT mAbs in the pneumonia model is associated with a reduced inflammatory response that can limit local tissue damage and promote bacterial removal. Example 7: Linkage and Competition Kinetics
[000326] Affinity measurements can be performed using surface plasmon resonance (SPR), to better characterize the mAbs that exhibited potent inhibitory activity. Purified IgG was captured in a sensor using anti-mouse rat IgG and the platelet was exposed to solutions with different concentrations of nAT. The association and dissociation rates were measured, from which the connection constants were determined. Antibodies 2A3.1, 10A7.5, 25E9.1 and 12B8.19 had similar affinities with KD values of 601, 504, 337 and 485 pM, respectively, while 28F6.1 exhibited a KD value of 13 nM, as shown in table below. KD was calculated as koff / kon.

[000327] Competition tests were also performed using SPR, the results of which suggest that antibodies 2A3.1, 10A7.5, 25E9.1 and 12B8.19 probably bind the same or similar epitope.
[000328] IC50 and Kd value are shown for mAbs QD20, LC10, QD33, QD37 and 2A3GL below

[000329] The IC50 was calculated using the RBC hemolytic assay with 0.1 mg / ml S. aureus alpha toxin. Example 8: Inhibitory mAbs Blocking of SDS-Resistant Heptamer
[000330] It is believed that S. aureus alpha-toxin (AT) does cell lysis in a multistage process in which a secreted soluble monomeric AT molecule binds to a cell surface receptor or does not specifically absorb cell membranes, oligomerized it occurs in a heptameric pre-pore on the cell surface and undergoes a conformational change that leads to the formation of a 14-chain transmembrane β roller that mediates the subsequent target cell lysis. The mechanism of inhibition by mAbs described here was further characterized to determine in which stage the inhibitory mAbs blocked the AT function. The ability of these mAbs to prevent AT binding to RBC ghosts of rabbits attached to a 96-well tissue culture plate was examined. 96-well ELISA plates were coated with RBC phantoms and blocked with 2% BSA. The ghosts were then incubated with nAT +/- at 20 molar excess of anti-AT IgG. The binding of nAT was then detected with rabbit anti-AT IgG and the% binding calculated; % binding = 100 x [100- (A490 nAT + mAb) / (A490 nAT without mAb)]. With a 20 molar excess of lgG, there was no inhibition of nAT binding to rabbit RBC membranes, as shown in Figure 11, indicating that these inhibitory mAbs were not acting at the receptor binding step.
[000331] In addition to cell membranes, AT and other pore-forming toxins have been shown to come together readily, forming pores in liposome membranes. Initially, the effect of anti-AT IgG on the formation of AT heptamer was tested on liposomes. Following the incubation of AT with a 10 molar excess of liposomes (lipid: AT, weight: weight), in the presence of IgG, heptamer formation was examined by western blot analysis, as shown in Figure 12. The samples were then solubilized in SDS-PAGE sample buffer at 37 ° C and heptamer formation was detected by western blot analysis. The presence of an SDS-resistant heptomer is readily apparent on top of the gel shown in Figure 12, in lanes 6 and 7 (for example, mAb9D7.3 and control track without IgG, respectively). All inhibitory mAbs eliminated heptamer formation, while an irrelevant isotype control (eg, lane 6; 9D7.3) had no effect. The inhibition of oligomerization activity was confirmed using mAbs 2A3.1, 10A7.5 and 12B8.19 in an oligomerization assay in rabbit RBC ghosts, as illustrated in the representative western blots shown in Figures 13A and 13B. AT was incubated with an IgG titration before incubation with rabbit erythrocyte phantoms and detection of heptomer formation by SDS-PAGE. mAbs 2A3.1, 10A7.5 and 12B8.19 effectively inhibited the formation of AT heptamer even at a 1: 1 IgG: toxin (mol: mol) ratio and the inhibition of oligomerization was titrated as the mAb levels were reduced (see Figures 13A and 13B; appearance of heptamers in molar ratios of 0.5: 1 and 0.25: 1). These results show that mAbs 2A3.1, 10A7.5 and 12B8.19 prevent AT-mediated cell lysis by inhibiting SDS-resistant heptomer formation. Example 9: Conversion to fully human lgG
[000332] A fully human IgG includes heavy (VH) and light (VL) variable domains of the chimeric mAbs described herein, fused genetically to the constant domains of a human IgG-1. The VH and VL of each of the chimeric mAbs was cloned, sequenced and fused to human VG IgG-1 and human kappa constant domains, respectively. The resulting fully human IgG-1s have been shown to retain the responsible variable human zone and binding properties of the mAb of interest. Fully human antibodies were expressed, purified and their activity compared with the chimeric mAbs isolated from the hybridomas of the VelocImmune mouse. The fully human mAbs exhibited potency similar to that of the original chimeras in inhibiting cell lysis RBC, A549 and THP-1, with the exception of 25E9.1hu, which became substantially more potent than the original 25E9.1 chimera. Figures 14-16 graphically illustrate the inhibition of LDH release, characteristic of cell lysis, in red blood cells (RBC; see Figure 14), A549 cells (see Figure 15) and THP-1 cells (see Figure 16). The increased potency of the human 25E9.1 mAb (eg 25E9.1hu) may have resulted from a mixed cell population in the original hybridoma that contained 2 distinct anti-AT IgG molecules, only one of which could have possessed the activity that inhibited the nAT function. As such, the molarity calculations and activity measurements for the original mAb chimera may not have a direct correlation. Example 10: Representative Amino Acid and Nucleotide Sequences for Antibodies that Specifically Bind to S. aureus Alpha-Toxin Table 1: VL CDR sequences for mAbs 2A3.1, 10A7.5, 12B8.19 and 25E9.1
Table 2: VL CDR sequences for mAB 28F6.1
Table 3: VH CDR sequences for mAb 2A3.1
Table 4: VH CDR sequences for 10A7.5 and 12B8.19 mAbs
Table 5: VH CDR sequences for 28F6.1 mAb
Table 6: VH CD R sequences for mAb 25E9.1
Table 7: VL and VH amino acid sequences for anti-alpha-toxin mAbs









