Immune checkpoint blockade therapy for treating Staphylococcus aureus infections

Abstract

The present disclosure provides methods for reducing the number of Staphylococcus aureus bacterial cells in a subject, and methods for treating or preventing a disease or disorder caused by S. aureus. The present disclosure also provides methods for diagnosing or prognosing a disease or disorder caused by S. aureus.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 482,322, filed January 31, 2023, and U.S. Provisional Patent Application No. 63 / 509,689, filed June 22, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under grants AR07200 and AI169736 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The present invention relates generally to immune checkpoint therapy for treating or preventing Staphylococcus aureus infections. [Background technology]

[0004] Staphylococcus aureus is a gram-positive cocci and a member of the Firmicutes family, frequently found in the nose, respiratory tract, and skin. It is a common cause of skin infections, respiratory infections such as sinusitis, and food poisoning. Pathogenic strains often promote infection by producing potent protein toxins and expressing cell surface proteins that bind and inactivate antibodies. The emergence of antibiotic-resistant strains of S. aureus, such as methicillin-resistant S. aureus (MRSA), is a global problem in clinical medicine. It is a substantial cause of illness and death in both humans and animals. Infections with S. aureus often result in the development of surface abscesses. Other cases of S. aureus infection can be much more serious. For example, invasion of the lymphatics and blood by S. aureus can lead to systemic infection, which can then cause complications such as endocarditis, arthritis, osteomyelitis, pneumonia, septic shock, and even death. Hospital-acquired S. aureus infections are common and particularly problematic, with S. aureus being the most common cause of hospital-acquired surgical site infections and pneumonia, and the second most common cause of cardiovascular and bloodstream infections. S. aureus is also the most common pathogen in orthopedic site infections, leading to sepsis and death in 10% of patients (Cram P, et al. JAMA 308-12, 1227-1236 (2012)). Antibiotic administration is the standard treatment for S. aureus infections. Unfortunately, antibiotic use is also fueling the development of antibiotic resistance in S. aureus.

[0005] Vaccines against S. aureus have been developed, and numerous antibody-based clinical trials have been conducted, but they have all failed. Most notably, a large-scale phase 2 clinical trial of an iron-regulatory surface determinant protein B (IsdB)-activated vaccine had to be terminated early due to postoperative sepsis and multiple organ failure (Fowler VG, et al. JAMA 2013;309:1368-78). Increased mortality has been observed in patients with S. aureus prosthetic joint infection (PJI) who had high anti-IsdB antibody titers (Nishitani K, et al. Clin Orthop Relat Res 473-9, 2735-49 (2015)).

[0006] Therefore, there is a need for improved methods for treating and preventing S. aureus infections and related disorders. Summary of the Invention

[0007] SUMMARY OF THE DISCLOSURE In some aspects, the present disclosure addresses the above-referenced needs.

[0008] In one aspect, the disclosure provides methods for reducing the number of pathogenic bacterial or fungal cells, such as S. aureus bacterial cells, in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of an inhibitor of an immune checkpoint molecule.

[0009] In some embodiments, the immune checkpoint molecule is selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1.

[0010] In some embodiments, the inhibitor comprises an antibody or antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof.

[0011] In some embodiments, the inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof. In some embodiments, the inhibitor comprises (i) an anti-LAG-3 antibody or an antigen-binding fragment thereof, (ii) a combination of an anti-PD-1 antibody or an antigen-binding fragment thereof and an anti-LAG-3 antibody or an antigen-binding fragment thereof, or (iii) a bispecific antibody that binds to PD-1 and LAG-3. In some embodiments, the anti-PD-1 antibody is nivolumab or the anti-LAG-3 antibody is leratolimab.

[0012] In another aspect, the present disclosure also provides methods for the diagnosis or prognosis of a disease or disorder caused by a pathogenic bacterium or fungus, such as S. aureus, in a subject in need thereof. In some embodiments, the method includes: (a) determining the level of each of a set of biomarkers in a sample from the subject, where the set of biomarkers includes an immune checkpoint molecule or a cytokine; (b) determining a change in the level of each of the set of biomarkers compared to a reference level for each of the set of biomarkers; and (c) assessing the presence of the disease or disorder or the state of the disease or disorder based on the change in the level of each of the set of biomarkers compared to the reference level for each of the set of biomarkers.

[0013] In some embodiments, the set of biomarkers comprises one or more of LAG-3, PD-1, CTLA-4, TIM-3, IFNγ, IL-2, TNFα, and IL-17.

[0014] In some embodiments, the set of biomarkers includes TIM-3. In some embodiments, the set of biomarkers includes TIM-3 and LAG-3. In some embodiments, the set of biomarkers includes TIM-3 and CTLA-4. In some embodiments, the set of biomarkers includes TIM-3 and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and CTLA-4. In some embodiments, the set of biomarkers includes TIM-3, CTLA-4, and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, CTLA-4, and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and CTLA-4. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and PD-1. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, and PD-1.

[0015] In some embodiments, the set of biomarkers comprises TIM-3 and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, CTLA-4, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, CTLA-4, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, PD-1, and IL-17.

[0016] In some embodiments, the change in the level of each of the set of biomarkers is an increase in the expression level of each of the set of biomarkers.

[0017] In yet another aspect, the present disclosure further provides methods of preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or pathogenic fungus (such as S. aureus) in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an inhibitor of an immune checkpoint molecule.

[0018] In some embodiments, the method further comprises selecting a subject having a chronic or acute disease or disorder caused by a pathogenic bacterium or pathogenic fungus according to a method described herein.

[0019] In some embodiments, the disease or disorder comprises an infection, e.g., a S. aureus infection. In some embodiments, the infection is bacteremia. In some embodiments, the infection is a bone infection. In some embodiments, the infection is osteomyelitis. In some embodiments, the disease or disorder comprises S. aureus-associated sepsis.

