阅读说明：本技术 减少定殖患者中的金黄色葡萄球菌感染 (Reduction of staphylococcus aureus infections in colonized patients ) 是由 M·埃泽尔 A·鲁金 H·贾夫里 K·舍马克 B·塞尔曼 喻利 于 2020-03-12 设计创作，主要内容包括：本披露涉及用抗α毒素抗体或其抗原结合片段治疗定殖有金黄色葡萄球菌的受试者的方法。这些方法可以降低该受试者中金黄色葡萄球菌的存在所伴随的感染的发生率。(The present disclosure relates to methods of treating a subject colonized with staphylococcus aureus with an anti-alpha toxin antibody or antigen-binding fragment thereof. These methods can reduce the incidence of infection associated with the presence of staphylococcus aureus in the subject.)

1. A method of treating a subject colonized with staphylococcus aureus, the method comprising administering to the subject an antibody, or antigen-binding fragment thereof, that binds to staphylococcus aureus Alpha Toxin (AT), wherein Polymerase Chain Reaction (PCR) has been used to detect the level of staphylococcus aureus in a sample obtained from the subject.

2. The method of claim 1, wherein the sample obtained from the subject has a level of Staphylococcus aureus that does not exceed a level of Staphylococcus aureus associated with a Polymerase Chain Reaction (PCR) cycle threshold (Ct) value.

3. The method of claim 1 or 2, wherein the method reduces the incidence of infection associated with the presence of staphylococcus aureus in the subject.

4. A method of preventing a staphylococcus aureus infection in a subject, comprising administering to the subject an antibody, or antigen-binding fragment thereof, that binds to staphylococcus aureus AT, wherein a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a PCR Ct value.

5. A method for reducing the incidence of staphylococcus aureus pneumonia in a subject, comprising administering sutolizumab to the subject, wherein the reduction is determined by clinical, microbiological and radiological measurements, optionally wherein the incidence is reduced by about 30%.

6. A method for reducing the incidence of pan-pneumonia in a subject, the method comprising administering to said subject secukinumab, wherein said reduction is determined by clinical, microbiological and radiological measurements, optionally wherein the incidence is reduced by about 30%.

7. The method of any one of claims 3 and 4, wherein the infection is determined by clinical, microbiological and radiological measurements.

8. The method of any one of claims 5, 6, and 7, wherein the clinical measurement comprises abnormal body temperature, abnormal white blood cell count, cough, purulent sputum, bronchial respiratory sounds, dyspnea, tachypnea (respiratory rate >30 breaths/minute), hypoxemia, or any combination thereof.

9. The method of any one of claims 5, 6, 7, and 8, wherein the microbiological measurement comprises a staphylococcus aureus positive breath sample, a blood culture, a pleural fluid aspirate, or a lung tissue culture.

10. The method of any one of claims 5, 6, and 7-9, wherein the radiological measurement comprises new or deteriorated infiltrates on chest X-rays.

11. The method of any one of claims 5-10, wherein PCR has been used to detect the level of staphylococcus aureus in a sample obtained from the subject.

12. The method of claim 11, wherein the sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a Polymerase Chain Reaction (PCR) cycle threshold (Ct) value.

13. The method of any one of claims 1-12, further comprising detecting the level of staphylococcus aureus in the sample obtained from the subject.

14. The method of any one of claims 1-4 and 11-13, wherein the sample obtained from the subject has a level of staphylococcus aureus that does not exceed the level of staphylococcus aureus associated with a PCR Ct value of 29 or more.

15. The method of claim 16, wherein a PCR Ct value of 29 corresponds to a concentration of staphylococcus aureus of about 1600 to about 1700 Colony Forming Units (CFU)/ml.

16. A method of treating a subject colonized with a staphylococcus aureus infection, the method comprising administering to the subject an antibody, or antigen-binding fragment thereof, that binds to staphylococcus aureus AT, wherein a sample obtained from the subject has a concentration of staphylococcus aureus of no more than 1700 CFU/ml.

17. The method of claim 18, wherein the method reduces the incidence of infection associated with the presence of staphylococcus aureus in the subject.

18. A method of preventing a staphylococcus aureus infection in a subject, the method comprising administering to the subject an antibody, or antigen-binding fragment thereof, that binds to staphylococcus aureus AT, wherein a sample obtained from the subject has a concentration of staphylococcus aureus of no more than 1700 CFU/ml.

19. The method of any one of claims 18-20, wherein the concentration of staphylococcus aureus AT is measured using PCR.

20. The method of any one of claims 4-17 and 20-21, wherein the subject is colonized with staphylococcus aureus.

21. The method of any one of claims 1-22, wherein the sample obtained from the subject has a level of staphylococcus aureus that correlates at least with a PCR Ct value.

22. The method of claim 23, wherein the sample obtained from the subject has a staphylococcus aureus level associated with a PCR Ct value of at least 3.

23. The method of any one of claims 1-24, wherein the staphylococcus aureus levels are detected in no more than 3 hours, preferably no more than 2 hours.

24. The method of any one of claims 1-4, 11-15, and 19-23, wherein the PCR detects staphylococcus aureus protein a.

25. The method of any one of claims 1-24, wherein the subject is ventilated, optionally wherein the subject is mechanically ventilated.

26. The method of any one of claims 1-25, wherein the subject is taking an antibiotic.

27. The method of any one of claims 1-4 and 11-26, wherein the sample is a skin or soft tissue sample.

28. The method of any one of claims 1-4 and 11-26, wherein the sample is obtained from the lower respiratory tract of the subject.

29. The method of any one of claims 1-4 and 11-26, wherein the sample is an intratracheal aspirate.

30. The method of any one of claims 1-4 and 11-26, wherein the sample is a tracheal sample.

31. The method of any one of claims 1-4 and 11-26, wherein the sample is a bronchial sample.

32. The method of any one of claims 1-4 and 11-31, wherein the sample contains bacteria that do not grow in culture to identify staphylococcus aureus.

33. The method of any one of claims 1-4 and 11-3213-34, wherein the sample contains bacteria that are not staphylococci.

34. The method of any one of claims 1-5 and 7-33, wherein the staphylococcus aureus is antibiotic resistant.

35. The method of any one of claims 1-5 and 7-34, further comprising determining whether the staphylococcus aureus is antibiotic-resistant.

36. The method of any one of claims 1-5 and 7-35, wherein the staphylococcus aureus is methicillin-resistant.

37. The method of any one of claims 1-5 and 7-36, further comprising determining whether the staphylococcus aureus is methicillin-resistant.

38. The method of any one of claims 34-37, wherein drug resistance is determined using PCR.

39. The method of any one of claims 3, 4, and 7-37, wherein the infection is pneumonia.

40. The method of any one of claims 3, 4, and 7-37, wherein the infection is Intensive Care Unit (ICU) pneumonia.

41. The method of any one of claims 1-40, wherein the subject is a human.

42. The method of any one of claims 1-4 and 7-41, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT binds the same epitope of Staphylococcus aureus AT as an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, the VL comprises the amino acid sequence of SEQ ID NO: 8.

43. The method of any one of claims 1-4 and 7-42, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT competitively inhibits binding of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, the VL comprises the amino acid sequence of SEQ ID NO: 8.

44. The method of any one of claims 1-4 and 7-43, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of MEDI 4893.

45. The method of any one of claims 1-4 and 7-44, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, a variable heavy chain (VH) Complementarity Determining Region (CDR)1 comprising the amino acid sequence of SEQ ID NO: 2, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4, a variable light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 5 and a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 6, VL CDR3 of the amino acid sequence of seq id No. 6.

46. The method of any one of claims 1-4 and 7-45, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7.

47. The method of any one of claims 1-4 and 7-46, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, VL of an amino acid sequence of seq id No. 8.

48. The method of any one of claims 1-4 and 7-47, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, or a light chain of the amino acid sequence of 9.

49. The method of any one of claims 1-4 and 7-48, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, or a light chain of the amino acid sequence of seq id No. 10.

50. The method of any one of claims 1-4, 7-47, and 49, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus AT further comprises a heavy chain constant region.

51. The method of claim 50, wherein the heavy chain constant region is selected from the group consisting of: human immunoglobulin IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂A heavy chain constant region.

52. The method of claim 51, wherein the heavy chain constant region is human IgG₁A constant region.

53. The method of any one of claims 1-4, 7-48, and 51-52, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus AT further comprises a light chain constant region.

54. The method of claim 53, wherein said light chain constant region is selected from the group consisting of human immunoglobulin IgG kappa and IgG lambda light chain constant regions.

55. The method of claim 54, wherein the light chain constant region is a human IgG kappa light chain constant region.

56. The method of any one of claims 1-4 and 7-47, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT is an IgG antibody or antigen-binding fragment thereof.

57. The method of any one of claims 1-4, 7-47, and 56, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT comprises an Fc region that has been engineered to improve half-life.

58. The method of any one of claims 1-4, 7-47, and 56, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT comprises an Fc region having YTE mutations.

59. The method of any one of claims 1-4 and 7-58, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus AT is a monoclonal antibody or antigen-binding fragment.

60. The method of any one of claims 1-4 and 7-59, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus AT is a full-length antibody.

61. The method of any one of claims 1-4 and 7-59, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus AT is an antigen-binding fragment.

62. The method of claim 61, wherein the antibody or antigen-binding fragment that binds Staphylococcus aureus AT, wherein the antigen-binding fragment comprises Fab, Fab ', F (ab')₂Single chain Fv (scFv), disulfide-linked Fv, intrabody, IgG Δ CH2, minibody, F (ab')₃Four-chain antibody, three-chain antibody, double-chain antibody, DVD-Ig, Fcab, mAb²、(scFv)₂Or scFv-Fc.