Table 8: VL and VH nucleotide sequences for anti-alpha-toxin mAbs




Table 9: Summary table VL and VH CDR
Table 10: Alpha-toxin amino acid sequences
Example 11: Mapping of Anti-alpha Staphylococcal Antibody Binding Zones
[000333] Chimeric variants, composed of portions of alpha-toxin and LukF-PV, were constructed to identify the fragment of alpha-toxin to which an antibody corresponding to mAb LC10 containing an Fc variant (LC10 YTE) binds. LukF-PV was chosen as the chimeric partner because it is not recognized by LC10 YTE (Figure 19), but shares a high structural similarity (Gouaux, E., M. Hobaugh, et al. "Alpha-Hemolysin, gamma-hemolysin, and leukocidin from Staphylococcus aureus: distant in sequence but similar in structure. "Protein Sci 6 (12): 2631-5 (1997); Meesters, C., A. Brack, et al." Structural characterization of the alpha-hemolysin monomer from Staphylococcus aureus. "Proteins 75 (1): 118-26 (2009)) and 25% sequence identity with the alpha toxin (Figure 20). A series of chimeric variants have been constructed by systematically replacing 50 alpha-toxin (aa) amino acids with their corresponding LukF-PV. Smaller zones within the 50 aa segments of interest have also been replaced (Table 11). A ProteOn instrument was used to analyze the binding affinity of LC10 YTE to these variants. The results of binding LC10 YTE to the variants are summarized in Table 11. Table 11: Linking LC10 YTE profiles to chimeric alpha-toxin / LukF-PV variants
* The 50nM poly anti-alpha-toxin binding signals were above 100RUs. ** The 50nM poly anti-alpha-toxin binding signals were below 100RUs.
[000334] All chimeric constructs can be expressed at the same level with the exception of KO_73-81, (Table 11). LC10 YTE did not bind to LukF-PV coding variants instead of aa 101- 110 of the alpha toxin (KO_52-110 and KO_101-110) or aa 224-231 (KO_204-241, KO_204-231 and KO_224-231 ). The LC10 YTE binding was significantly impaired or completely interrupted by replacing aa 248-277 (KO_248-277) or its largest segment aa 248-293 (KO_248-293), respectively.
[000335] In certain cases, a complete lack of binding to LC10 YTE, can be explained by individual alpha-toxin / LukF-PV variants exhibiting an incorrect fold. A general inability to make the correct fold can also explain the apparent lack of expression of KO 73-81. In addition, the homology of the amino acid sequence between alpha toxin and LukF-PV varies substantially in different zones. For example, a segment corresponding to aa 179-193 of alpha-toxin shares 67% identity with LukF-PV, while the rest of the sequence shares 25% identity. Thus, although no effect on LC10 YTE binding was observed when switching zones of high homology sequence, these sequences could potentially contain additional sequences to which the LC10 YTE antibody binds.
[000336] The results of the above mutagenesis indicate that the replacement of any of the three zones of aa 101-110, aa 224-231, and 248-293 of the alpha-toxin with LukF-PV residues impaired the LC10 YTE bond, while substitution of the remaining aa zones had no impact.
[000337] These three zones represent two different locations in the three-dimensional structure of the alpha toxin. Segments corresponding to aa 101-110 and 224-231 are in spatial proximity and located on one side of an interleaved Beta domain, while seed corresponding to aa 248-277 is mainly located in the "Rebordo" domain (Song, L., MR Hobaugh , et al., "Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore." Science 274 (5294): 1859-66 (1996)) (Figure 21).
[000338] The segment corresponding to aa 248-277 revealed an impact on the connection and also contained structural X-ray contact residues aa 261-272 (where T263, N264, and K266 are actual contact residues) (Figure 20). In addition, the crystal structure revealed another segment corresponding to aa 173-201 in which D183, W187, and N188 are real contact residues (Figure 20). The chimeric variant that contained this particular segment (KO_148-205) still exhibited good connection to LC10 YTE. This is probably attributable to the high homology sequence of that particular zone (52% identity and 63% similarity) between alpha-toxin and LukF-PV. The amino acids surrounding the contact residues (aa 179-193) share an even higher homology (67% identity), while, in contrast, the total sequence shares only 25% identity.
[000339] Using the mutagenesis-based approach, the segment corresponding to aa 248-277 was identified as important for the binding of LC10 YTE. This was further confirmed by the structural analysis of the LC10 YTE / alpha-toxin complex structure. Structural analysis also revealed certain contact residues within the aa 248-277 fragment that are present within aa 261-272.
[000340] As mentioned above, X-ray crystallography experiments were performed to determine the contact residues of LC10 YTE mAb. Purified alpha-toxin residues (residues 1 to 293) and LC10 YTE Fab were concentrated separately. Almost equimolar amounts of these proteins were mixed with each other and the solution was subjected to Gel-Filtration chromatography on a Sephadex S75 column (GE Healthcare). The eluted peak contained both protein molecules linked to each other. Continued concentration and crystallization produced crystals that diffracted to 2.5Â.
[000341] The complex structure was resolved using the molecular substitution method. The Fab structure (D25) previously determined with a removed complementarity from zone determination was used as a model for LC10 YTE Fab. The alpha-toxin molecule monomer derived from a heptameric complex (PDB Id. 7AHL) with some truncations was used as a model for the alpha-toxin molecule. The sequence of the alpha-toxin molecule used for crystallographic research corresponds to that of SEQ ID NO: 39. Two complexes per asymmetric unit of the LC10 YTE-alpha-toxin complex were identified using a Phaser program from the CCP4 suite of programs. The structure model was further refined using a Refmac program from the CCP4 suite of programs. The elaboration of the manual and improvements in the iterative model were carried out using the crystallographic program "O".
[000342] Both the Fab heavy and light chains have been shown to be in contact with the alpha-toxin molecule (Fig. 22). In particular, crystallographic studies determined contact residues within the alpha-toxin molecule that corresponded to the following for both heavy and light chains: N177, W179, G180, P181, Y182, D183, D185, S186, W187, N188, P189, V190 , Y191 and R200. In addition, it was determined that the light chain has contact with T261, T263, N264, K266 and K271. Paráppos were identified as: LC - W32 (CDR1), K50 (CDR2), Y91, A92, N93, Y94, W95 (CDR3); HC - D33 (CDR1), T53, A54, D56, Y58 (CDR2), D98, Y100, P102, T103, G104, H105, Y106 (CDR3).
[000343] Molecular modeling incorporating structural data from crystallographic analysis revealed that most of the alpha-toxin structure remained unchanged during the transition from the monomeric to the heptameric state. However, a critical binding zone (where contact residues were identified as T261, T263, N264, K266 and K271) was found to correspond to a portion of the alpha-toxin molecule that participates in the formation of heptamers. It is this critical zone that is compactly folded when the alpha-toxin molecule is in a monomeric state, which extends like a cycle before insertion into a host cell membrane (Fig. 23b), and ends up forming the base of the structure mushroom type after assembly in the heptameric state (Fig. 23a). In the heptameric state, that zone, as shown in Fig. 24a, would be predicted to be protected from binding by the LC10 YTE antibody molecule. Example 12: Therapeutic Efficacy of Staphylococcal Anti-alpha-toxin Antibodies