[0020] In some embodiments, the infection is a prosthetic joint infection, a fracture-related infection, a diabetic foot infection, hematogenous osteomyelitis, or a spinal infection.

[0021] In some embodiments, the immune checkpoint molecule is selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1.

[0022] In some embodiments, the inhibitor comprises an antibody or antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof.

[0023] In some embodiments, the inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the inhibitor comprises (i) an anti-LAG-3 antibody or an antigen-binding fragment thereof, (ii) a combination of an anti-PD-1 antibody or an antigen-binding fragment thereof and an anti-LAG-3 antibody or an antigen-binding fragment thereof, or (iii) a bispecific antibody that binds to PD-1 and LAG-3. In some embodiments, the anti-PD-1 antibody is nivolumab or the anti-LAG-3 antibody is leratolimab.

[0024] In some embodiments, the method includes administering to the subject an additional therapeutic agent. In some embodiments, the additional therapeutic agent includes a second inhibitor of a second immune checkpoint molecule, an antibiotic, an anti-pathogen antibody specific for a pathogenic bacterium or fungus, or a combination thereof. In some embodiments, the anti-pathogen antibody specifically binds to Staphylococcus aureus. In some embodiments, the inhibitor and the additional therapeutic agent are contained in the same composition.

[0025] In some embodiments, the second immune checkpoint molecule is selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1.

[0026] In some embodiments, the second inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an antigen-binding fragment thereof, or a combination thereof.

[0027] In some embodiments, the antibiotic has antibacterial activity against S. aureus.

[0028] In some embodiments, the additional therapeutic agent is administered simultaneously with the inhibitor, hi some embodiments, the additional therapeutic agent is administered before or after the inhibitor.

[0029] In some embodiments, the inhibitor or additional therapeutic agent is administered to the subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, sustained release, controlled release, delayed release, as a suppository, or sublingually.

[0030] In some embodiments, the subject has undergone or is about to undergo surgery or an implant. In some embodiments, the subject has undergone surgery or an implant within the last 1 week, 2 weeks, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months. In some embodiments, the subject has undergone surgery or an implant within the last 1 week, 2 weeks, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

[0031] In some embodiments, the surgery is selected from the group consisting of orthopedic surgery, cardiothoracic surgery, plastic surgery, neurosurgery, oral surgery, total joint replacement, open reduction and internal fixation (ORIF), debridement for an open fracture, spinal surgery, median sternotomy, modified total joint arthroplasty, modified ORIF, drainage of a soft tissue abscess, or organ transplant surgery.

[0032] In some embodiments, the present disclosure provides: [1] A method for reducing the number of pathogenic bacterial or fungal cells in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of an inhibitory immune checkpoint molecule; [2] The method according to [1], wherein the administered inhibitor inhibits an inhibitory immune checkpoint molecule selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1; [3] The method of [1] or [2], wherein the administered inhibitor comprises an antibody or an antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof; [4] The method according to any one of [1] to [3], wherein the administered inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an antigen-binding fragment thereof, or a combination thereof; [5] The method according to any one of [1] to [4], wherein the administered inhibitor comprises (i) an anti-LAG-3 antibody or an antigen-binding fragment thereof, (ii) a combination of an anti-PD-1 antibody or an antigen-binding fragment thereof and an anti-LAG-3 antibody or an antigen-binding fragment thereof, or (iii) a bispecific antibody that binds to PD-1 and LAG-3; [6] The method according to [5], wherein the administered anti-PD-1 antibody is nivolumab and the administered anti-LAG-3 antibody is leratolimab, or the administered bispecific antibody comprises an antigen-binding fragment of nivolumab and an antigen-binding fragment of leratolimab; [7] A method for diagnosing or prognosing a disease or disorder caused by a pathogenic bacterium or a pathogenic fungus in a subject in need thereof, comprising: determining a level of each of a set of biomarkers in a sample from the subject, wherein the set of biomarkers includes an inhibitory immune checkpoint molecule or a cytokine; determining a change in the level of each of the set of biomarkers compared to a reference level for each of the set of biomarkers; assessing the presence of a disease or disorder, or a disease or disorder status, based on a change in the level of each of the set of biomarkers compared to a reference level for each of the set of biomarkers; [8] The set of biomarkers (a) LAG-3, PD-1, CTLA-4, TIM-3, IFNγ, IL-2, TNFα, and IL-17, or (b) the method described in [7], which comprises one or more of LAG-3, TIM-3, and CXCL13; [9] The set of biomarkers was: (a) TIM-3, (b) TIM-3 and LAG-3, (c) TIM-3 and CTLA-4, (d) TIM-3 and PD-1, (e) TIM-3 and IL-17, (f) TIM-3, LAG-3, and PD-1, (g) TIM-3, LAG-3, and CTLA-4, (h) TIM-3, CTLA-4, and PD-1, (i) TIM-3, LAG-3, CTLA-4, and PD-1, (j) TIM-3, LAG (k) TIM-3, CTLA-4, and IL-17, (l) TIM-3, PD-1, and IL-17, (m) TIM-3, LAG-3, PD-1, and IL-17, (n) TIM-3, LAG-3, CTLA-4, and IL-17, (o) TIM-3, CTLA-4, PD-1, and IL-17, or (p) TIM-3, LAG-3, CTLA-4, PD-1, and IL-17;

[10] The method according to any one of [7] to [9], wherein the set of biomarkers includes TIM-3, LAG-3, and CXCL13;

[11] The method according to any one of [7] to

[10] , wherein the sample is a bone marrow or PBMC sample;

[12] The method according to any one of [7] to

[11] , wherein the sample is a serum sample;

[13] The method according to any one of [7] to

[12] , wherein the change in the level of the set of biomarkers is an increase in the expression level of each of the biomarkers in the set;

[14] A method for preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or pathogenic fungus in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an inhibitor of an inhibitory immune checkpoint molecule;

[15] A method for preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or a pathogenic fungus in a subject in need thereof, the method comprising administering a therapeutically effective amount of an inhibitor of an inhibitory immune checkpoint molecule, wherein the subject has been determined to have a disease or disorder caused by a pathogenic bacterium or a pathogenic fungus according to the method described in any one of [7] to

[10] .