63. The method of any one of claims 1-4 and 7-62, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT has an affinity of 80-100pM for Staphylococcus aureus AT.

64. The method of any one of claims 1-4 and 7-63, wherein the antibody or antigen-binding fragment thereof is sumatrizumab.

65. The method of any one of claims 1-4 and 7-64, wherein 2000mg of the antibody or antigen-binding fragment is administered.

66. The method of any one of claims 1-4 and 7-64, wherein 5000mg of the antibody or antigen-binding fragment is administered.

67. The method of any one of claims 1-4, 7-15, and 18-66, wherein the preventing S.aureus infection comprises toxin neutralization, induction of opsonophagocytosis, inhibition of thromboembolic lesion formation, inhibition of S.aureus-associated sepsis, or any combination of the foregoing.

68. An antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT for use in treating a subject colonized with staphylococcus aureus (S aureus), wherein a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a Polymerase Chain Reaction (PCR) cycle threshold (Ct) value.

69. The antibody or antigen-binding fragment of claim 68, wherein the treatment reduces the incidence of infection associated with the presence of Staphylococcus aureus in the subject.

70. An antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT for use in preventing a staphylococcus aureus infection in a subject colonized with staphylococcus aureus, wherein a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a PCR Ct value.

71. The antibody or antigen-binding fragment thereof of any one of claims 68-70, wherein the sample obtained from the subject has a level of Staphylococcus aureus that does not exceed the level of Staphylococcus aureus associated with a PCR Ct value of 29.

72. The antibody or antigen-binding fragment of any one of claims 68-70, wherein the antibody or antigen-binding fragment thereof is not administered to the subject if a level of Staphylococcus aureus that does not exceed a level of Staphylococcus aureus associated with a PCR Ct value is detected in a sample obtained from the subject.

73. An in vitro method of identifying a subject colonized with staphylococcus aureus as being responsive to an antibody, or antigen-binding fragment thereof, to alloy staphylococcus aureus AT, the method comprising detecting a level of staphylococcus aureus in a sample obtained from the subject, wherein a level of staphylococcus aureus that does not exceed the level of staphylococcus aureus associated with a PCR Ct value indicates that the subject is responsive to the antibody, or antigen-binding fragment thereof.

74. The method of claim 73, wherein the PCR Ct value is 29.

Background

Bacterial pneumonia occurring in hospitalized or Intensive Care Unit (ICU) populations is a clinically significant and serious disease with significant impact on morbidity and mortality. This constitutes the second major type of nosocomial infection in the united states and the leading cause of death for nosocomial infections (Spellberg and Talbot, 2010). Staphylococcus aureus is the leading cause of nosocomial pneumonia. A recent study by european ICU reported that 23% of mechanically ventilated ICU patients develop pneumonia caused by staphylococcus aureus, more than half of which are caused by methicillin-resistant staphylococcus aureus (MRSA) (esperati et al, 2010).

S. aureus also causes a variety of other diseases including skin and soft tissue infections, endocarditis, osteomyelitis, pneumonia and bacteremia (Lowy, f.d., n.engl.j.med. [ new england journal of medicine ], 339 (8): 520-32 (1998)). During infection, staphylococcus aureus releases many toxins, of which Alpha Toxin (AT) is the most prevalent virulence factor causing tissue invasion and necrosis (Wilke and Bubeck Wardenburg, 2010). The key role of AT in the pathogenesis of s.aureus is supported by animal models (skin necrosis, pneumonia, sepsis, endocarditis and mastitis) and observation studies in humans, where the presence of anti-AT antibodies during severe infections correlates with improved outcomes.

Preclinical studies have shown that monoclonal antibody-based methods are promising for the prevention and adjuvant treatment of staphylococcus aureus infections (see, e.g., Hazenbos et al, PLoS Pathog [ scientific public library & etiology ], 9 (10): e1003653. doi: 10.1371/journel.p. 1003653.doi (2013); Rouha, h., MAbs [ monoclonal antibodies ], 7 (1): 243. 254 (2015); Foletti et al, j.mol. biol. [ journal of molecular biology ], 425 (10): 1641. biol 4 (2013); Karauzum et al, JBiol Chem. [ journal of biochemistry ], 287 (30): 03-15 (2012); and Hua et al, Antimicrob chemicotherapy (58) [ antimicrobial and 58): 17): 2014). anti-AT antibodies show promising results in terms of their ability to treat and prevent s. MEDI4893 (or sutomozumab) is a human monoclonal antibody with an extended half-life that binds AT with high affinity and effectively blocks the formation of AT pores in the target cell membrane. Preclinical results demonstrate that prevention with anti-AT antibodies comprising MEDI4893 binding regions reduces disease severity in murine models of skin necrosis, pneumonia, and fatal bacteraemia/sepsis infection (see, e.g., WO 2012/109285 and WO 2014/074540, each of which is incorporated herein by reference in its entirety).

However, staphylococcus aureus infections (e.g., pneumonia) can develop very rapidly in patients colonized with staphylococcus aureus, and methods are therefore needed to identify AT-risk patients who will receive the greatest benefit from anti-AT antibodies.

Disclosure of Invention

Staphylococcus aureus (s.aureus) pneumonia is a life-threatening complication that occurs early in Intensive Care Unit (ICU) patients who receive mechanical ventilation despite infection control and antibiotics. As demonstrated herein, anti-Alpha Toxin (AT) antibodies were evaluated for prevention of staphylococcus aureus pneumonia and shown to correlate with clinically meaningful efficacy (> 25% reduction in relative risk) and acceptable safety. In particular, a 32% reduction in staphylococcus aureus pneumonia was observed in patients receiving anti-AT antibodies, and there were no safety concerns. In addition, even greater efficacy was observed in certain patient subgroups. Accordingly, provided herein are methods of identifying patients AT risk of developing a staphylococcus aureus infection that would benefit from receiving an anti-AT antibody.

Provided herein are methods of treating a subject colonized with staphylococcus aureus (s.aureus), the methods comprising administering to the subject an antibody, or antigen-binding fragment thereof, that binds to staphylococcus aureus Alpha Toxin (AT), wherein Polymerase Chain Reaction (PCR) has been used to detect the level of staphylococcus aureus in a sample obtained from the subject. In certain instances, a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a Polymerase Chain Reaction (PCR) cycle threshold (Ct) value. In certain instances, the method reduces the incidence of infection associated with the presence of staphylococcus aureus in the subject.

Provided herein are methods of preventing a staphylococcus aureus infection in a subject, the methods comprising administering to the subject an antibody, or antigen-binding fragment thereof, that binds to staphylococcus aureus AT, wherein a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a PCR Ct value.

Provided herein are methods for reducing the incidence of staphylococcus aureus pneumonia in a subject, the methods comprising administering sumatrizumab to the subject, wherein the reduction is determined by clinical, microbiological and radiological measurements, optionally wherein the incidence is reduced by about 30%.

Provided herein are methods for reducing the incidence of pan-pneumonia in a subject, the methods comprising administering to the subject secukinumab, wherein the reduction is determined by clinical, microbiological and radiological measurements, optionally wherein the incidence is reduced by about 30%.

In some cases, the infection is determined by clinical, microbiological and radiological measurements.

In some cases, these clinical measurements include abnormal body temperature, abnormal white blood cell counts, coughing, purulent sputum, bronchial respiratory sounds, dyspnea, shortness of breath (respiratory rate >30 breaths/minute), hypoxemia, or any combination thereof.

In certain instances, the microbiological measurement comprises a breath sample positive for staphylococcus aureus, a blood culture, a pleural fluid aspirate, or a lung tissue culture.

In some cases, the radiological measurements include new or deteriorated infiltrates on chest X-rays.

In some cases, PCR has been used to detect the level of staphylococcus aureus in a sample obtained from the subject. In certain instances, a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a Polymerase Chain Reaction (PCR) cycle threshold (Ct) value.

In certain instances, the methods provided herein further comprise detecting the level of staphylococcus aureus in a sample obtained from the subject.

In certain instances, the sample obtained from the subject has a level of staphylococcus aureus that does not exceed the level of staphylococcus aureus associated with a PCR Ct value of 29 or higher. In certain instances, a PCR Ct value of 29 corresponds to a concentration of staphylococcus aureus of about 1600 to about 1700 Colony Forming Units (CFU)/ml.

Provided herein are methods of treating a subject colonized with a staphylococcus aureus infection, the methods comprising administering to the subject an antibody, or antigen-binding fragment thereof, that binds to staphylococcus aureus AT, wherein a sample obtained from the subject has a concentration of staphylococcus aureus of no more than 1700 CFU/ml. In certain instances, the method reduces the incidence of infection associated with the presence of staphylococcus aureus in the subject.

Provided herein are methods of preventing a staphylococcus aureus infection in a subject, the methods comprising administering to the subject an antibody, or antigen-binding fragment thereof, that binds to staphylococcus aureus AT, wherein a sample obtained from the subject has a concentration of staphylococcus aureus of no more than 1700 CFU/ml.

In some cases, the concentration of staphylococcus aureus AT is measured using PCR.

In certain instances, the subject is colonized with staphylococcus aureus.

In certain instances, the sample obtained from the subject has a level of staphylococcus aureus that correlates at least with the PCR Ct value. In certain instances, the sample obtained from the subject has a staphylococcus aureus level associated with a PCR Ct value of at least 3.

In some cases, the level of Staphylococcus aureus is measured in no more than 3 hours, preferably no more than 2 hours.