[000344] The results presented above describe the efficacy of anti-AT mAbs used in prophylaxis. To test the possibility that these mAbs could also function therapeutically, the effectiveness of LC10 was tested in a therapeutic environment in the models of dermonecrosis and pneumonia. In the model of dermonecrosis, LC10 IV was administered 24 hours before the bacterial challenge (prophylaxis) and 1, 3 or 6 hours after intradermal infection (therapy). The size of the animals' lesion was monitored for 6 days. Prophylaxis (-24hr) and treatment 1 or 3 hours after infection resulted in a reduction in the size of the lesion in relation to the negative control (R347) (Fig.24). The strong treatment benefit was lost when LC10 was administered 6 hours after infection in this model. These results indicate that LC10 can function as an effective therapy for staph skin and soft tissue infections.
[000345] Similar experiments were conducted in the pneumonia model in which LC10 was administered to mice (IV) either in prophylaxis or 1, 3 or 6 hours after intranasal infection. As expected, prophylactic mAb administration resulted in total survival. (Fig. 25) Although total survival did not result when LC10 was administered 1, 3, or 6 hours after infection, there was a time-of-death benefit over a negative control when LC10 was used for treatment 1 hour after infection at the highest LC10 doses. Given the requirement of the high-dose model of infection and the rapid advent of death, these improvements in survival indicate that therapeutic improvement may occur during a human infection. Example 13: Efficacy of Vancomycin in Combination with LC-10 anti-alpha-toxin mAb
[000346] Studies have been conducted to evaluate the potential of LC-10 anti-alpha-toxin mAb for use in auxiliary therapy with vancomycin in a murine pneumonia model by comparison of anti-alpha-toxin mAb and vancomycin monotherapy for combination therapy .
[000347] Seven week old female C57BL / 6J mice were infected intranasally with 2e8 cfu (LD100) of USA300 methicillin resistant Staphylococcus aureus. Vancomycin and LC-10 were individually titrated to determine optimal and sub-effective doses. For mAb evaluation in monotherapy or dual therapy with vancomycin, mice were treated one hour after infection with a single LC-10 intraperitoneal, or negative control antibody R347 (15 mg / kg). Vancomycin treatment in mono or dual therapy was started 1 hour after infection and administered subcutaneously BID 3 days. The percentage of survival of all treated groups was determined after seven days. Survival curves were analyzed using the Mandel-Cox test.
[000348] Treatment with vancomycin at 200 or 40 mg / kg / day resulted in survival of 90% and 43%, respectively. Monotherapy after infection with LC-10 at 45 or 15 mg / kg protected 50% and 33% of the mice. (Fig. 26A and B) Combination therapy with a single sub-effective dose of LC-10 (15 mg / kg) and BID dosing of vancomycin at 40 mg / kg resulted in the survival of 75% of the animals. 90% of the mice survived with the combination of 40 mg / kg / day of vancomycin with 45 mg / kg of LC-10. The survival differences between vancomycin monotherapy and combined therapy with 15 mg / kg or 45 mg / kg LC-10 were statistically significant. (p = 0.026 and p = 0.015 respectively). The co-administration of vancomycin and LC-10 produced a synergistic effect by an isobologram analysis (Fig. 27). Example 14: Examples of specific modalities
[000349] Non-limiting examples of certain modalities are now provided.
[000350] A1. An isolated antibody or antigen-binding fragment that immunospecifically binds a Staphylococcus aureus alpha-toxin polypeptide and includes: (a) a VH CDR1 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 substitutions amino acid residues related to SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 8, 11, 14, 17, 70 or 75 and (c) a VH CDR3 comprising an amino acid sequence identical to, or containing, 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78.
[000351] A2. The A1 modality antibody or antigen binding fragment, where VH CDR1, VH CDR2 and VH CDR3 are represented by SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 13, 14 and 15; SEQ ID NOs: 7, 17 and 18; SEQ ID NOs: 7, 8 and 16; SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7, 8 and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs: 69, 70 and 71; SEQ ID NOs: 7, 8 and 72; SEQ ID NOs: 69, 75 and 71; SEQ ID NOs: 69, 75 and 76; or SEQ ID NOs: 69, 70 and 71.
[000352] A3. An isolated antibody or antigen-binding fragment in which the isolated antibody or its antigen-binding fragment immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide and includes: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70 or 75; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 4; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74.
[000353] A4. The antibody or its antigen-binding fragment of the A3 modality in which VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 correspond to the amino acid sequences of SEQ ID NOs: 7, 8, 9, 1, 2 and 3; SEQ ID NOs: 10, 11, 12, 1, 2 and 3; SEQ ID NOs: 13, 14, 15, 4, 5 and 6; SEQ ID NOs: 7, 17, 18, 1, 2 and 3; SEQ ID NOs: 7, 8, 16, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 64; SEQ ID NOs; 7, 8, 66, 1, 2 and 64; SEQ ID NOs: 7, 8, 67, 1, 2 and 68; SEQ ID NOs: 7, 8, 67, 1, 2 and 64; SEQ ID NOs: 7, 8, 78, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 68; SEQ ID NOs: 69, 70, 71, 1, 2 and 68; SEQ ID NOs: 7, 8, 72, 1, 73 and 74; SEQ ID NOs: 69, 75, 71, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 77 and 74; SEQ ID NOs: 69, 70, 71, 1, 77 and 74.
[000354] A5. The isolated antibody or its antigen binding fragment of the A1 modality wherein the isolated antibody or its antigen binding fragment (i) comprises a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs; (ii) and that immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide in which the three VH chain domain CDRs comprise: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13 or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70 or 75; and (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78.
[000355] A6. The antibody or its antigen-binding fragment of the A5 modality in which VH CDR1, VH CDR2 and VH CDR3 correspond to the amino acid sequences of SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 13, 14 and 15; SEQ ID NOs: 7, 17 and 18; SEQ ID NOs: 7, 8 and 16; SEQ ID NOs: 7, 8 and 65; SEQ ID NOs: 7, 8 and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8 and 78; SEQ ID NOs: 69, 70 and 71; SEQ ID NOs: 7, 8 and 72; SEQ ID NOs: 69, 75 and 71; SEQ ID NOs: 69, 75 and 76; or SEQ ID NOs: 69, 70 and 71.
[000356] A7. The isolated antibody or its antigen binding fragment of the A1 modality wherein the isolated antibody or its antigen binding fragment (i) comprises a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs; (ii) and which immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide in which the three CDRs of the VL chain domain comprise: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 4; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and (c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74.
[000357] A8. The antibody or its antigen-binding fragment of the A7 modality in which VL CDR1, VL CDR2 and VL CDR3 correspond to the amino acid sequences of SEQ ID NOs: 1, 2 and 3; SEQ ID NOs: 4, 5 and 6; SEQ ID NOs: 1, 2 and 64; SEQ ID NOs: 1, 2 and 68; SEQ ID NOs: 1, 73 and 74; or SEQ ID NOs: 1, 77 and 74.
[000358] A9. An isolated antibody or antigen-binding fragment that (i) immunospecifically binds to a Staphylococcus aureus alpha-toxin polypeptide, (ii) containing a heavy chain variable domain comprising at least 90% identity with the sequence of amino acids of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and (iii) comprises a variable domain of light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.
[000359] A10. The antibody or its antigen-binding fragment of the A9 modality in which VH and VL correspond to the amino acid sequences of SEQ ID NOs: 20 and 19; SEQ ID NOs; 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ ID SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; SEQ ID SEQ ID NOs: 53 and 54; SEQ ID SEQ ID NOs: 59 and 60; SEQ ID SEQ ID NOs: 62 and 63; SEQ ID NOs: 79 and 63.
[000360] A11. The isolated antibody or an antigen-binding fragment thereof where the isolated antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain of SEQ ID NO 20, 22, 24, 26, 28, 41, 43, 45 , 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and a light chain variable domain of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48 , 50, 52, 54, 56, 58, 60 or 63.