[16] The method according to any one of [7] to

[15] , wherein the disease or disorder includes an infectious disease;

[17] The method according to

[16] , wherein the infection is bacteremia, bone infection, osteomyelitis, or sepsis.

[18] The method according to

[16] , wherein the infection is a prosthetic joint infection, a fracture-related infection, a diabetic foot infection, hematogenous osteomyelitis, or a spinal infection;

[19] The method according to any one of

[14] to

[18] , wherein the administered inhibitor inhibits an inhibitory immune checkpoint molecule selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1;

[20] The method according to any one of

[14] to

[19] , wherein the administered inhibitor comprises an antibody or an antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof;

[21] The method according to any one of

[14] to

[20] , wherein the administered inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an antigen-binding fragment thereof, or a combination thereof;

[22] The method according to any one of

[14] to

[21] , wherein the administered inhibitor comprises (i) an anti-LAG-3 antibody or an antigen-binding fragment thereof, (ii) a combination of an anti-PD-1 antibody or an antigen-binding fragment thereof and an anti-LAG-3 antibody or an antigen-binding fragment thereof, or (iii) a bispecific antibody that binds to PD-1 and LAG-3;

[23] The method according to

[22] , wherein the anti-PD-1 antibody is nivolumab and the anti-LAG-3 antibody is leratolimab, or the bispecific antibody is an anti-PD-1 and anti-LAG-3 bispecific antibody (e.g., a bispecific antibody comprising an antigen-binding fragment of nivolumab and an antigen-binding fragment of leratolimab);

[24] The method according to any one of [1] to [6] and

[14] to

[23] , further comprising administering an additional therapeutic agent to the subject;

[25] The method of

[24] , wherein the additional therapeutic agent comprises a second inhibitor of a second inhibitory immune checkpoint molecule, an antibiotic, an anti-pathogen antibody specific for a pathogenic bacterium or a pathogenic fungus, or a combination thereof;

[26] The method of

[25] , wherein the additionally administered therapeutic agent is an anti-pathogen antibody that specifically binds to Staphylococcus aureus;

[27] The method of

[25] , wherein the additionally administered therapeutic agent is a second inhibitory immune checkpoint molecule selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1;

[28] The method of

[27] , wherein the additional administered therapeutic agent comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof;

[29] The method of

[24] , wherein the additional administered therapeutic agent is an antibiotic having antibacterial activity against Staphylococcus aureus;

[30] The method according to any one of

[24] to

[29] , wherein the additional therapeutic agent is administered simultaneously with the inhibitor;

[31] The method according to any one of

[24] to

[29] , wherein the additional therapeutic agent is administered before or after the inhibitor;

[32] The method of

[24] or

[30] , wherein the inhibitor and the additional therapeutic agent are contained in the same composition;

[33] The method according to any one of

[24] to

[32] , wherein the inhibitor and / or additional therapeutic agent is administered to the subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, by sustained release, controlled release, delayed release, as a suppository, or sublingually;

[34] The method according to any one of [7] to

[33] , wherein the subject has undergone or is about to undergo surgery or an implant;

[35] A method according to any one of [7] to

[34] , wherein the subject is undergoing surgery;

[36] A method according to any one of [7] to

[34] , wherein the subject is about to undergo surgery;

[37] The method according to any one of

[34] to

[36] , wherein the surgery is selected from the group consisting of orthopedic surgery, cardiothoracic surgery, plastic surgery, neurosurgery, oral surgery, total joint replacement, open reduction and internal fixation (ORIF), debridement for an open fracture, spinal surgery, median sternotomy, modified total joint replacement, modified ORIF, drainage of a soft tissue abscess, or organ transplant surgery;

[38] The method of any one of

[34] ,

[35] or

[37] , wherein the subject has received an implant;

[39] The method of any one of

[34] or

[36] , wherein the subject is about to receive an implant;

[40] The method according to any one of

[34] to

[39] , wherein the subject has received or is about to receive an orthopedic implant;

[41] The method according to any one of [1] to

[40] , wherein the bacterium is selected from the group consisting of Staphylococcus aureus, S. epidermidis, S. lugdunensis, Cutibacterium acnes, group B Streptococcus, and Enterobacteria;

[42] The method according to

[41] , wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-susceptible Staphylococcus aureus (MSSA);

[43] The method according to any one of [1] to

[42] , wherein the subject is a mammal;

[44] The method according to any one of [1] to

[43] , wherein the mammal is a human;

[45] Use of one or more inhibitors of inhibitory immune checkpoint molecules in the manufacture of a medicament for the method according to any one of [1] to [6] and

[14] to

[44] ;

[46] One or more inhibitors of inhibitory immune checkpoint molecules for use in the methods described in any one of [1] to [6] and

[14] to

[45] ;

[47] A method for reducing the number of pathogenic bacterial or fungal cells in a subject in need thereof, comprising: (i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) administering to a subject determined to have a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by methods described herein or known in the art), an effective amount of an inhibitor of an inhibitory immune checkpoint molecule;

[48] ​​A method for preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or a pathogenic fungus in a subject in need thereof, comprising: (i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) administering a therapeutically effective amount of an inhibitor of an inhibitory immune checkpoint molecule to a subject determined to have a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by methods described herein or known in the art);

[49] The method of

[48] , wherein the disease or disorder comprises an infection, and the infection is a prosthetic joint infection, a fracture-related infection, a diabetic foot infection, hematogenous osteomyelitis, or a spinal infection;