In some cases, the PCR detects staphylococcus aureus protein a.

In certain instances, the subject is ventilated, optionally wherein the subject is mechanically ventilated. In some cases, the subject is taking an antibiotic.

In some cases, the sample is a skin or soft tissue sample. In certain instances, the sample is obtained from the lower respiratory tract of the subject. In some cases, the sample is an endotracheal aspirate. In some cases, the sample is a tracheal sample. In some cases, the sample is a bronchial sample.

In some cases, the sample contains bacteria that do not grow in culture to identify Staphylococcus aureus. In some cases, the sample contains bacteria that are not staphylococci. In certain instances, the staphylococcus aureus is antibiotic resistant. In certain instances, the methods provided herein further comprise determining whether the staphylococcus aureus is antibiotic resistant.

In some cases, the staphylococcus aureus is methicillin-resistant. In certain instances, the methods provided herein further comprise determining whether the staphylococcus aureus is methicillin-resistant.

In some cases, resistance is determined using PCR.

In some cases, the infection is pneumonia. In some cases, the infection is Intensive Care Unit (ICU) pneumonia.

In certain instances, the subject is a human.

In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT binds the same staphylococcus aureus AT epitope as an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, the VL comprises the amino acid sequence of SEQ ID NO: 8. In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT competitively inhibits binding of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, the VL comprises the amino acid sequence of SEQ ID NO: 8.

In certain instances, the antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of MEDI 4893. In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT comprises a heavy chain variable region comprising SEQ ID NO: 1, a variable heavy chain (VH) Complementarity Determining Region (CDR)1 comprising the amino acid sequence of SEQ ID NO: 2, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4, a variable light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 5 and a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 6, VL CDR3 of the amino acid sequence of seq id No. 6.

In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT comprises a heavy chain variable region comprising SEQ ID NO: 7. In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT comprises a heavy chain variable region comprising SEQ ID NO: 8, VL of an amino acid sequence of seq id No. 8.

In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT comprises a heavy chain variable region comprising SEQ ID NO: 9, or a light chain of the amino acid sequence of 9. In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT comprises a heavy chain variable region comprising SEQ ID NO: 10, or a light chain of the amino acid sequence of seq id No. 10.

In certain instances, the antibody or antigen-binding fragment that binds to staphylococcus aureus AT further comprises a heavy chain constant region. In some cases, the heavy chain constant region is selected from the group consisting of: human immunoglobulin IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂A heavy chain constant region. In some cases, the heavy chain constant region is a human IgG₁A constant region.

In certain instances, the antibody or antigen-binding fragment that binds to staphylococcus aureus AT further comprises a light chain constant region. In some cases, the light chain constant region is selected from the group consisting of human immunoglobulin IgG κ and IgG λ light chain constant regions. In some cases, the light chain constant region is a human IgG kappa light chain constant region.

In certain instances, the antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT is an IgG antibody or antigen-binding fragment thereof.

In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT comprises an Fc region that has been engineered to improve half-life. In certain instances, the antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT comprises an Fc region having a YTE mutation.

In certain instances, the antibody or antigen-binding fragment that binds to staphylococcus aureus AT is a monoclonal antibody or antigen-binding fragment.

In certain instances, the antibody or antigen-binding fragment that binds to staphylococcus aureus AT is a full-length antibody. In certain instances, the antibody or antigen-binding fragment that binds to staphylococcus aureus AT is an antigen-binding fragment. In certain instances, the antigen-binding fragment comprises Fab, Fab ', F (ab')₂Single chain Fv (scFv), disulfide-linked Fv, intrabody, IgG Δ CH2, minibody, F (ab')₃Four-chain antibody, three-chain antibody, double-chain antibody, DVD-Ig, Fcab, mAb²、(scFv)₂Or scFv-Fc.

In certain instances, an antibody or antigen-binding fragment thereof that binds to staphylococcus aureus AT has an affinity of 80-100pM for staphylococcus aureus AT.

In certain instances, the antibody or antigen-binding fragment thereof is sumatrizumab.

In some cases, 2000mg of the antibody or antigen-binding fragment is administered. In some cases, 5000mg of the antibody or antigen-binding fragment is administered.

In certain instances, preventing a staphylococcus aureus infection comprises toxin neutralization, induction of opsonophagocytosis, inhibition of thromboembolic lesion formation, inhibition of staphylococcus aureus-associated sepsis, or any combination of the foregoing.

Provided herein are antibodies, or antigen-binding fragments thereof, that bind to staphylococcus aureus AT for use in treating a subject colonized with staphylococcus aureus (s. In certain instances, the treatment reduces the incidence of infection associated with the presence of staphylococcus aureus in the subject.

Provided herein are antibodies, or antigen-binding fragments thereof, that bind to staphylococcus aureus AT for use in preventing a staphylococcus aureus infection in a subject colonized with staphylococcus aureus, wherein a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a PCR Ct value.

In certain instances, the sample obtained from the subject has a level of staphylococcus aureus that does not exceed the level of staphylococcus aureus associated with a PCR Ct value of 29. In certain instances, the antibody or antigen-binding fragment thereof is not administered to the subject where a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a PCR Ct value is detected in a sample obtained from the subject.

Provided herein are in vitro methods of identifying a subject colonized with staphylococcus aureus as being responsive to an antibody, or antigen-binding fragment thereof, to alloy staphylococcus aureus AT, the methods comprising detecting a level of staphylococcus aureus in a sample obtained from the subject, wherein a level of staphylococcus aureus that does not exceed the level of staphylococcus aureus associated with a PCR Ct value indicates that the subject is responsive to the antibody, or antigen-binding fragment thereof. In some cases, the PCR Ct value is 29.

Drawings

FIG. 1 is a schematic diagram illustrating the rapid identification of patients using the Staphylococcus aureus PCR test. The PCR test yields a cycle threshold (Ct) value that represents the number of PCR cycles (represented by the horizontal line in the right graph of the figure) required to reach a threshold signal. Ct values are inversely related to bacterial load: a higher number of staphylococcus aureus in the sample requires fewer cycles to reach the threshold level and therefore has a low Ct value (curve on the left of the graph), while a lower number of staphylococcus aureus in the sample requires more cycles to reach the threshold level and therefore has a higher Ct value (curve on the right of the graph). (see example 1.)

Figure 2 is a histogram showing Ct values obtained from patients at phase II clinical trial screening. A large number of patients had low Ct values (high bacterial load) at screening. (see example 2.)

Fig. 3 is a graph showing the Ct value as a function of time with a mechanical ventilator. Ct values have a wide distribution even in the first few days. (see example 2.)

Figure 4 shows the results of 3 different types of culture assays: quantitative, semi-quantitative (culture score, where mild ═ or + +; moderate ═ + +, and severe ═ + + +), and qualitative (presence/absence) measurements of CFU/ml were measured. More than half of the test subjects had high bacterial loads (by at least 10)⁵CFU/ml (in quantitative assays) or by moderate or severe culture score (in semi-quantitative assays). (see example 3.)

Figure 5 provides a graph showing Ct values obtained from samples with mild, moderate, or severe scores in semi-quantitative measurements. (see example 3.)

FIG. 6 provides a graph showing the consistency and inconsistency between PCR and culture assays for Staphylococcus aureus. Of the 209 samples tested positive in the PCR assay, 162 tested positive in the culture assay as well (77.5% identity), while 47 of them tested negative in the culture assay (22.5% non-identity). All inconsistencies were caused by samples that tested negative in the culture assay and positive in the PCR assay. Thus, PCR assays are more sensitive than culture assays. (see example 3.)

Figure 7 provides a graph showing that antibiotic use negatively affected the culture results. The percentage of patients taking antibiotics was significantly higher in the group of patients who were positive for staphylococcus aureus by the PCR test but negative for staphylococcus aureus by the culture test, compared to the group of patients who were positive for staphylococcus aureus by both the PCR and the culture test. (see example 3.)

Figure 8 provides a graph showing that historical antibiotic use had no effect on the culture results. (see example 3.)

FIG. 9 provides a graph showing the distribution of PCR Ct values for culture positive and culture negative samples. 85% of the culture negative samples have a Ct value higher than 29 and 80% of the culture positive samples have a Ct value lower than 29. Thus, a Ct value of 29 effectively distinguishes culture positive samples from culture negative samples. (see example 4.)

FIG. 10 provides a graph showing the relationship between Ct values measured in a phase 2 clinical trial and Staphylococcus aureus concentration in the sample (colony Forming units (CFU)/ml). There was a statistically significant negative correlation between Ct values and CFU/ml counts. Most samples with Ct values greater than 29 have a Ct value less than or equal to 10³Staphylococcus aureus load of CFU/ml. Most samples with Ct values less than 29 have Ct values greater than 10³Staphylococcus aureus load of CFU/ml. The Ct value of 29 corresponds to about 3.2log10 CFU/ml (about 1600-1700CFU/ml) and effectively distinguishes low from high. (see example 4.)

FIG. 11 provides a graph showing the relationship between Ct values in tracheal (left panel) and bronchial (right panel) samples and the concentration of Staphylococcus aureus in the samples (colony Forming units (CFU)/ml). These results demonstrate that Ct values in both tracheal and bronchial samples are inversely related to staphylococcus aureus load. (see example 4.)

FIG. 12 provides a graph showing the relationship between Ct values and the concentration of Staphylococcus aureus (colony Forming units (CFU)/ml) in samples with (left panel) and without (right panel) non-staphylococcal growth. These results show that the Ct values are inversely related to the staphylococcus aureus load, whether non-staphylococcus is present or absent in the sample, demonstrating that the PCR test is specific for staphylococcus aureus. (see example 4.)