[000361] A12. The antibody or its antigen-binding fragment of the A11 modality in which VH and VL correspond to the amino acid sequences of SEQ ID NOs: 20 and 19; SEQ ID NOs; 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; SEQ ID NOs: 51 and 52; SEQ ID NOs: 51 and 52; SEQ ID NOs: 53 and 54; SEQ ID NOs: 55 and 56; SEQ ID NOs: 57 and 58; SEQ ID NOs: 59 and 60; SEQ ID NOs: 61 and 58; SEQ ID NOs: 62 and 58; SEQ ID NOs: 62 and 63; SEQ ID NOs: 79 and 63.
[000362] A13. The antibody or its antigen-binding fragment according to any of modalities A1 to A12 wherein the isolated antibody or its antigen-binding fragment binds immunospecifically to an alpha toxin polypeptide of Staphylococcus aureus and has one or more of the following selected characteristics of the group consisting of: (a) affinity constant (KD) for alpha toxin of about 13 nM or less; (b) binds to alpha-toxin monomers, but does not inhibit the binding of the alpha-toxin to the alpha-toxin receptor; (c) inhibits the formation of alpha-toxin oligomers by at least 50%, 60%, 70%, 80%, 90% or 95%; (d) reduces the alpha-toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (for example, as determined by cell lysis and hemolysis assays); and (e) reduces cell infiltration and release of pro-inflammatory cytokine (for example, in a model of animal pneumonia).
[000363] A14. The antibody or its antigen-binding fragment according to any of embodiments A1 to A13 wherein the isolated antibody or its antigen-binding fragment comprises an additional agent.
[000364] A15. The antibody or its antigen-binding fragment according to modality A14 in which the additional agent is an antibiotic.
[000365] A16. The antibody or its antigen-binding fragment according to modality A14 wherein the isolated antibody or its antigen-binding fragment is linked to the therapeutic agent by means of a ligand.
[000366] A17. The antibody or its antigen-binding fragment according to any of embodiments A1 to A13 wherein the isolated antibody or its antigen-binding fragment further comprises a diagnostic agent.
[000367] A18. The antibody or its antigen-binding fragment according to modality A17 wherein the diagnostic agent comprises an imaging agent.
[000368] A19. The antibody or its antigen-binding fragment according to modality A17 wherein the diagnostic agent comprises a detectable marker.
[000369] A20. The antibody or its antigen-binding fragment according to modality A17 wherein the isolated antibody or its antigen-binding fragment is linked to the diagnostic agent by means of a ligand.
[000370] A21. The antibody or its antigen-binding fragment according to any of modalities A1 to A20 wherein the alpha-toxin polypeptide from Staphylococcus aureus is a native toxin polypeptide.
[000371] A22. The antibody or its antigen-binding fragment according to any of modalities A1 to A20 wherein the alpha-toxin polypeptide of Staphylococcus aureus includes an amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40.
[000372] A23. The antibody or its antigen-binding fragment according to modality A13, in which the cell is blood or lung.
[000373] A24. The antibody or its antigen-binding fragment according to modality A23 in which the blood cell is a red blood cell.
[000374] A25. The antibody or its antigen-binding fragment according to any of the modalities A13, A23 or A24 in which cell lysis is determined by an in vitro hemolytic assay or an in vitro lactate dehydrogenase assay.
[000375] A26. The antibody or antigen-binding fragment according to any of A1 to A25 wherein the isolated antibody or its antigen-binding fragment binds immunospecifically to an alpha toxin polypeptide of Staphylococcus aureus and comprises a VH CDR3 comprising a amino acid sequence identical to, or containing 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78, where antibody or antigen binding fragment neutralizes the Staphylococcus aureus alpha toxin polypeptide.
[000376] A27. The antibody or antigen-binding fragment according to any of A1 to A26 wherein the isolated antibody or its antigen-binding fragment binds immunospecifically to an alpha toxin polypeptide of Staphylococcus aureus and comprises a CDR3 VL comprising a amino acid sequence identical to, or containing 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 3, 6, 64, 68 or 74, wherein the antibody or antigen-binding fragment neutralizes the alpha-toxin polypeptide of Staphylococcus aureus.
[000377] A28. The antibody or antigen-binding fragment according to any of A1 to A27 wherein the isolated antibody or its antigen-binding fragment binds immunospecifically to a Staphylococcus aureus alpha-toxin polypeptide and comprises a VH CDR3 comprising a amino acid sequence identical to, or containing 1, 2 or 3 amino acid residue substitutions for SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76 or 78, where antibody or antigen-binding fragment inhibits oligomerization of the Staphylococcus aureus alpha-toxin polypeptide.
[000378] A29. The antibody or antigen-binding fragment according to any of A1 to A28 wherein the isolated antibody or its antigen-binding fragment binds immunospecifically to an alpha toxin polypeptide of Staphylococcus aureus and comprises a VL CDR3 comprising a amino acid sequence identical to, or containing 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 3, 6, 64, 68 or 74, wherein the antibody or antigen binding fragment inhibits the oligomerization of the alpha polypeptide -toxin of Staphylococcus aureus.
[000379] A30. The antibody or antigen binding fragment according to any of the modalities A13, A28 and A29 in which the inhibition of oligomerization is determined by in vitro binding and / or electrophoretic mobility assay.
[000380] D1. A composition comprising the antibody or antigen binding fragment according to any of embodiments A1 to A30.
[000381] B1. A kit, comprising (a) an antibody or antigen-binding fragment according to any of modalities A1 to A30 or the composition of modality D1; (b) instructions for using the composition or directions for instructions for using the composition.
[000382] B2. The kit according to modality B1 in which the antibody of the composition is attached to a solid support.
[000383] B3. The kit according to the B2 mode in which the solid support is a sphere.
[000384] B4. The kit according to modality B3 in which the sphere is a sepharose sphere.
[000385] B5. The kit according to any of the modalities B1 to B4, in which the instructions for use include one or more of isolating, purifying, detecting and quantifying a Staphylococcus aureus alpha-toxin polypeptide.
[000386] B6. The kit according to any of the modalities B1 to B5 comprising a plug, a solid support or a plug and a solid support.
[000387] B7. The kit according to modality B6 in which the solid support is one or more of a sphere, filter, membrane and multi-well plate.
[000388] B8. The kit according to modality B6, which includes a buffer and membrane suitable for a Western blot.
[000389] B9. The kit according to modality B6, which includes a loading buffer and an elution buffer.
[000390] B10. The kit according to modality B6, which includes a buffer suitable for an enzyme linked immunosorbent assay (ELISA).
[000391] C1. A method to prevent, treat or manage pneumonia in a patient, comprising:
[000392] administer an antibody or antigen-binding fragment according to any of modalities A1 to A30 or with the composition of modality D1 to a patient in need in an amount effective to prevent, treat or manage pneumonia.
[000393] C2. The method according to modality C1, which is a method to prevent pneumonia.
[000394] C3. The method according to the C1 or C2 modality, wherein the antibody or antigen binding fragment binds immunospecifically to a conformational epitope within SEQ ID NO: 39.
[000395] C4. A method to prevent, treat or manage a skin infection condition in a patient, comprising: administering an antibody or an antigen-binding fragment that immunospecifically binds a Staphylococcus aureus alpha-toxin polypeptide to a patient in need. an effective amount to prevent, treat or manage the condition of skin infection.