[50] A method for treating or preventing bone infection or bone loss in a subject in need thereof, comprising administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule;

[51] The method according to

[50] , wherein the method treats or prevents bone infection caused by pathogenic bacteria or pathogenic fungi;

[52] The method according to

[51] , wherein the bone infection is osteomyelitis;

[53] The method according to

[51] or

[52] , wherein the method prevents bone loss associated with infection by pathogenic bacteria or pathogenic fungi;

[54] A method for treating or preventing sepsis associated with pathogenic bacteria or pathogenic fungi in a subject in need thereof, comprising administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule;

[55] The method of

[54] , wherein the method treats or prevents S. aureus-associated sepsis;

[56] A method of treating or preventing the development of bacteremia, biofilm formation, or antimicrobial-resistant infection in a subject in need thereof, comprising administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule;

[57] Prior to administration of the inhibitor, the subject: (i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) the method of any one of [1]-[6],

[49] -

[56] , wherein the subject is determined to have a serum titer of soluble TIM-3 of at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by a method described herein or known in the art);

[58] Prior to surgery or receiving an implant (e.g., within 1 year, 9 months, 6 months, 3 months, 1 month, or 2 weeks), the subject: (i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) the method of any one of

[49] to

[56] , wherein the subject is determined to have a serum titer of soluble TIM-3 of at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by a method described herein or known in the art);

[59] The method according to any one of [1] to [6] and

[42] to

[58] , wherein the subject is administered the inhibitor before surgery or before receiving an implant;

[60] The method according to any one of [1] to [6] and

[42] to

[57] , wherein the subject is administered the inhibitor after surgery or after receiving an implant;

[61] The method according to any one of [1] to [6] and

[42] to

[59] , wherein the subject undergoes surgery within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months after administration of an inhibitor of an inhibitory immune checkpoint molecule;

[62] The method according to any one of [1] to [6],

[42] to

[57] , and

[60] , wherein the subject has undergone surgery within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months prior to administration of an inhibitor of an inhibitory immune checkpoint molecule;

[63] The method according to any one of

[57] to

[62] , wherein the surgery is selected from the group consisting of orthopedic surgery, cardiothoracic surgery, plastic surgery, neurosurgery, oral surgery, total joint replacement, open reduction and internal fixation (ORIF), debridement for an open fracture, spinal surgery, median sternotomy, modified total joint replacement, modified ORIF, drainage of a soft tissue abscess, or organ transplant surgery;

[64] The method of any one of [1]-[6],

[42] -

[59] , [63

[61] , and

[63] , wherein the subject receives the implant within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months after administration of an inhibitor of an inhibitory immune checkpoint molecule;

[65] The method according to any one of [1]-[6],

[42] -

[57] ,

[60] ,

[62] , and

[63] , wherein the subject has received an implant within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months prior to administration of an inhibitor of an inhibitory immune checkpoint molecule;

[66] The method according to any one of

[59] to

[65] , wherein the implant is an orthopedic implant;

[67] The method according to any one of

[14] to

[44] ,

[47] to

[66] , wherein the administered inhibitor comprises an antibody or an antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof;

[68] The method of any one of [1]-[6],

[14] -

[44] , and

[47] -

[68] , wherein the serum titer of soluble TIM-3 in the subject is determined to be elevated compared to a serum titer of TIM-3 previously measured in the subject;

[69] The method of any one of [1]-[6],

[14] -

[44] , and

[47] -

[68] , wherein the serum titer of soluble TIM-3 in the subject is determined to be at least 10% higher in the subject than in healthy control subjects;

[70] The method of [1]-[6],

[14] -

[44] , and

[47] -

[69] , wherein the serum titer of soluble TIM-3 in the subject is determined to be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (2-fold) higher in the subject than in healthy control subjects;

[71] The method of any one of [1] to [6],

[14] to

[44] , and

[47] to

[70] , wherein the serum titer of soluble TIM-3 in the subject is determined to be at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by the methods described in the Examples);

[72] The method of any one of [1]-[6],

[14] -

[44] , and

[47] -

[71] , wherein the serum titer of soluble TIM-3 in the subject is determined to be at least 3000 pg / ml (e.g., as determined by the methods described herein);

[73] The method according to any one of [1] to [6],

[14] to

[44] , and

[47] to

[72] , wherein the soluble TIM-3 is differentially spliced ​​soluble TIM-3 and / or shed TIM-3;

[74] The method of any one of

[47] -

[49] and

[57] -

[70] , wherein the serum titer of soluble TIM-3 in healthy control subjects is the median or average titer of soluble TIM-3 in at least 10, 50, or 100 subjects;

[75] A method for reducing the number of pathogenic bacterial or fungal cells in a subject in need thereof, comprising administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule to: (i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule to a subject who has been determined to have a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml (e.g., as determined by methods described herein or known in the art);

[76] A method for preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or a pathogenic fungus in a subject in need thereof, comprising: (i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) administering a therapeutically effective amount of an inhibitor of an inhibitory immune checkpoint molecule to a subject who has been determined to have a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml (e.g., as determined by methods described herein or known in the art);

[77] The method of

[76] , wherein the disease or disorder comprises an infection, and the infection is a prosthetic joint infection, a fracture-related infection, a diabetic foot infection, hematogenous osteomyelitis, or a spinal infection;

[78] A method of treating or preventing bone infection or bone loss in a subject in need thereof, comprising administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule;

[79] The method according to

[78] , wherein the method treats or prevents bone infection caused by pathogenic bacteria or pathogenic fungi;

[80] The method according to

[78] or

[79] , wherein the bone infection is osteomyelitis;

[81] The method according to any one of

[78] to

[80] , wherein the method prevents bone loss associated with infection by pathogenic bacteria or pathogenic fungi;