Detailed Description

The present disclosure relates to methods of preventing staphylococcus aureus infections in patients with low levels of staphylococcus aureus colonization, and methods of identifying patients colonized with staphylococcus aureus and who will benefit from alpha toxin antibodies.

I. Definition of

The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Unless otherwise indicated herein or clearly contradicted by context, the use of the term "at least one" followed by a list of one or more items (e.g., "at least one of a and B") should be understood to mean one item selected from the listed items (a or B) or any combination of two or more of the listed items (a and B).

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

As used herein, the term "alpha toxin" or "AT" refers to bacterial alpha toxin polypeptides, including but not limited to native alpha toxin polypeptides and isoforms of alpha toxin polypeptides. "alpha toxin" encompasses full-length, unprocessed alpha toxin polypeptides as well as forms of alpha toxin polypeptides produced by intracellular processing. The term "s.aureus alpha toxin" as used herein refers to a polypeptide comprising the amino acid sequence of adsdiniktgtdigsnttvtdgdldlvtikedkumkengmhkckvmkvckvfysfiddhnknkllvrtktgtiagqyrvyyseegankglwpsafkqllqlnednnevassignynvingldngldnplaynsylkeymstvmglvstrytfgfnfgfnglvpldvfggnvkgvqldvkdlktkkvckvkvkvglkvvmjnmvnnmvgnfgldcpdrcpvmgnqgnvqgnglcnvkgvkgvglnkvkgvjnflnkvjdvjdssglssglssglvjdvjditssglvqglvjdvqkgatvqkdvqldvqldvjdtdldvjdtsqldvjdtsqldvjdtsekatgsemstkentn (SEQ ID NO: 12).

The Staphylococcus aureus alpha toxin H35L mutant has the sequence adsdiniktgtdsnttvktdgdlvtikedgkinkmlklkkvfkvffysfiddnkhnkllvirtkvintgiagqyrvyseeganksglawpsafkqlqlqlnpdnevaqyipdyngnssidtkentgngfntgngvtglgtvkkkatgkvkiggligvsigvqvqqqqpdftlpkwkvwkvvmgnvmjnvnnnmvgngdngqpgrdndspwnpvgnqgnqlffrglcktrnkvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvklvjdkqklvjdngtsvjdkwktkwhtkwldkwldkwldkwssgungsemtn (SEQ ID: 13).

By "alpha toxin polynucleotide", "alpha toxin nucleotide" or "alpha toxin nucleic acid" is meant a polynucleotide that encodes an alpha toxin.

The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds a target (e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination of the foregoing) through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecule, so long as the antibody exhibits the desired biological activity. The antibody may be any one of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), are referred to as α, δ, ε, γ, and μ, respectively, based on the properties of their heavy chain constant domains. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. The antibodies may be naked or conjugated to other molecules such as toxins, radioisotopes, and the like.

As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of a B cell and that binds to the same epitope. In contrast, the term "polyclonal antibody" refers to a population of antibodies produced by different B cells and binding to different epitopes of the same antigen.

The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of an intact antibody that binds an antigen. An antigen-binding fragment can contain an antigenic determining region (e.g., a Complementarity Determining Region (CDR)) of an intact antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab ', F (ab') 2, and Fv fragments, linear antibodies, and single chain antibodies. Antigen-binding fragments of antibodies can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be produced artificially.

Intact antibodies typically consist of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains an N-terminal Variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains an N-terminal Variable (VL) region and a C-terminal Constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of the antibody. The VH and VL regions have the same general structure, with each region comprising four framework regions whose sequences are relatively conserved. As used herein, the term "framework region" refers to relatively conserved amino acid sequences within the variable regions, which are located between hypervariable regions or Complementarity Determining Regions (CDRs). There are four framework regions in each variable domain, these are designated FR1, FR2, FR3, and FR 4. The framework regions form the beta sheet of the structural framework that provides the variable regions (see, e.g., c.a. janeway et al (editors), Immunobiology [ Immunobiology ], 5 th edition, Garland Publishing [ gayland press ], New York, NY [ New York city, New York ] (2001)). The three CDRs (designated CDR1, CDR2, and CDR3) form the "hypervariable region" of the antibody, which is responsible for antigen binding.

The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

The term "Kabat numbering" and similar terms are art-recognized and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain aspects, the CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA and Wu TT (1971) Ann NY Acad Sci [ New York academy of sciences ] 190: 382 Across 391 and Kabat EA et al, (1991) Sequences of proteins of immunological Interest [ protein Sequences of immunological Interest ], fifth edition, U.S. department of Health and Human Services [ U.S. department of Health and public Services ], NIH publication No. 91-3242). Using the kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (optionally one or two additional amino acids following position 35 (referred to as 35A and 35B in the kabat numbering scheme)) position (CDR1), amino acid positions 50 to 65(CDR2), and amino acid positions 95 to 102(CDR 3). Using the kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34(CDR1), amino acid positions 50 to 56(CDR2), and amino acid positions 89 to 97(CDR 3). In a particular embodiment, the CDRs of the antibodies described herein have been determined according to the kabat numbering scheme.

In contrast, Georgia (Chothia) refers to the position of the structural loops (Chothia and Lesk, J.mol.biol. [ J.M.biol. ] 196: 901-917 (1987)). The ends of the Gerocia CDR-H1 loops when numbered using the kabat numbering convention vary between H32 and H34 depending on the length of the loop (since the kabat numbering scheme places the insertions at H35A and H35B; the loop end point at 32 if neither 35A nor 35B is present; the loop end point at 33 if only 35A is present; the loop end point at 34 if both 35A and 35B are present). The AbM hypervariable regions represent a compromise between the kabat CDRs and the georgia structural loops and are used by the AbM antibody modeling software of the Oxford molecule (Oxford Molecular).

As used herein, the terms "constant region" or "constant domain" are interchangeable and have their ordinary meaning in the art. The constant region is a portion of an antibody, e.g., the carboxy-terminal portion of a light and/or heavy chain, that is not directly involved in binding of the antibody to an antigen, but may exhibit various effector functions, such as interaction with an Fc receptor. The constant regions of immunoglobulin molecules typically have more conserved amino acid sequences relative to immunoglobulin variable domains.

As used herein, the term "heavy chain" when used in reference to an antibody, the amino acid sequence based on the constant domain can refer to any of a variety of types, e.g.α, δ, ε, γ and μ, which respectively generate IgA, IgD, IgE, IgG and IgM classes of antibodies, comprise subclasses of IgG, e.g. IgG₁、IgG₂、IgG₃And IgG₄. Heavy chain amino acid sequences are well known in the art. In a specific embodiment, the heavy chain is a human heavy chain.

As used herein, the term "light chain" when used in reference to an antibody, the amino acid sequence based on the constant domain can refer to any of the different types, e.g., κ or λ. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

"chimeric" antibody refers to an antibody or fragment thereof comprising both human and non-human regions. A "humanized" antibody is an antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as a rodent (e.g., a mouse or a rat). A humanized antibody may comprise one, two or three CDRs obtained or derived from a non-human antibody. Fully human antibodies do not contain any amino acid residues obtained or derived from non-human animals. It is understood that fully human and humanized antibodies are at lower risk of inducing an immune response in humans than mouse or chimeric antibodies (see, e.g., Harding et al, mAbs [ monoclonal antibodies ], 2 (3): 256-26 (2010)).

As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope), or an epitope can be, for example, derived together from two or more non-contiguous regions of one or more polypeptides (conformational, non-linear, non-contiguous, or non-contiguous epitopes). In certain embodiments, the epitope to which the antibody or antigen-binding fragment thereof binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange-coupled mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Gieger et al, (1994) Acta Crystallogr D Biol Crystallogr [ Proc. Natl. Acad. D. Natl. crystallography: Biocrystallography ]50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem [ European J. Biochem ] 189: 1-23; Chayen NE (1997) Structure [ 5: 1269-1274; McPherson A (1976) J Biol Chem [ J. Biochem ] 251: 6300-6303). Antibody/antigen binding fragment thereof: the antigen crystals can be studied using well known X-ray diffraction techniques and can be refined using computer software, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol [ methods of enzymology ] (1985) volumes 114 and 115, ed Wyckoff HW et al; U.S.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr [ Crystal proceedings D: Biocrystallography ]49(Pt 1: 37-60; Bricogne G (1997) Meth Enzymol [ methods of enzymology ] A: 361; 423, edited Carter CW; Rovers P et al, (2000) Acta D Crystaygogron D [ crystallography ] D: crystal: 13256 (Pt 3: 1316). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques (including alanine scanning mutagenesis techniques), see, e.g., Champe M et al, (1995) J Biol Chem [ journal of biochemistry ] 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science [ Science ] 244: 1081-1085.

An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that binds to the same amino acid residues as the reference antibody. The ability of an antibody to bind the same epitope as a reference antibody can be determined by hydrogen/deuterium exchange assay (see, Coales et al, Rapid Commun. Mass Spectrum 2009; 23: 639-647) or x-ray crystallography.

As used herein, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" are similar terms in the context of an antibody or antigen-binding fragment thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain and that binding requires some complementarity between the antigen-binding domain and the epitope. Thus, for example, an antibody that "specifically binds" to a first s.aureus leukotoxin can also bind to other s.aureus leukotoxins, but binds to an unrelated, non-leukotoxin protein to less than about 10% of the extent of the antibody's binding to the first s.aureus leukotoxin (as measured, for example, by Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), BiaCore, or octant binding assay).