[000396] C5. The method according to modality C4, in which the skin infection condition is dermonecrosis.
[000397] C6. The method according to modality C4 or C5 in which the condition of the skin infection includes an infection of the skin by Staphylococcus aureus.
[000398] C7. The method according to any of the modalities C4 to C6, which is a method to prevent a skin infection condition.
[000399] C8. A method to prevent, treat or manage a condition associated with a Staphylococcus aureus infection, comprising administering an antibody or an antigen-binding fragment that immunospecifically binds a Staphylococcus aureus alpha-toxin polypeptide to a needy patient in a effective amount to reduce oligomerization of the toxin polypeptide.
[000400] C9. The method according to modality C8, which is a method to prevent the condition associated with infection by Staphylococcus aureus.
[000401] C10. A method to prevent, treat or manage a condition associated with a Staphylococcus aureus infection, comprising administering an antibody or an antigen-binding fragment that immunospecifically binds a Staphylococcus aureus alpha-toxin polypeptide to a needy patient in a effective amount to reduce red cell lysis.
[000402] C11. The method according to modality C10, which is a method to prevent the condition associated with infection by Staphylococcus aureus.
[000403] C12. The method according to modality C10 or C11, in which the erythrocyte is a blood or lung cell.
[000404] C13. The method according to any of the modalities C4 to C12, in which the antibody or an antigen-binding fragment thereof has one or more of the characteristics selected from the group consisting of: (a) affinity constant (KD) for alpha-toxin of about 13 nM or less; (b) binds to alpha-toxin monomers, but does not inhibit the binding of the alpha-toxin to the alpha-toxin receptor; (c) inhibits the formation of alpha-toxin oligomers by at least 50%, 60%, 70%, 80%, 90% or 95%; (d) reduces the alpha-toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (for example, as determined by cell lysis and hemolysis assays); and (e) reduces cell infiltration and release of pro-inflammatory cytokine (for example, in a model of animal pneumonia).
[000405] C14. The method according to modality C13, in which the antibody or its antigen-binding fragment binds to a conformational epitope within SEQ ID NO: 39.
[000406] C15. The method according to any of modalities C1 to C14, wherein the antibody or antigen binding fragment or composition administered to the patient is in accordance with any of modalities A1 to A30 or D1.
[000407] C16. One method, comprising:
[000408] administering an antibody or antigen-binding fragment according to any of modalities A1 to A30 or the composition of modality D1 to cells; and
[000409] detecting the presence, absence or quantity of a biological effect associated with the administration of the composition to the cells.
[000410] C17. One method, comprising:
[000411] administering an antibody or antigen-binding fragment according to any of modalities A1 to A30 or the composition of modality D1 to a patient; and
[000412] detecting the presence, absence or quantity of a biological effect in the patient associated with the administration of the composition.
[000413] C18. One method, comprising:
[000414] administering an antibody or antigen-binding fragment according to any of modalities A1 to A30 or the composition of modality D1 to a patient; and
[000415] monitor the patient's condition.
[000416] C19. A method of neutralizing a Staphylococcus aureus alpha-toxin polypeptide by administering to a patient in need an effective amount of an antibody or antigen-binding fragment according to any of modalities A1 to A30 or the composition of modality D1 to neutralize the polypeptide of toxin.
[000417] C20. A method to prevent, treat or manage a condition mediated by an alpha toxin of Staphylococcus aureus in a needy patient, the method comprising administering to the patient an effective amount of an antibody or antigen-binding fragment according to any of the A1 modalities to A30 or with the composition of modality D1 to prevent, treat or manage the condition.
[000418] C21. A method for treating, preventing or alleviating the symptoms of a disorder mediated by Staphylococcus aureus alpha toxin in a needy patient, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment according to any of the A1 modalities to A30 or with the composition of modality D1 to treat, prevent or alleviate symptoms.
[000419] C22. A method for diagnosing a condition mediated by Staphylococcus aureus alpha toxin in a patient, comprising selecting a patient who needs a diagnosis and administering to the patient an effective amount of an antibody or antigen-binding fragment according to any of the modalities A1 to A30 or with the composition of the D1 modality.
[000420] C23. The method according to any of the modalities C1 to C22, in which the patient is a domestic animal.
[000421] C24. The method according to any of the modalities C1 to C22, in which the patient is a human.
[000422] C25. A method of inhibiting the formation of alpha-toxin oligomers of Staphylococcus aureus in at least 50%, 60%, 70%, 80%, 90% or 95% of an antibody or antigen-binding fragment according to any of the modalities A1 to A30 or with the composition of modality D1.
[000423] C26. The method according to modality C25, in which the inhibition of the formation of alpha-toxin oligomers of Staphylococcus aureus inhibits the formation of the active pore formation complex.
[000424] C27. A method of reducing the cytolytic activity of Staphylococcus aureus alpha toxin by at least 50%, 60%, 70%, 80%, 90% or 95% of an antibody or antigen-binding fragment according to any of the modalities A1 to A30 or with the composition of modality D1, in which cytolytic activity is determined by cell lysis and / or hemolysis assays. * * *
[000425] The totality of each patent, patent application, publication and document referenced here is incorporated by reference. The citation of the above patents, patent applications, publications and documents is not an admission that any of the above is a relevant prior technique, nor does it constitute any admission in relation to the contents or data of those publications or documents.
[000426] Modifications can be made to the above without abandoning the basic aspects contained herein. Although the technology has been described in substantial detail with reference to one or more specific modalities, technicians in the field will recognize that changes may be made to the modalities specifically revealed in this application, although these modifications and improvements remain under the scope and spirit of the technology.
[000427] The technology illustratively described here can be practiced in the absence of any or any element (s) not specifically disclosed here. Thus, for example, in each case here any of the terms "comprising", "consisting essentially of" and "consisting of" can be replaced by the other two terms. The terms and expressions that have been used are used as terms of description and not of limitation, and the use of such terms and expressions does not exclude any equivalents of the characteristics presented and described or their parts, being possible several modifications within the scope of the claimed technology. The term "one" or "one" may refer to one of several elements that it modifies (for example, "a reagent" may mean one or more reagents) unless it is clear from the context whether one of the elements is being described or more than one of them. It should be understood that although the present technology has been specifically revealed by representative modalities and optional features, the modification and variation of the concepts disclosed here can be used by technicians in the field, and such modifications and variations are considered to fall within the scope of this technology.
[000428] Certain embodiments disclosed herein are presented in the following claim (s).