[82] A method for treating or preventing sepsis associated with pathogenic bacteria or pathogenic fungi in a subject in need thereof, comprising administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule;

[83] The method of

[82] , wherein the method treats or prevents S. aureus-associated sepsis;

[84] A method of treating or preventing the development of bacteremia, biofilm formation, or antimicrobial-resistant infection in a subject in need thereof, comprising administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule;

[85] Prior to administration, the subject: (i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) the method of any one of

[77] to

[84] , wherein the subject is determined to have a serum titer of soluble LAG-3 of at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml (e.g., as determined by methods described herein or known in the art);

[86] The method of any one of

[75] to

[85] , wherein the serum titer of soluble LAG-3 in the subject is determined to be elevated compared to a serum titer of LAG-3 previously measured in the subject;

[87] The method according to any one of

[75] to

[86] , wherein the serum titer of soluble LAG-3 in the subject is determined to be at least 10% higher in the subject than in healthy control subjects;

[88] The method of any one of [1]-[6],

[14] -

[44] , and

[47] -

[87] , wherein the serum titer of soluble LAG-3 in the subject is determined to be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (2-fold) higher in the subject than in healthy control subjects;

[89] The method of any one of [1]-[6],

[14] -

[44] , or

[47] -

[88] , wherein the serum titer of soluble LAG-3 in the subject is determined to be at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml (e.g., as determined by the methods described in the Examples);

[90] The method of any one of [1]-[6],

[14] -

[44] , and

[47] -

[89] , wherein the serum titer of soluble LAG-3 in the subject is determined to be at least 90,000 pg / ml (e.g., as determined by the methods described herein or an accepted standard);

[91] The method according to any one of

[75] to

[90] , wherein the soluble LAG-3 is differentially spliced ​​soluble LAG-3 and / or shed LAG-3;

[92] The method of any one of

[75] ,

[76] ,

[85] ,

[87] , or

[88] , wherein the serum titer of soluble LAG-3 in healthy control subjects is the median or average titer of soluble LAG-3 in at least 10, 50, or 100 subjects;

[93] A method for reducing the number of pathogenic bacterial or fungal cells in a subject in need thereof, comprising: (i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) administering an effective amount of an inhibitor of an inhibitory immune checkpoint molecule to a subject who has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml (e.g., as determined by methods described herein or known in the art);

[94] A method for preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or a pathogenic fungus in a subject in need thereof, comprising: (i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) administering a therapeutically effective amount of an inhibitor of an inhibitory immune checkpoint molecule to a subject who has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml (e.g., as determined by methods described herein or known in the art);

[95] Prior to administration of the inhibitor, the subject may further (i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) the method of any one of

[93] or

[94] , further determined to have a serum titer of soluble TIM-3 (e.g., as determined by methods described herein or known in the art) of at least 2100, 2200, 2300, 2400, or 2500 pg / ml;

[96] Prior to administration of the inhibitor, the subject: (i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) the method of any one of

[93] to

[95] , further determined to have a serum titer of soluble LAG-3 of at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml (e.g., as determined by a method described herein or known in the art);

[97] The disease or disorder is: (a) an infection such as a bone infection (e.g., a bone infection caused by a pathogenic bacteria or a pathogenic fungus), osteomyelitis, a prosthetic joint infection, a fracture-related infection, a diabetic foot infection, a hematogenous osteomyelitis, or a spinal infection; (b) Sepsis associated with pathogenic bacteria or pathogenic fungi, such as S. aureus-associated sepsis; (c) bacteremia; (d) biofilm formation, or (e) the development of an antimicrobial-resistant infection, including the method according to any one of

[94] to

[96] ;

[98] Prior to administration of the inhibitor, the subject: (a)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) a serum titer of CXCL13 that is at least at least 50, 55, 60, 65, 70, or 75 pg / ml (e.g., as determined by methods described herein or known in the art); (b)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined serum titer of TIM-3; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 of at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by methods described herein or known in the art), and / or (c)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml (e.g., as determined by methods described herein or known in the art); or any combination of (a) to (c);

[99] Prior to surgery or receiving an implant (e.g., within 1 year, 9 months, 6 months, 3 months, 1 month, or 2 weeks), the subject: (a)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) a serum titer of CXCL13 of at least 50, 55, 60, 65, 70, or 75 pg / ml (e.g., as determined by methods described herein or known in the art); (b)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined serum titer of TIM-3; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 of at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by methods described herein or known in the art), and / or (c)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml (e.g., as determined by methods described herein or known in the art); or any combination of (a) to (c);

[0100] The method of

[99] , wherein the subject has undergone surgery within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months prior to administration of an inhibitor of an inhibitory immune checkpoint molecule;

[0101] The method according to

[99] or

[0100] , wherein the subject has received an implant within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months prior to administration of an inhibitor of an inhibitory immune checkpoint molecule;

[0102] After surgery or receiving an implant, the subject: (a)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) a serum titer of CXCL13 of at least 50, 55, 60, 65, 70, or 75 pg / ml (e.g., as determined by methods described herein or known in the art); (b)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined serum titer of TIM-3; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 of at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by methods described herein or known in the art), and / or (c)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml (e.g., as determined by methods described herein or known in the art); or any combination of (a) to (c);

[0103] The method according to

[0102] , wherein the subject undergoes surgery within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months after administration of the inhibitor of an inhibitory immune checkpoint molecule;

[0104] The method according to

[0102] or

[0103] , wherein the subject receives the implant within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months after administration of the inhibitor of an inhibitory immune checkpoint molecule;

[0105] The method according to any one of

[99] to

[0104] , wherein the surgery is selected from the group consisting of orthopedic surgery, cardiothoracic surgery, plastic surgery, neurosurgery, oral surgery, total joint replacement, open reduction and internal fixation (ORIF), debridement for open fractures, spinal surgery, median sternotomy, modified total joint replacement, modified ORIF, drainage of a soft tissue abscess, or organ transplant surgery;