An antibody is considered to "competitively inhibit" the binding of a reference antibody to a given epitope or an overlapping epitope if it preferentially binds to that epitope to the extent that it blocks the binding of the reference antibody to that epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody can be considered to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

The term "nucleic acid sequence" is intended to encompass a polymer (i.e., a polynucleotide) of DNA or RNA that may be single-stranded or double-stranded and that may contain non-natural or altered nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to a polymeric form of nucleotides of any length, either Ribonucleotides (RNA) or Deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and thus include double-and single-stranded DNA, as well as double-and single-stranded RNA. These terms include as equivalents RNA or DNA analogs made from nucleotide analogs and modified polynucleotides (such as, but not limited to, methylated polynucleotides and/or capped polynucleotides). Nucleic acids are typically linked via phosphate linkages to form nucleic acid sequences or polynucleotides, although many other linkages (e.g., phosphorothioate, boranophosphate, etc.) are known in the art.

As used herein, "transfection", "transformation" or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J (ed.), Methods in Molecular Biology, vol 7, Gene Transfer and Expression Protocols, Humana Press [ lima Press ] (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment (Johnston, Nature [ Nature ], 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al, mol. cell Biol. [ molecular cell biology ], 7: 2031-. After the infectious particles are grown in suitable packaging cells, many of which are commercially available, the phage or viral vector can be introduced into the host cell.

As used herein, the term "treating" or the like refers to a measure (e.g., administering to a subject an antibody or antigen-binding fragment thereof provided herein) that cures, slows, alleviates, and/or halts the progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder. By "therapeutically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result (e.g., treatment of a staphylococcus aureus infection).

A Prophylactic measure refers to a measure that prevents and/or slows the development of a targeted pathological condition or disorder (e.g., administration of an antibody or antigen-binding fragment thereof provided herein to a subject). Thus, those in need of prophylactic measures include those patients having a predisposition to develop the disorder and those patients for whom the disorder is to be prevented. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of S.aureus infection or onset of disease).

A subject colonized with Staphylococcus aureus is one in which the Staphylococcus aureus is present in or on the body. Colonization may be determined, for example, by detecting staphylococcus aureus in a sample obtained from the subject. Staphylococcus aureus can be detected, for example, by culture or by Polymerase Chain Reaction (PCR). Infection caused by or accompanied by the presence of staphylococcus aureus in or on a subject's body shows radiological and/or clinical signs of bacteria. Staphylococcus aureus infections may occur, for example, as skin or soft tissue infections (SSTIs) or bacteremia. Staphylococcus aureus bacteria can spread through the bloodstream and infect sites in the body, resulting in pneumonia, ICU pneumonia, bone or joint infections, instrument infections, wound infections, surgical site infections, or osteomyelitis. Radiological signs include, for example, X-rays showing infiltration. Clinical signs include, for example, abnormal body temperature, abnormal white blood cell counts, coughing, purulent sputum, bronchial respiratory sounds, dyspnea, shortness of breath (respiratory rate >30 breaths/minute), and/or hypoxemia.

As used herein, the term "administering (administer, administering, administeration)" or the like refers to a method useful to enable delivery (e.g., intravenous administration) of a drug (e.g., a combination of anti-staphylococcus aureus antibodies or antigen-binding fragments thereof) to a desired biological site of action. Administration techniques that may be used with The agents and methods described herein may be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current versions, Pergamon; and Remington's, Pharmaceutical Sciences [ Remington Pharmaceutical science ], current edition, Mack Publishing co, Easton, Pa [ Easton macpublishing company, Pa ].

Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) or sequential administration in any order.

The term "or" as used herein is to be understood as being inclusive unless explicitly stated or otherwise apparent from the context. The term "and/or" as used herein in phrases such as "a and/or B" is intended to include "a and B", "a or B", "a" and "B". Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

Anti-staphylococcus aureus alpha toxin antibodies

As provided herein, antibodies and antigen-binding fragments thereof (e.g., monoclonal antibodies and fragments) that bind to staphylococcus aureus alpha toxin can be used to avoid staphylococcus aureus infections in subjects colonized with low levels of staphylococcus aureus.

Alpha Toxin (AT) is a key virulence factor in several S.aureus diseases including pneumonia, Skin and Soft Tissue Infections (SSTI) and bacteremia (Bubeck Wardenburg, J. and O.Schneeland, J.Exp.Med. [ J.EXPERIMEDIENT MEDICAL ], 205: 287-294 (2008); Inoshima et al, J.invest.Dermatol. [ J.Dermatology ], 132: 1513-1516 (2012); and Foletti et al, supra). Immunity with anti-AT monoclonal antibodies reduced disease severity in models of pneumonia and skin necrosis (Hua et al, Antimicrob. Agents Chemothers [ antimicrobial agents and chemotherapy ], 58: 1108-. AT promotes multiple aspects of Staphylococcus aureus pathogenesis during bacteremia and sepsis, including stimulation of the high inflammatory response characteristic of sepsis, and activation of ADAM 10-mediated cleavage of endothelial tight junctions, leading to loss of vascular integrity (Powers et al, J Infect. Dis. [ J.Infect., 206: 352-. AT has also been shown to target platelets, which prevent repair of damaged endothelial barriers and promote organ dysfunction through platelet-neutrophil aggregate formation (Powers et al, CellHost Microbe [ cell host and microorganism ], 17: 775-. Alpha toxin structures and functions are described in detail, for example, in Bhakdi, s, and j.tranum-Jensen, microbiol.mol.biol.rev. [ review in microbiology and molecular biology ], 55 (4): 733 (1991).

Monoclonal and polyclonal antibodies that bind AT are known in the art (see, e.g., Hua et al, antibodies. agents chemither. [ antimicrobial agents and chemotherapy ], 58 (2): 1108-. Exemplary antibodies that bind to AT are disclosed in, for example, WO 2012/109285 and WO 2014/074540 (both of which are incorporated herein by reference in their entirety).

In one example, an antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) that specifically binds to staphylococcus aureus Alpha Toxin (AT) comprises, consists essentially of, or consists of: (i) a heavy chain polypeptide; and (ii) a light chain polypeptide comprising SEQ ID NO: 1, the CDR1 amino acid sequence of SEQ ID NO: 2, and the CDR2 amino acid sequence of SEQ ID NO: 3, and a light chain polypeptide comprising the amino acid sequence of CDR3 of SEQ ID NO: 4, the CDR1 amino acid sequence of SEQ ID NO: 5, and the CDR2 amino acid sequence of SEQ ID NO: 6 CDR3 amino acid sequence. In another example, the heavy chain polypeptide of an antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) that specifically binds to staphylococcus aureus AT comprises the amino acid sequence of SEQ ID NO: 7, consisting essentially of, or consisting of the amino acid sequence of the variable region of seq id no. In another example, the light chain polypeptide of an antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) that specifically binds to staphylococcus aureus AT comprises the amino acid sequence of SEQ ID NO: 8, consisting essentially of, or consisting of the amino acid sequence of a variable region of seq id no. In another example, an antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) that specifically binds to staphylococcus aureus AT comprises, consists essentially of, or consists of: a variable heavy and light chain variable region comprising SEQ ID NO: 7, the light chain variable region comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 8, consisting essentially of, or consisting of. In another example, an antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) that specifically binds to staphylococcus aureus AT comprises, consists essentially of, or consists of: a heavy and/or light chain variable region, the heavy chain comprising SEQ ID NO: 9, the light chain variable region comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 10, consisting essentially of, or consisting of.

The sequences of exemplary anti-AT antibodies are provided below. Additional anti-AT antibodies are provided, for example, in U.S. patent No. 9,527,905, which is incorporated herein by reference in its entirety. In certain examples, an antibody or antigen-binding fragment thereof described herein binds to AT and comprises six CDRs of an antibody listed in two tables below (i.e., the three VH CDRs of an antibody listed in the first table and the three VL CDRs of the same antibody listed in the second table).

anti-AT antibody MEDI4893 (also known as sutoxizumab) is a human monoclonal antibody with an extended half-life that binds AT with high affinity and effectively blocks the formation of AT pores in the target cell membrane. Half-life extension is achieved by introducing three amino acid substitutions (M252Y/S254T/T256E; referred to as YTE) in the Fc domain to increase binding to the neonatal Fc receptor (FcRn) and thus increase serum half-life. Because YTE mutations reduce antibody half-life and exposure in mice, the same version of MEDI4893 (except YTE modifications) designated MEDI4893 or "LC 10" has been used to demonstrate efficacy in preclinical models, as previously described in international patent application publications WO 2012/109285 and WO 2014/074540 (both incorporated herein by reference in their entirety). MEDI4893 (or sutosuzumab) contains a peptide having the sequence of SEQ ID NO: 9 and a light chain having the amino acid sequence set forth in SEQ ID NO: 10, or a light chain of the amino acid sequence set forth in seq id No. 10. The same CDR, VH and VL sequences for MEDI4893 (sutosuzumab) and MEDI4893 are provided in the table below.

VH CDR amino acid sequence

VL CDR amino acid sequences

In certain examples, an antibody or antigen-binding fragment thereof described herein binds to AT and comprises a VH of an antibody listed in the table below, e.g., in combination with a VL.

Variable heavy chain (VH) amino acid sequence

In certain examples, an antibody or antigen-binding fragment thereof described herein binds to AT and comprises the VL of an antibody listed in the table below, e.g., in combination with a VH (optionally of the same antibody listed in the table above).