权利要求:
Claims (14)
[0001]
1. Isolated antibody or its antigen-binding fragment, characterized by the fact that the isolated antibody or its antigen-binding fragment immunospecifically binds to an alpha toxin polypeptide of Staphylococcus aureus and comprises VH CDR1, VH CDR2, VH sequences CDR3, VL CDR1, VL CDR2 and VL CDR3 that correspond to the amino acid sequences of SEQ ID Nos: 69, 70, 71, 1, 2 and 68; SEQ ID NOs: 7, 8, 9, 1, 2 and 3; SEQ ID NOs: 10, 11, 12, 1, 2 and 3; SEQ ID NOs: 13, 14, 15, 4, 5 and 6; SEQ ID NOs: 7, 17, 18, 1, 2 and 3; SEQ ID NOs: 7, 8, 16, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 64; SEQ ID NOs; 7, 8, 66, 1, 2 and 64; SEQ ID NOs: 7, 8, 67, 1, 2 and 68; SEQ ID NOs: 7, 8, 67, 1, 2 and 64; SEQ ID NOs: 7, 8, 78, 1, 2 and 64; SEQ ID NOs: 7, 8, 65, 1, 2 and 68; SEQ ID NOs: 7, 8, 72, 1, 73 and 74; SEQ ID NOs: 69, 75, 71, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 2 and 68; SEQ ID NOs: 69, 75, 76, 1, 77 and 74; or SEQ ID NOs: 69, 70, 71, 1, 77 and 74.
[0002]
2. Isolated antibody or its antigen-binding fragment according to claim 1, characterized in that the isolated antibody or its antigen-binding fragment comprises VH CDR1, VH CDR2, VH CDR3, VL CDR2, VL CDR2 sequences and VL CDR3 that correspond to the amino acid sequences of SEQ ID Nos: 69, 70, 71, 1, 2 and 68, respectively.
[0003]
3. Antibody or its antigen-binding fragment, according to claim 1, characterized by the fact that VH and VL correspond to the amino acid sequences of SEQ ID NOs: 57 and 58; SEQ ID NOs: 20 and 19; SEQ ID Nos: 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 62 and 58; SEQ ID NOs: 62 and 63; or SEQ ID NOs: 79 and 63.
[0004]
4. Antibody or its antigen-binding fragment, according to claim 3, characterized by the fact that VH and VL correspond to the amino acid sequences of SEQ ID NOs: 57 and 58.
[0005]
Antibody or its antigen-binding fragment according to any one of claims 1 to 4, characterized in that the isolated antibody or its antigen-binding fragment binds immunospecifically to an alpha-toxin polypeptide from Staphylococcus aureus e has one or more of the selected characteristics of the group consisting of: (a) affinity constant (KD) for alpha toxin of 13 nM or less; (b) binds to alpha-toxin monomers, but does not inhibit the binding of the alpha-toxin to the alpha-toxin receptor; . (c) inhibits the formation of alpha-toxin oligomers by at least 50%, 60%, 70%, 80%, 90% or 95%; (d) reduces the alpha-toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (for example, as determined by cell lysis and hemolysis assays); and (e) reduces cell infiltration and release of pro-inflammatory cytokine (for example, in a model of animal pneumonia).
[0006]
6. Composition, characterized in that the composition comprises the antibody or antigen binding fragment as defined in any one of claims 1 to 5.
[0007]
7. Composition according to claim 6, characterized in that the composition comprises an additional agent, wherein the additional agent is an antibiotic.
[0008]
8. Kit, characterized in that the kit comprises: (a) an antibody or antigen-binding fragment as defined in any one of claims 1 to 5 or the composition as defined in claim 6 or 7; (b) instructions for using the antibody, antigen-binding fragment or composition or directions for using the antibody, antigen-binding fragment or composition.
[0009]
9. Use of an effective amount of an antibody or its antigen-binding fragment that binds immunospecifically to an alpha-toxin polypeptide of Staphylococcus aureus, as defined in any of claims 1 to 5, characterized in that it is to prepare a pharmaceutical composition to prevent, treat or manage pneumonia and / or to prevent, treat or manage a condition of skin infection in an individual.
[0010]
10. Method for inhibiting the formation of Staphylococcus aureus alpha-toxin oligomers, characterized in that the isolated antibody or antigen-binding fragment as defined in any of claims 1 to 5 or the composition as defined in claims 6 or 7 inhibit the formation of alpha-toxin oligomers by at least 50%, 60%, 70%, 80%, 90% or 95%.
[0011]
11. Use of an antibody or its antigen-binding fragment, as defined in any one of claims 1 to 5, characterized in that it is to prepare a pharmaceutical composition to inhibit the formation of Staphylococcus aureus alpha-toxin oligomers in at least minus 50%, 60%, 70%, 80%, 90% or 95%.
[0012]
12. Composition according to claim 7, characterized by the fact that the antibiotic is vancomycin.
[0013]
13. Isolated antibody or its antigen-binding fragment, characterized by the fact that the isolated antibody or its antigen-binding fragment binds to a Staphylococcus aureus alpha-toxin polypeptide and comprises a variant Fc zone, in which the Isolated antibody comprises SEQ ID NO: 91 and SEQ ID NO: 92.
[0014]
14. Pharmaceutical composition, characterized by the fact that the pharmaceutical composition comprises the antibody or antigen-binding fragment as defined in claim 13.