[0106] The method according to any one of

[99] to

[0105] , wherein the implant is an orthopedic implant;

[0107] the method according to any one of

[68] to

[0106] , wherein the administered inhibitor comprises an antibody or an antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof;

[0108] the method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0107] , wherein the serum titer of soluble CXCL13 in the subject is determined to be elevated compared to a serum titer of CXCL13 previously measured in the subject;

[0109] The method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0108] , wherein the serum titer of CXCL13 in the subject is determined to be at least 10% higher in the subject than in a healthy control subject;

[0110] The method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0109] , wherein the serum titer of CXCL13 in the subject is determined to be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (2-fold) higher in the subject than in a healthy control subject;

[0111] The method according to any one of

[11] to [6],

[14] to

[44] , and

[46] to

[0110] , wherein the serum titer of CXCL13 in the subject is determined to be at least 2100, 2200, 2300, 2400, or 2500 pg / ml (e.g., as determined by the methods described in the Examples);

[0112] The method of any one of [1] to [6],

[14] to

[44] , and

[46] to

[0111] , wherein the serum titer of CXCL13 in the subject is determined to be at least 3000 pg / ml (e.g., as determined by the methods described herein);

[0113] the method according to any one of

[98] to

[11] , wherein the serum titer of CXCL13 in the healthy control subjects is the median or average titer of CXCL13 in at least 10, 50, or 100 subjects;

[0114] The method according to any one of

[46] to

[0113] , wherein the administered inhibitor is a LAG-3 inhibitor, a TIM-3 inhibitor, a TIGIT inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor:

[0115] the method according to any one of

[46] to

[0114] , wherein the administered inhibitor is an antibody or an antigen-binding fragment thereof;

[0116] The method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0115] , wherein the administered inhibitor is a LAG-3 inhibitor;

[0117] the method according to

[0116] , wherein the administered inhibitor is a LAG-3 antibody or an antigen-binding fragment thereof;

[0118] the method according to

[0117] , wherein the administered LAG-3 antibody or antigen-binding fragment thereof is an antibody or an antigen-binding fragment thereof selected from the group consisting of BMS986016, MK-4280 (28G-10), REGN3767, GSK283l781, IMP73l (H5L7BW), BAP050, IMP-701 (LAG-5250), TSR-033, LAG525, BI754111, and FS-118;

[0119] the method according to

[0117] , wherein the administered LAG-3 antibody or antigen-binding fragment thereof is an antibody or an antigen-binding fragment thereof selected from the group consisting of leratolimab, BMS-986016, and GSK2831781;

[0120] The method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0115] , wherein the administered inhibitor is a TIM-3 inhibitor;

[0121] the method according to

[0120] , wherein the administered inhibitor is a TIM-3 antibody or an antigen-binding fragment thereof;

[0122] the method according to

[0121] , wherein the administered TIM inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of TSR-022, LY3321367, EPZ005687, and DZNep;

[0123] the method according to

[0121] , wherein the administered TIM-3 inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of TSR-022 and LY3321367;

[0124] The method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0115] , wherein the administered inhibitor is a TIGIT inhibitor;

[0125] the method according to

[0124] , wherein the administered inhibitor is a TIGIT antibody or an antigen-binding fragment thereof;

[0126] the method according to

[0125] , wherein the administered TIGIT inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of BMS-986207, AB154, COM902 (CGEN-15137), or OMP-313M32;

[0127] the method according to

[0125] , wherein the administered TIGIT inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of BMS-986207 and AB154;

[0128] The method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0115] , wherein the administered inhibitor is a PD-1 inhibitor;

[0129] the method according to

[0128] , wherein the administered inhibitor is a PD-1 antibody or an antigen-binding fragment thereof;

[0130] the method according to

[0129] , wherein the administered PD-1 inhibitor is an antibody or an antigen-binding fragment thereof selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, nivolumab, PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS00I, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI754091, and SHR-1210;

[0131] the method according to

[0129] , wherein the administered PD-1 inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, nivolumab, BGB-A317, AMP-224, PDR001, and MEDI0680 (AMP-514);

[0132] The method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0115] , wherein the administered inhibitor is a PD-L1 inhibitor;

[0133] the method of

[0132] , wherein the administered inhibitor is a PD-L1 antibody or an antigen-binding fragment thereof;

[0134] the method according to

[0133] , wherein the administered PD-L1 inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of atezolizumab, durvalumab, BMS-936559, avelumab, LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MPDL3280A, MEDI4736, MSB0010718C, and MDX-1105;

[0135] the method according to

[0133] , wherein the administered PD-L1 inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of atezolizumab, durvalumab, BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, and avelumab;

[0136] The method according to

[0132] , wherein the administered inhibitor is a small molecule;

[0137] 136. The method of claim 136, wherein the administered inhibitor is a small molecule selected from the group consisting of BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, JQI, and I-BET151;

[0138] the method according to any one of [1] to [6],

[14] to

[44] , and

[46] to

[0137] , wherein the administered inhibitor is a bispecific antibody or a soluble immune checkpoint molecule-binding ligand (e.g., a CTLA4-Fc fusion protein) that binds to at least one immune checkpoint inhibitory molecule;

[0139] The method of claim 1, wherein the administered bispecific antibody binds to two different inhibitory immune checkpoint molecules;

[0140] the method of claim 1, wherein the bispecific antibody binds to PD-1 and LAG-3, or PD-1 and TIM-3;

[0141] The method of

[0139] , wherein the bispecific antibody comprises antigen-binding fragments of nivolumab and leratolimab;

[0142] The method according to any one of

[46] to

[0141] , further comprising administering to the subject an additional therapeutic agent;