Variable light chain (VL) amino acid sequences

In certain examples, an antibody or antigen-binding fragment thereof described herein binds to AT and comprises the heavy chain of an antibody listed in the table below, e.g., in combination with the light chain.

Full length heavy chain amino acid sequence

In certain examples, an antibody or antigen-binding fragment thereof described herein binds to AT and comprises a light chain of an antibody listed in the tables below, e.g., in combination with a heavy chain (optionally of the same antibody listed in the tables above).

Full length light chain amino acid sequence

In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Johnasia numbering scheme, which refers to the location of the immunoglobulin structural loops (see, e.g., Chothia C and Lesk AM, (1987), J Mol Biol 196: 901. 917; Al-Lazikani B et Al, (1997) J Mol Biol 273: 927. 948; Chothia C et Al, (1992) J Mol Biol 227: 799. 817; Tramotano A et Al, (1990) J Mol Biol 215(1) 175-82; and U.S. Pat. No. 7,709,226). Typically, when using the kabat numbering convention, the georgia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the georgia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the georgia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the georgia CDR-L1 loop is present at light chain amino acids 24 to 34, the georgia CDR-L2 loop is present at light chain amino acids 50 to 56, and the georgia CDR-L3 loop is present at light chain amino acids 89 to 97. The ends of the Gerocia CDR-H1 loops when numbered using the kabat numbering convention vary between H32 and H34 depending on the length of the loop (since the kabat numbering scheme places the insertions at H35A and H35B; the loop end point at 32 if neither 35A nor 35B is present; the loop end point at 33 if only 35A is present; the loop end point at 34 if both 35A and 35B are present).

In certain aspects, provided herein are combinations of antibodies and antigen-binding fragments thereof comprising the geodesia VH and VL CDRs of the MEDI4893 antibody. In certain embodiments, the antibody or antigen-binding fragment thereof comprises one or more CDRs wherein the geodesia and kabat CDRs have the same amino acid sequence. In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof comprising a combination of kabat CDRs and georgia CDRs.

In certain aspects, The immune response may be determined according to, for example, Lefranc M-P, (1999) The Immunologist [ Immunologist ] 7: 132-136 and Lefranc M-P et al, (1999) Nucleic Acids Res [ Nucleic Acids research ] 27: 209-212 to determine the CDRs of the antibody or antigen-binding fragment thereof. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97. In particular embodiments, provided herein are combinations of antibodies and antigen-binding fragments thereof comprising the IMGT VH and VL CDRs of MEDI4893, e.g., as described in Lefranc M-P (1999) supra and Lefranc M-P et al (1999) supra.

In certain aspects, the expression may be determined according to MacCallum RM et al, (1996) J Mol Biol [ journal of molecular biology ] 262: 732-745 determining the CDRs of the antibody or antigen-binding fragment thereof. See also, for example, Martin A. "Protein Sequence and Structure analysis of Antibody Variable Domains [ Protein Sequence and Structure analysis of Antibody Variable Domains ]," in Antibody engineering [ Antibody engineering ], Kontermann and Dubel editions, Chapter 31, p. 422-439, Springer-Verlag, Berlin Springs [ Berlin Springs ] (2001). In particular embodiments, provided herein are combinations of antibodies or antigen-binding fragments thereof comprising the VH and VL CDRs of the MEDI4893 antibody determined by the method of MacCallum RM et al.

In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers to the hypervariable regions of AbM that represent a compromise between the kabat CDRs and the structural loops of the johnsonia, and used by the AbM antibody modeling software of the Oxford molecule (Oxford Molecular Group, Inc.). In particular embodiments, provided herein are combinations of antibodies or antigen-binding fragments thereof comprising the VH and VL CDRs of MEDI4893 antibodies as determined by the AbM numbering scheme.

In another aspect, an antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody or fragment) described herein can comprise a constant region (Fc) of any suitable class (e.g., IgG, IgA, IgD, IgM, and IgE) that has been modified in order to improve the half-life of the antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment). For example, an antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody or fragment) described herein can comprise an Fc that comprises a mutation that results in an increased half-life relative to the same antibody without the mutation.

Fc region engineering is widely used in the art to extend the half-life of therapeutic antibodies and to avoid degradation in vivo. In some embodiments, the Fc region of an IgG antibody or antigen binding fragment can be modified to increase the affinity of the IgG molecule for neonatal Fc receptor (FcRn), thereby mediating IgG catabolism and avoiding IgG molecule degradation. Suitable amino acid substitutions or modifications of the Fc region are known in the art and include, for example, the ternary substitutions M252Y/S254T/T256E (referred to as "YTE") (see, e.g., U.S. Pat. No. 7,658,921; U.S. patent application publication 2014/0302058; and Yu et al, antimicrob. agents chemicotherapy, [ antimicrobial agents and chemotherapy ], 61 (1): e01020-16 (2017)). In certain aspects, an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that binds to staphylococcus aureus AT comprises an Fc region comprising YTE mutations.

The antibodies or antigen-binding fragments (e.g., monoclonal antibodies or fragments) described herein can be or can be obtained from human, humanized, non-human, or chimeric antibodies. In one aspect, an antibody or antigen-binding fragment thereof described herein is a fully human antibody.

Human, non-human, chimeric, or humanized antibodies can be obtained by any means, including via in vitro sources (e.g., hybridomas or cell lines that recombinantly produce antibodies) and in vivo sources (e.g., rodents, human tonsils). Methods for producing antibodies are known in the art and described, for example, inAnd Milstein, eur.j.immunol.[ European journal of immunology],5: 511-519 (1976); harlow and Lane (editors), Antibodies: the antigen Manual [ antibody: laboratory manual]CSH Press](1988) (ii) a And Janeway et al (ed.), Immunobiology [ Immunobiology]5 th edition, Garland Publishing, Garland Press]New York, n.y. [ New York city, New York, n.y. ]](2001) In (1). In certain embodiments, transgenic animals (e.g., mice) can be used to produce human or chimeric antibodies in which one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, Medarex HUMAB-MOUSE^TMKylin group (Kirin) TC MOUSE^TMKyowa Kirin KM-MOUSE^TM(see, e.g., Lonberg, nat. Biotechnol. [ Nature Biotechnology ]],23(9): 1117-25(2005), and Lonberg, handbb.Handb.exp.Pharmacol. [ handbook of Experimental pharmacology],181: 69-97(2008)). Humanized Antibodies can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to clinical [ Therapeutic Monoclonal Antibodies: From laboratory to clinical)]，John Wiley&Sons, Inc. [ john william son company]Hoboken, N.J. [ Hobock, N.J. ]](2009) Including, for example, grafting of non-human CDRs onto human antibody scaffolds (see, e.g., Kashmiri et al, Methods [ Methods ]],36(1): 25-34 (2005); and Hou et al, J.biochem. [ J.Biochem],144(1): 115-120(2008)). In one embodiment, humanized antibodies can be generated using the methods described in, for example, U.S. patent application publication 2011/0287485 a 1.

Nucleic acids, vectors and host cells

Also provided herein are one or more isolated nucleic acid sequences encoding an antibody or antigen-binding fragment thereof that binds to AT (optionally wherein the antibody or antigen-binding fragment is a monoclonal antibody or fragment).

The disclosure further provides one or more vectors comprising one or more nucleic acid sequences encoding an antibody or antigen-binding fragment thereof that binds to AT (optionally wherein the antibody or antigen-binding fragment is a monoclonal antibody or fragment). The vector may be, for example, a plasmid, episome, cosmid, viral vector (e.g., retroviral vector or adenoviral vector), or phage.

One or more vectors comprising one or more nucleic acids encoding an antibody or antigen-binding fragment thereof that binds to AT (optionally wherein the antibody or antigen-binding fragment is a monoclonal antibody or fragment) can be introduced into a host cell, including any suitable prokaryotic or eukaryotic cell, capable of expressing the polypeptide encoded thereby. Accordingly, the present disclosure provides an isolated cell comprising a vector. Host cells that may be used include those that are: these host cells can be grown easily and reliably, have a reasonably fast growth rate, have well characterized expression systems, and can be transformed or transfected easily and efficiently.

The nucleic acid sequence encoding an amino acid in any of the antibodies or antigen-binding fragments (optionally monoclonal antibodies or fragments) described herein can be introduced into a cell by transfection, transformation, or transduction.

Pharmaceutical compositions and methods of administering anti-AT antibodies

The present disclosure provides a composition comprising an effective amount of any of the anti-AT antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier. The amount is effective to reduce the risk of infection in a subject colonized with staphylococcus aureus.

In another aspect, the composition can comprise a nucleic acid sequence encoding an AT-binding antibody or antigen-binding fragment. The nucleic acid sequence may be present in a vector or a combination of vectors.

In one aspect, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition comprising a carrier (such as a pharmaceutically acceptable (e.g., physiologically acceptable) carrier) and one or more AT-binding antibody or antigen-binding fragment nucleic acid sequences, or one or more vectors.

Any suitable carrier may be used within the context of the present disclosure, and such carriers are well known in the art. The choice of carrier will be determined in part by the particular site at which the composition can be administered and the particular method used to administer the composition. The composition optionally may be sterile. The compositions may be frozen or lyophilized for storage and reconstitution in a suitable sterile vehicle prior to use. The compositions may be produced according to conventional techniques described, for example, in Remington: the Science and Practice of Pharmacy [ Remington: pharmaceutical science and practice ], 21 st edition, Lippincott Williams & Wilkins [ Ri Ke Te Williams Wilkins publishing Co ], Philadelphia, PA [ Philadelphia, Pa. (2001).