类似技术:

---

公开号 | 公开日 | 专利标题

---

JP6723293B2|2020-07-15|Antibodies that specifically bind to Staphylococcus aureus alpha toxin and methods of use

---

AU2019261828B2|2021-06-10|Neutralizing anti-influenza a antibodies and uses thereof

---

KR102218494B1|2021-02-19|Anti-hepcidin antibodies and uses thereof

---

CN111629754A|2020-09-04|anti-CD 47 antibodies that do not cause significant red blood cell agglutination

---

KR20180101436A|2018-09-12|How to treat influenza A

---

KR20160138220A|2016-12-02|Compositions and methods for treatment of diabetic macular edema

---

KR20150132473A|2015-11-25|Anti-plasma kallikrein antibodies

---

CN106573154B|2021-06-15|Neutralizing anti-influenza b antibodies and uses thereof

---

US10457721B2|2019-10-29|Anti-OSPA antibodies and methods of use

同族专利:

---

公开号 | 公开日

---

PL2673373T3|2019-04-30|

---

MX2013009127A|2014-01-31|

---

PT2673373T|2018-12-05|

---

CA2826566A1|2012-08-16|

---

SG192212A1|2013-09-30|

---

HRP20182017T1|2019-02-08|

---

JP2019011329A|2019-01-24|

---

RU2620065C2|2017-05-22|

---

CN107474133A|2017-12-15|

---

TR201817932T4|2019-02-21|

---

ES2709065T7|2021-12-09|

---

SI2673373T1|2019-03-29|

---

JP2014508748A|2014-04-10|

---

EP2673373B3|2021-06-02|

---

HK1248257A1|2018-10-12|

---

JP6367393B2|2018-08-01|

---

LT2673373T|2019-01-25|

---

SG10201600899PA|2016-03-30|

---

BR112013020086A2|2016-11-22|

---

AU2012214531C1|2021-07-29|

---

CN103443285B|2017-06-23|

---

KR20140019344A|2014-02-14|

---

PL2673373T6|2021-09-27|

---

US20140072577A1|2014-03-13|

---

JP6723293B2|2020-07-15|

---

EP2673373A4|2015-06-17|

---

EP3470526A2|2019-04-17|

---

US9527905B2|2016-12-27|

---

CN103443285B9|2018-04-24|

---

EP2673373B1|2018-08-29|

---

DK2673373T6|2021-08-23|

---

JP2020172489A|2020-10-22|

---

HRP20182017T4|2021-08-20|

---

RS58190B2|2021-08-31|

---

JP2017128575A|2017-07-27|

---

DK2673373T3|2019-01-02|

---

CN103443285A|2013-12-11|

---

ES2709065T3|2019-04-15|

---

SG10201913690SA|2020-03-30|

---

WO2012109285A3|2012-10-04|

---

MX345285B|2017-01-24|

---

HUE040734T2|2019-03-28|

---

WO2012109285A2|2012-08-16|

---

EP3470526A3|2019-07-17|

---

RU2013141134A|2015-03-20|

---

EP2673373A2|2013-12-18|

---

AU2012214531B2|2017-03-16|

---

CY1121389T1|2020-05-29|

---

KR101993839B1|2019-06-28|

---

CN107474133B|2021-10-15|

---

RS58190B1|2019-03-29|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

IL85035D0|1987-01-08|1988-06-30|Int Genetic Eng|Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same|

---

DE69233254T2|1991-06-14|2004-09-16|Genentech, Inc., South San Francisco|Humanized Heregulin antibody|

---

AT357663T|1996-04-25|2007-04-15|Genicon Sciences Corp|PARTICULAR LABEL USING ANALYTASSAY|

---

DK1522590T3|2000-06-28|2009-12-21|Glycofi Inc|Process for Preparation of Modified Glycoproteins|

---

US6596541B2|2000-10-31|2003-07-22|Regeneron Pharmaceuticals, Inc.|Methods of modifying eukaryotic cells|

---

ES2727425T3|2000-12-12|2019-10-16|Medimmune Llc|Molecules with prolonged half-lives, compositions and uses thereof|

---

US20030226155A1|2001-08-30|2003-12-04|Biorexis Pharmaceutical Corporation|Modified transferrin-antibody fusion proteins|

---

AT458006T|2003-05-14|2010-03-15|Kenta Biotech Ag|HUMAN MONOCLONAL ANTIBODY SPECIFIC TO LEPOPOLYSACCHARIDES OF SEROTYPE IAT O6 BY PSEUDOMONAS AERUGINOSA|

---

CN1324049C|2003-08-04|2007-07-04|中国疾病预防控制中心病毒病预防控制所|Human source anti SARS coronavirus gene engineering antibody|

---

ES2403055T3|2004-04-13|2013-05-13|F. Hoffmann-La Roche Ag|Anti-P-selectin antibodies|

---

EP2305294B1|2004-09-22|2015-04-01|GlaxoSmithKline Biologicals SA|Immunogenic composition for use in vaccination against staphylococcei|