[0143] The method of claim 1, wherein the additional therapeutic agent is a second inhibitor of a second inhibitory immune checkpoint molecule, an antibiotic, an anti-pathogen antibody specific for a pathogenic bacterium or a pathogenic fungus, or a combination thereof;

[0144] The method of claim 1, wherein the additionally administered therapeutic agent is an anti-pathogen antibody that specifically binds to Staphylococcus aureus;

[0145] the method of claim 143, wherein the additionally administered therapeutic agent is a second administered inhibitory immune checkpoint molecule selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1;

[0146] the method of

[0143] or

[0145] , wherein the second administered inhibitor of an inhibitory immune checkpoint molecule is an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof;

[0147] The method of claim 1, wherein the additionally administered therapeutic agent is an antibiotic having antibacterial activity against Staphylococcus aureus;

[0148] The method of any one of

[0143] to

[0147] , wherein the additional therapeutic agent is administered simultaneously with the inhibitor;

[0149] The method of any one of

[0143] to

[0147] , wherein the additional therapeutic agent is administered before or after the inhibitor;

[0150] The method according to any one of

[0143] to

[0148] , wherein the inhibitor and the additional therapeutic agent are contained in the same composition;

[0151] The method according to any one of

[0143] to

[0150] , wherein the inhibitor and / or additional therapeutic agent is administered to the subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, by sustained release, controlled release, delayed release, as a suppository, or sublingually;

[0152] The method according to any one of

[0142] to

[0151] , wherein the subject has undergone or is about to undergo surgery or an implant;

[0153] the method according to any one of

[46] to

[0152] , wherein the bacterium is selected from the group consisting of Staphylococcus aureus, S. epidermidis, S. lugdunensis, Cutibacterium acnes, group B Streptococcus, and Enterobacteria;

[0154] The method according to

[0153] , wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-susceptible Staphylococcus aureus (MSSA);

[0155] The method according to any one of

[47] to

[0154] , wherein the subject is a mammal;

[0156] The method according to any one of

[47] to

[0155] , wherein the mammal is a human;

[0157] Use of one or more inhibitors of inhibitory immune checkpoint molecules in the manufacture of a medicament for the method according to any one of

[47] to

[0156] ; and / or

[0158]

[0156] One or more inhibitors of inhibitory immune checkpoint molecules for use in the methods described in any one of

[47] to

[0156] .

[0033] In some embodiments, the bacterium is selected from the group consisting of Staphylococcus aureus, S. epidermidis, S. lugdunensis, Cutibacterium acnes, Group B Streptococcus, and Enterobacteria. In some embodiments, the bacterium is selected from the group consisting of S. saprophyticus, S. haemolyticus, S. caprae, and S. simiae. In some embodiments, the S. aureus is methicillin-resistant S. aureus (MRSA) or methicillin-susceptible S. aureus (MSSA). In some embodiments, the S. aureus is resistant to one or more b-lactam antimicrobial agents. In some embodiments, the S. aureus is vancomycin-resistant.

[0034] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0035] Also within the scope of the present disclosure is (a) the use of one or more inhibitors of immune checkpoint molecules in the manufacture of a medicament for the methods described herein, and (b) one or more inhibitors of immune checkpoint molecules for use in the methods described herein.