The composition desirably comprises the AT-binding antibody or antigen-binding fragment in an amount effective to reduce the risk of a staphylococcus aureus infection in a patient colonized with staphylococcus aureus. To this end, the disclosed methods comprise administering a therapeutically effective amount, or a prophylactically effective amount, of an AT-binding antibody or antigen-binding fragment thereof, or a composition of the foregoing antibodies or fragments (including monoclonal antibodies or fragments).

For repeated administrations over several days or longer, depending on the condition, the treatment can be repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the present disclosure. The desired dose may be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

An effective amount of an anti-AT antibody or antigen-binding fragment thereof can be administered to a subject (e.g., a human) using standard administration techniques, including intravenous, intraperitoneal, subcutaneous, and intramuscular routes of administration. The anti-AT antibody or antigen-binding fragment thereof may be suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous and intraperitoneal administration. In some embodiments, the anti-AT antibody or antigen-binding fragment thereof is administered to the subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery.

The AT-binding antibody or antigen-binding fragment or composition comprising the same may be administered alone or in combination with other drugs conventionally used to treat staphylococcus aureus infections (e.g., as adjuvants). Compositions comprising AT-binding antibodies or antigen-binding fragments can be used in combination with, for example, one or more antibiotics, such as penicillinase-resistant beta-lactam antibiotics (e.g., oxacillin (oxacillin) or flucloxacillin (flucloxacillin))). Gentamicin (Gentamicin) can be used to treat severe infections such as endocarditis. However, today most staphylococcus aureus strains are penicillin-resistant and two of 100 people carry methicillin-resistant staphylococcus aureus strains (MRSA). MRSA infections are typically treated with vancomycin (vancomycin), and minor skin infections may be treated with triple antibiotic ointments.

Compositions comprising AT-binding antibodies or antigen-binding fragments can be used in combination with, for example, one or more anti-staphylococcus aureus antibodies.

Methods of identifying patients who would benefit from anti-AT antibodies

As demonstrated herein, administration of an anti-AT antibody or antigen-binding fragment thereof to a subject colonized with low levels of staphylococcus aureus (e.g., levels of staphylococcus aureus that do not exceed a threshold level of staphylococcus aureus) can reduce the incidence of infection in the subject that accompanies the presence of staphylococcus aureus.

The amount of staphylococcus aureus in a subject can be determined based on the amount of staphylococcus aureus in a sample obtained from the subject. The sample may be, for example, a skin or soft tissue sample. The sample may be obtained, for example, from the lower respiratory tract of a subject. The sample may be, for example, an intratracheal aspirate, a tracheal sample, or a bronchial sample.

In certain aspects, an anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a concentration of Staphylococcus aureus that does not exceed a certain threshold estimated to be 3.2log10 CFU/ml (about 1600-1700 CFU/ml).

The amount of staphylococcus aureus in a sample obtained from a subject can be quantified using Polymerase Chain Reaction (PCR). For example, the amount of staphylococcus aureus in a sample obtained from a subject can be an amount that can be quantified based on the number of PCR cycles (referred to herein as a "cycle threshold" or "Ct value") required to reach a threshold signal. A high Ct value (i.e., a large number of PCR cycles required to reach the threshold signal) indicates a low level of staphylococcus aureus, while a low Ct value (i.e., a small number of PCR cycles required to reach the threshold signal) indicates a high level of staphylococcus aureus.

PCR is a particularly advantageous method for quantifying the amount of Staphylococcus aureus in a sample because it can be performed quickly and consistently. For example, PCR can detect the amount of Staphylococcus aureus in a sample in 2 hours or less. Although laboratories vary greatly in the way bacteria are cultured, they will use the same type of instrument for PCR testing, so that the PCR output is consistent from laboratory to laboratory.

PCR can be used to determine the amount of staphylococcus aureus in a sample based on amplification of a single staphylococcus aureus gene or a combination of staphylococcus aureus genes. For example, PCR can be used to detect the Staphylococcus aureus protein A gene. Detection of Staphylococcus aureus protein A all Staphylococcus aureus were detected regardless of their susceptibility to methicillin. However, PCR can also be used to determine whether staphylococcus aureus is antibiotic resistant, e.g., methicillin resistant. For example, PCR can be used to detect the presence of methicillin resistance determinants (mecA) and staphylococcal chromosomal cassettes (SCCmec). Detection of mecA and SCCmec indicates that Staphylococcus aureus is methicillin resistant.

In certain aspects, the anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a maximum level of staphylococcus aureus associated with a PCR Ct value. In certain aspects, an anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a PCR Ct value of about 29. In certain aspects, an anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of staphylococcus aureus that does not exceed a level of staphylococcus aureus associated with a PCR Ct value between 29 and 36.

In certain aspects, the anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of staphylococcus aureus that is AT least the lowest level of staphylococcus aureus associated with a PCR Ct value. In certain aspects, an anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of staphylococcus aureus associated with a PCR Ct value of about 3 to about 29. In certain aspects, an anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of staphylococcus aureus associated with a PCR Ct value of 3 to 29.

The PCR Ct value of 29 is estimated to correspond to a concentration of staphylococcus aureus of about 1600 to 1700 Colony Forming Units (CFU)/ml.

The methods provided herein for identifying a subject who would benefit from receiving an anti-AT antibody or antigen-binding fragment thereof may be particularly useful for mechanically ventilated subjects. Thus, the subject may be a mechanically ventilated subject. Although the overall incidence is relatively low (about 1% -2%), the incidence of staphylococcus aureus pneumonia is much higher (above 20%) in patients with staphylococcus aureus colonization. In mechanically ventilated subjects, staphylococcus aureus pneumonia is an early event that typically occurs within the first week after the onset of ventilation, and thus early administration of anti-AT antibodies or antigen-binding fragments thereof may be critical to avoid pneumonia. PCR-based techniques for assessing the level of staphylococcus aureus in a patient can be completed in about 2 hours, while culture techniques are much slower (and also lack the precise quantitative nature of PCR-based techniques).

PCR-based techniques are also more advantageous than culture-based techniques for detecting colonization of staphylococcus aureus, as PCR-based techniques can be more sensitive in detecting the presence of staphylococcus aureus, particularly in subjects taking antibiotics. Thus, the subject may be taking an antibiotic. In addition, in certain aspects of the methods provided herein, PCR analysis of a sample obtained from a subject indicates that the subject would benefit from receiving an anti-AT antibody or antigen-binding fragment thereof, while the sample does not contain bacteria that would grow in a culture assay for determining the presence of staphylococcus aureus.

The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

Example 1

Staphylococcus aureus (s.aureus) pneumonia is an early event in mechanically ventilated patients that typically occurs within the first week after ventilation. Thus, rapid identification of patients at risk of developing such infections may be life-saving. Although the overall incidence is relatively low (about 1% -2%), it is much higher (over 20%) in patients colonized with staphylococcus aureus.

In a phase 2 clinical trial of mechanically ventilated patients in Intensive Care Units (ICU), analytical rapid Polymerase Chain Reaction (PCR) was performed as an assay for identifying at-risk patients with colonisation of staphylococcus aureus in their Lower Respiratory Tract (LRT). According to the product agreement useMRSA/SA SSTI assay (Cepheid, Sunnyvale, Calif.) for PCR. The assay is a rapid, automated DNA test for the simultaneous detection of methicillin-resistant staphylococcus aureus (MRSA) and Staphylococcus Aureus (SA) directly from skin and soft tissue samples. Samples were collected on double swabs and placed in tubes containing the elution reagent. After a short vortex, the eluted material and the two single-use reagents provided with the assay (reagent 1 and reagent 2) were transferred to different, uniquely labeled chambers of a disposable fluidic cartridge (Xpert MRSA/SA cartridge). Placing the box inDX system instrumentIn the platform, the platform does not interfere with real-time multiplex PCR in order to detect DNA. In this platform, additional sample preparation, amplification and real-time detection are all fully automated and fully integrated.

Primers and probes in the Xpert MRSA/SA assay detect nucleic acid sequences of staphylococcus aureus protein a (spa), MecA-mediated oxacillin-resistance gene (MecA), and staphylococcal cassette chromosome (SCCmec) inserted into attB site of SA chromosome. The test includes sample handling control to control proper handling of the target bacteria and to monitor for the presence of inhibitors in the PCR assay. The Probe Check Control (Probe Check Control) verifies reagent rehydration, PCR tube filling in the cassette, Probe integrity and dye stability.

The assay is depicted in figure 1: an intratracheal aspirate (ETA) sample is obtained from the patient and a swab of the sample is inserted into the elution reagent. The mixture was vortexed and dispensed into a cassette. The cartridge is then inserted into a PCR instrument to initiate the test. The total operating time of the test was less than 2 minutes and the entire test was completed in 75 minutes.

The PCR test yields a cycle threshold (Ct) value that represents the number of PCR cycles required to reach a threshold signal. Ct values are inversely related to bacterial load: higher numbers of staphylococcus aureus in the sample required fewer cycles to reach the threshold level and therefore had a low Ct value, while lower numbers of staphylococcus aureus in the sample required more cycles to reach the threshold level and therefore had a higher Ct value.

Example 2

The rapid PCR test discussed in example 1 was used to detect the presence or absence of staphylococcus aureus and to determine whether staphylococcus aureus is methicillin-resistant staphylococcus aureus (MRSA) by observing three molecular targets. The first target is staphylococcus aureus protein a (spa), which detects all staphylococcus aureus regardless of their methicillin-sensitive status. The second and third targets are the methicillin resistance determinant (mecA) and the staphylococcal chromosomal cassette (SCCmec) for detection of MRSA.