---

WO2007084922A2|2006-01-17|2007-07-26|Biolex Therapeutics, Inc.|Compositions and methods for humanization and optimization of n-glycans in plants|

---

TW200813091A|2006-04-10|2008-03-16|Amgen Fremont Inc|Targeted binding agents directed to uPAR and uses thereof|

---

US20090053235A1|2006-06-12|2009-02-26|Nabi Biopharmaceuticals|Use of alpha-toxin for treating and preventing staphylococcus infections|

---

BRPI0811193A2|2007-05-31|2014-11-11|Merck & Co Inc|ISOLATED ANTIGEN BINDING PROTEIN, NUCLEIC ACID, RECOMBINANT CELL, METHODS FOR PRODUCTING A PROTEIN AND FOR PROTECTING OR TREATING AGAINST S. AUREUS INFECTION IN PATIENT, PHARMACEUTICAL COMPOSITION, LYNAL PROTEIN AND POLYTEIN USE|

---

TWI595005B|2007-08-21|2017-08-11|安健股份有限公司|Human c-fms antigen binding proteins|

---

WO2009029831A1|2007-08-31|2009-03-05|University Of Chicago|Methods and compositions related to immunizing against staphylococcal lung diseases and conditions|

---

US8172115B1|2008-04-10|2012-05-08|Spencer Forrest, Inc.|Hair building solids dispenser for one handed operation|

---

MX2011000041A|2008-07-02|2011-05-23|Emergent Product Dev Seattle|Tnf-î± antagonist multi-target binding proteins.|

---

EP2208787A1|2009-01-19|2010-07-21|Université de Liège|A recombinant alpha-hemolysin polypeptide of Staphylococcus aureus, having a deletion in the stem domain and heterologous sequences inserted|

---

GB2467939A|2009-02-20|2010-08-25|Univ Sheffield|Removal of bacteria from media|

---

EP2445522B1|2009-06-22|2017-08-09|Wyeth LLC|Immunogenic compositions of staphylococcus aureus antigens|

---

PL2917360T3|2012-11-06|2020-06-29|Medimmune, Llc|Antibodies to s. aureus surface determinants|EP2718320B1|2011-06-10|2018-01-10|MedImmune Limited|Anti-pseudomonas psl binding molecules and uses thereof|

---

MX2014005566A|2011-11-07|2014-10-14|Medimmune Llc|Combination therapies using anti- pseudomonas psl and pcrv binding molecules.|

---

US9988439B2|2011-12-23|2018-06-05|Nicholas B. Lydon|Immunoglobulins and variants directed against pathogenic microbes|

---

EP2793944A4|2011-12-23|2015-09-02|Nicholas B Lydon|Immunoglobulins and variants directed against pathogenic microbes|

---

JOP20200236A1|2012-09-21|2017-06-16|Regeneron Pharma|Anti-cd3 antibodies, bispecific antigen-binding molecules that bind cd3 and cd20, and uses thereof|

---

EP2917236A2|2012-11-06|2015-09-16|Medlmmune, LLC|Combination therapies using anti-pseudomonas psl and pcrv binding molecules|

---

PL2917360T3|2012-11-06|2020-06-29|Medimmune, Llc|Antibodies to s. aureus surface determinants|

---

KR102272744B1|2012-11-06|2021-07-06|메디뮨 엘엘씨|Methods of treating s. aureus-associated diseases|

---

EP3057989A1|2013-10-17|2016-08-24|ARSANIS Biosciences GmbH|Cross-reactive staphylococcus aureus antibody sequences|

---

WO2015109012A1|2014-01-14|2015-07-23|Integrated Biotherapeutics, Inc.|Targeting immunological functions to the site of bacterial infections using cell wall targeting domains of bacteriolysins|

---

TWI719938B|2014-06-19|2021-03-01|美商麥迪紐有限責任公司|Treatment of polybacterial infections|

---

CA2956429A1|2014-08-12|2016-02-18|Arsanis Biosciences Gmbh|Predicting s. aureus disease|

---

KR20170089881A|2014-11-17|2017-08-04|리제너론 파아마슈티컬스, 인크.|Methods for tumor treatment using cd3xcd20 bispecific antibody|

---

EP3353317A1|2015-09-24|2018-08-01|H. Hoffnabb-La Roche Ag|Alpha-hemolysin variants|

---

WO2017075188A2|2015-10-30|2017-05-04|Medimmune, Llc|Methods of using anti-alpha toxin antibody|

---

TW201837054A|2017-01-03|2018-10-16|美商再生元醫藥公司|Human antibodies to s. aureus hemolysin a toxin|

---

US11267886B2|2017-02-09|2022-03-08|Bluefin Biomedicine, Inc.|Anti-ILT3 antibodies and antibody drug conjugates|

---

CN109384844B|2017-08-04|2020-12-18|中国人民解放军军事医学科学院基础医学研究所|Anti-staphylococcus aureus alpha hemolysin monoclonal antibody and application thereof|

---

EP3840838A2|2018-07-24|2021-06-30|MedImmune, LLC|Antibody directed against s.aureus clumping factor a |

---

WO2020041532A1|2018-08-21|2020-02-27|Sf17 Therapeutics, Inc.|Compositions and methods for treating progressive neurodegenerative diseases|

---

TW202035443A|2018-10-09|2020-10-01|美商麥迪紐有限責任公司|Combinations of anti-staphylococcus aureus antibodies|

---

JP2022512646A|2018-10-09|2022-02-07|メディミューン，エルエルシー|Staphylococcus aureus Antibodies directed against leukotoxin|

---

TW202100549A|2019-03-13|2021-01-01|美商麥迪紐有限責任公司|Decreasing staphylococcus aureus infections in colonized patients|

---

WO2020210650A1|2019-04-12|2020-10-15|Medimmune, Llc|Neutralization of tgf-beta or alpha-v-beta-8 integrin for s. aureus infections|

---

CN113444173A|2020-03-25|2021-09-28|兴盟生物医药有限公司|Staphylococcus aureus alpha-toxin specific antibody and application thereof|

法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2018-07-24| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

---

2019-08-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2020-04-22| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

---

2020-08-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2020-12-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/02/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

US201161440581P| true| 2011-02-08|2011-02-08|

---

US61/440,581|2011-02-08|

---

PCT/US2012/024201|WO2012109285A2|2011-02-08|2012-02-07|Antibodies that specifically bind staphylococcus aureus alpha toxin and methods of use|

---