At the time of phase 2 clinical trial screening, 720 patients were subjected to a rapid PCR test. Of these patients 299 (41.5%) were colonised with staphylococcus aureus. This number is higher than the ordinary public staphylococcus aureus colonization rate (25% -30%), but is a reasonable number for mechanically ventilated patients in the ICU. Among 299 patients colonized with staphylococcus aureus, 277 (92.6%) carried methicillin-sensitive staphylococcus aureus (MSSA) and 22 (7.4%) carried MRSA.

FIG. 2 shows Ct values of 295 of 299 patients colonized with Staphylococcus aureus. (4 PCR + patients with a Ct value equal to zero or missing this value are not shown.) the mean Ct value for these patients was 25.7, and a large number of patients had low Ct values (high bacterial load) at the time of screening.

Screening occurred at various times after the start of mechanical ventilation. Therefore, Ct values were compared to the number of mechanical ventilation days. The results shown in fig. 3 demonstrate that Ct values have a broad distribution regardless of the number of mechanical ventilation days. Thus, the number of days of mechanical ventilation cannot be used to predict staphylococcus aureus colonization or Ct values.

Example 3

This example demonstrates that rapid PCR is more sensitive than culture assays in detecting staphylococcus aureus colonization. Culture assays vary significantly between laboratories and typically involve the inoculation or streaking of undiluted or serially diluted samples onto agar plates containing agar. After incubation of the plates for 24-48 hours, the colonies of S.aureus colonies were counted (quantitative culture method) or the number of growth quadrants and growth density of S.aureus were evaluated (semi-quantitative culture method). Qualitative culture does not provide quantification and only assesses the presence or absence of staphylococcus aureus. (see fig. 4.) thus, the status of the culture (positive or negative for s.aureus infection) may be different for different culture methods and, if inaccurate, may produce a negative bias to the treatment regimen.

A random subset of 299 patients (N209) was cultured. Of these 209 patients, only 162 (77.5%) produced positive culture results, while 47 (22.5%) produced negative culture results. Thus, the culture assay will lack 22.5% of subjects colonized with staphylococcus aureus. In other words, there was a 77.5% agreement and 22.5% disagreement between the rapid PCR and culture assays. The reason for all inconsistencies was samples that were positive by the rapid PCR test and negative by the culture test. (see FIG. 6.)

A further review of the results demonstrates that a significantly higher percentage of patients whose samples were positive by the PCR test and negative by the culture test were concurrently using antibiotics than patients whose samples were positive by the PCR and by the culture test. (see FIG. 7.) these data indicate that the use of antibiotics negatively affected the culture results. However, historical antibiotic use had no effect on culture results. (see FIG. 8.)

In addition, the sensitivity of culture assays varies in different laboratories due to differences in inoculation (e.g., sample dilution, inoculum volume, and plate number). The laboratory investigations are summarized in the table below.

Laboratory	Detection limit (CFU/ml)	Laboratory	Detection limit (CFU/ml)
				1	100-200	6	1,000
2	100	7	1,000
				3	1,000	8	1,000-10,000
4	100,000	9	10,000
				5	3.3

In these nine laboratories, the detection limits were 3.3 and 10⁵CFU/ml. Thus, the low culture sensitivity in some laboratories also leads to inconsistencies between PCR and culture assays.

The Ct cut-off was examined as to whether the cultures were classified as mild, moderate or severe. Most cultures classified as severe had PCR Ct values below 29. However, a significant fraction of cultures classified as medium also have PCR Ct values below 29. In fact, some cultures classified as mild were also below the PCR Ct value of 29. Therefore, the evaluation of these cultures was inconsistent. (see FIG. 5.)

The methicillin sensitivity assay was consistent using rapid PCR or culture assays. Of 162 samples tested by PCR and culture, 9 (5.6%) were identified as MRSA and 153 (94.4%) as MSSA, whichever method was used.

These results demonstrate that the culture assay and the PCR assay are equally effective in detecting methicillin sensitivity, but that the PCR assay is more sensitive in detecting colonization of Staphylococcus aureus.

Example 4

Ct values were analyzed in culture positive and culture negative samples. The Ct distributions for the culture positive and culture negative samples are shown in fig. 9, and the results are also summarized in the following table.

	Ct≤29	Ct＞29
			Negative in culture	14.9％	85.1％
Positive in culture	78.4％	21.6％

Based on these results, the Ct value of 29 best distinguished between culture positive and culture negative samples.

Ct values also correlate with staphylococcus aureus concentrations. The results shown in FIG. 10 demonstrate that there is a statistically significant negative linear correlation between Ct values and CFU/ml counts. Most samples with Ct values greater than 29 had low Staphylococcus aureus loads (< 10)³CFU/ml) and Ct values less than 29, have high staphylococcus aureus loads (> 10)⁴CFU/ml). The Ct value of 29 corresponds to about 3.2log10 CFU/ml (about 1600-1700 CFU/ml).

Similar correlations between Ct values and staphylococcus aureus concentrations were detected in bronchial and tracheal cultures. (see FIG. 11.)

Similar correlations between Ct values and staphylococcus aureus concentrations were also examined, whether or not non-staphylococcal growth was observed. (see FIG. 12.)

These results demonstrate that PCR is a robust way to quantify staphylococcus aureus colonization, and that the Ct cutoff of 29 effectively distinguishes low and high staphylococcus aureus colonization.

Example 5

The efficacy of anti-staphylococcus aureus antibody MEDI4893 in preventing staphylococcus aureus pneumonia in mechanically ventilated ICU patients was compared in patients with low and high levels of staphylococcus aureus colonization (using a Ct cutoff of 29).

A subject is considered mechanically ventilated when: (i) when intubated and supported by positive airway pressure via endotracheal or nasal endotracheal tubes, or (ii) not intubated via endotracheal or nasal endotracheal tubes, but requires at least 8 hours of positive airway pressure over the past 24 hours (e.g., patients with tracheostomy, continuous positive airway pressure [ CPAP ], etc.).

Staphylococcus aureus pneumonia was diagnosed in patients who were mechanically ventilated at the time of diagnosis when they met the following radiological, clinical and microbiological criteria, which were not due to any apparent non-infectious cause.

Radiology standards:new or worsening infiltrations consistent with pneumonia on chest X-rays obtained within 24 hours of the event (diagnosed by a qualified radiologist) and

clinical criteria are as follows:at least 2 of the following minor or 1 major new signs or symptoms of respiration:

minor criteria

O signs of systemic infection (one or more of the following): abnormal body temperature (oral or tympanic > 38 ℃ or core body temperature ≧ 38.3 ℃ or hypothermia, defined as core body temperature < 35 ℃) and/or abnormal White Blood Cells (WBCs) (WBC count >10,000 cells/mm 3, WBC count < 4500 cells/mm 3 or > 15% band-shaped neutrophils);

production of purulent tracheal secretions

O physical examination results consistent with pneumonia/lung sthenia (pulmonary) (e.g., rale, dry rale, bronchial breath sounds) or percussion voiced sounds

Major criteria

An acute change in the ventilatory support system to enhance oxygenation as determined by:

a PaO2/FiO2 ratio < 240mmHg for at least 4 hours, or

The reduction of PaO2/FiO2 by not less than 50mmHg is maintained for at least 4 hours

And

and (3) microbiological confirmation:at least one of the following (obtained within 24 hours of the event onset):

culture of breath samples positive for staphylococcus aureus. Including samples of respiratory secretions obtained by intratracheal aspiration or by bronchoscopy and bronchoalveolar lavage (BAL) or Protective Sample Brush (PSB) sampling in intubated subjects. Expectorated sputum samples were acceptable in subjects who were not intubated but who met the definition of the mechanical ventilation protocol

Staphylococcus aureus positive blood cultures (and no significant major source of infection outside the lungs)

Pleural fluid aspirate or lung tissue culture positive for staphylococcus aureus during the onset of pneumonia

The results are shown in the table below.

Reduced relative risk (MEDI48935000mg versus placebo); a 90% Confidence Interval (CI); and p-values based on poisson regression with robust variance.

A82.6% relative risk reduction of Staphylococcus aureus pneumonia alone (90% CI: -1.0%, 97.0%), a 30.6% relative risk reduction of all-cause pneumonia (90% CI: -4.9%, 54.0%) and a 23.1% relative risk reduction of all-cause pneumonia or death (90% CI: 23.1% -4.9%, 43.6%) were also observed.

The interaction p-values were obtained from poisson regression with robust variance, including treatment groups, subgroups tested, and treatment terms interacting through subgroups. Relative risk reduction (MEDI48935000mg versus placebo) and 90% Confidence Interval (CI) are unconditional confidence intervals based on a ratio of proportions.

Patients with Ct values of at least 29 (low staphylococcus aureus) had a 33.3% prevalence and MEDI4893 produced a statistically significant Relative Risk Reduction (RRR) of about 67%. In contrast, the overall population had a 26% prevalence, and MEDI4893 produced a statistically insignificant 32% RRR. Those with Ct values less than 29 (high staphylococcus aureus) had a 22.2% prevalence and an RRR of about 2.5%, which was not statistically significant.

These results demonstrate that immunoprophylaxis using anti-alpha toxin antibody MEDI4893 shows increased efficacy in patients with low staphylococcus aureus colonization.

Example 6

The effect of anti-staphylococcus aureus antibody MEDI4893 on health resource utilization savings was also analyzed. The key results are summarized in the table below.

These results demonstrate that MEDI4893 shortens the duration of hospitalization, the duration of stay ICU, and the duration of mechanical ventilation.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.