[0036] The foregoing summary is not intended to define all aspects of the present disclosure; additional aspects are described in other sections, such as the following detailed description. It should be understood that the entire specification is intended to be related as a unified disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the specification. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0037] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0038] [Figure 1A-K]Figure 1A shows that humanized NSG-SGM3 BLT mice have exacerbated susceptibility to S. aureus compared with murinized NSG-SGM3 and C57BL / 6 WT mice. Figure 1A shows that humanized NSG-SGM3 BLT mice were generated by engrafting CD34+ human hematopoietic cells from three different human donors, autologous human fetal liver, and thymus. Murinized NSG-SGM3 BLT mice were generated using CD34+ murine hematopoietic cells from three different C57BL / 6 WT mice. Figure 1B shows a schematic diagram of the experimental design for the in vivo experiment. Twenty-week-old humanized NSG-SGM3 BLT mice, murinized NSG-SGM3 mice, and C57BL6 (WT) mice were subjected to transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 LAC::lux). Figures 1C and 1D show longitudinal assessment of in vivo S. aureus growth via bioluminescence imaging, revealing increased in vivo S. aureus growth in humanized NSG-SGM3 BLT mice. Figure 1E shows MRSA spread from the infection site to distal viscera. Figures 1F, 1G, 1H, and 1I show that 14 days after surgery, huNSG-SGM3 BLT mice and control animals were euthanized, and ex vivo CFU quantification was performed on the implant, tibia, soft tissue surrounding the tibia, and viscera (heart, liver, kidney, and spleen). Interestingly, CFU quantification revealed that huNSG-SGM3 BLT mice exhibited exacerbated susceptibility and increased sepsis due to S. aureus osteomyelitis compared with murinized NSG-SGM3 and C57BL6 (WT) animals (n = 25 (3 human donors), ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001). After euthanasia, MRSA-infected and sterile implant-inserted tibiae from a subset of huNSG-SGM3 BLT, NSG-SGM3, and C57BL / 6 animals were demineralized, sectioned, and processed for histopathological analysis.Figures 1J and 1K show that Blau-Brenn (B&B) staining to identify bacteria revealed numerous SAC formations and increased SAC area across the tibia region in humanized BLT mice compared to control animals (n = 4-5, ANOVA, *p < 0.05). [Figure 2A-F] Single-cell RNA sequencing reveals significant human T cell heterogeneity in numbers and gene expression resulting from S. aureus osteomyelitis in humanized BLT mice. (Figure 2A) Schematic showing the experimental setup for single-cell RNA sequencing of humanized NSG-SGM3 BLT mice engrafted with three different human donor tissues. Bone marrow (BM) cells were isolated from the tibiae of humanized NSG-SGM3 BLT mice operated with or without bioluminescent MRSA-contaminated transtibial implants on day 14 after surgery. Isolated BM cells were FACS-sorted into human CD45+CD19+ B cells and CD45+CD3+ T cells. Equal proportions of B and T cells were subjected to sc-RNA sequencing and sc-TCR / BCR repertoire analysis, as shown here. (Figure 2B) UMAP plot of single-cell gene expression of T and B cells in all approximately 30,000 BM cells from the tibiae of humanized NSG-SGM3 BLT mice. (Figure 2C) Feature plot of the pan-T cell marker CD3E and the B cell marker CD19 in all implanted BM cells. (Figures 2D-2E) UMAP and DEG clustering analysis of hCD45+CD3+ T cells identified 24 T cell clusters. (Figure 2F) Bar plot showing the percentage of cell numbers in each cluster between the sterile sham-operated and infected implant groups. Interestingly, the number of Th1 / Th17 cells (clusters 8 and 20) was significantly increased in infected animals compared to sham-operated (sterile) animals. [Figure 3A-E]We show that immune checkpoint proteins are elevated in CD4+ Th1 / Th17 cells in the tibiae of S. aureus-infected humanized BLT mice (Figure 3A). The Th1 / Th17 cells (clusters 8 and 20) in Figure 3 were subjected to UMAP and differential gene expression (DEG) subclustering analysis, revealing seven clusters (Figure 3B). Bar plot analysis demonstrated that these cells were of the Th1 / Th17 phenotype. Some Th1 / Th17 clusters showed significantly increased expression of immune checkpoint molecules LAG3, TIM-3 (HAVCR2), and, to a lesser extent, other immunosuppressive genes such as CTLA-4 and TIGIT. (Figure 3C) DEG analysis of transcription factors (TCF7, TOX1-2, EOMES, NR4A1), cytokines, and chemokines (IL-1, IL-17, CXCL13, CXCR5) associated with functional T cell exhaustion, long-term antigen stimulation (CD40L), and proliferation (MKi67). Note that lower expression of TCF7, MKi67, IL-1, and IL-17 genes, and higher expression of CXCL13 and TOX2, indicate transcriptional reprogramming of these cells to a terminally functionally exhausted state (*p<0.05). (Figure 3D-E) Immunofluorescence analysis of tibia sections from sham-operated controls and humanized BLT mice demonstrates high accumulation of LAG3+, TIM-3+, and / or PD-1+ T cells near the infection site in MRSA-infected BLT mice. In contrast, human T cell accumulation was scarce in uninfected BLT mice, with minimal evidence of exhaustion observed near the infection site. (Figure 3E) A multicolor spectroscopic flow cytometry assay was developed, optimized, and performed on tibia bone marrow (BM) cells from uninfected and MRSA-infected BLT mice. Live human CD45+ / CD3+ / T cells and their subpopulations (CD4+, CD8+, Tregs) were analyzed for immune checkpoint expression (LAG3, TIM-3, and PD-1) and proliferation (Ki67). Note that the frequency of human CD3+CD4+ T cells expressing TIM-3, LAG3, and PD-1 in bone marrow cells from MRSA-infected BLT mice was significantly higher than in controls (n = 4–8 mice, *p < 0.05, t-test). [Figure 4A-B]These results demonstrate that splenic and bone marrow CD4+ T cells expressing TIM-3 and LAG3 checkpoint proteins exhibit reduced proliferative capacity due to S. aureus infection. Multicolor spectroscopic flow cytometry was performed on uninfected and MRSA-infected BLT mice (Figure 4A) spleen cells and (Figure 4B) tibial bone marrow cells using the protocol described herein. Subsequently, CD4+ TIM-3+ and CD4+ LAG3+ cells had significantly lower frequencies of proliferative Ki67+ cells in the spleen and tended to have lower amounts of proliferative Ki67+ cells in the bone marrow of infected BLT mice, suggesting functional exhaustion and dysfunction (n = 4–9 mice, *p < 0.05, ANOVA). [Figure 5A-C] Serum immune checkpoint proteins are highly prognostic for adverse events in patients with S. aureus osteomyelitis. Figure 5A shows serum samples collected from individuals undergoing total hip / knee arthroplasty (n=15) and orthopedic patients with culture-confirmed S. aureus osteomyelitis at 1 year (n=37, 12 - adverse event (AD), 11 - infection control (IC), 14 - inconclusive). Immune checkpoint proteins LAG-3, TIM-3, CTLA-4, PD-1, and cytokines (IFN-γ, IL-2, TNFα, IL-17A, IL-17F) were assessed by multiplex Luminex assay. Data are presented as mean + / - SEM for each experimental group. Receiver operating characteristic (ROC) curve analysis was performed using individual protein levels, either alone or in combination, to distinguish between acute and chronic S. aureus infections (Figure 5B) and to generate area under the curve (AUC) for prognostic prediction of outcome (Figure 5C). No correlation was observed between immune checkpoint protein levels and clinical duration-based case classification of acute versus chronic. On the other hand, immune checkpoint proteins, particularly TIM-3, were highly predictive of adverse outcomes in these patients (*p<0.05, **p<0.01, ****p<0.00001). [Figure 6A-E]Preliminary feasibility data demonstrate a trend toward efficacy of the immune checkpoint blockade drug OPDUALAG™ (anti-PD-1 (nivolumab) + anti-LAG3 (relatolimab) mAb cocktail) in reducing S. aureus burden in humanized BLT mice. (Figure 6A) Schematic of the experimental design. 20-week-old humanized NSG-SGM3 BLT mice were subjected to transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 LAC::lux) and treated with OPDUALAG™ or a saline placebo control. The concentrations of anti-PD1 and anti-LAG3 mAbs used in the study are also outlined. (Figure 6B) Longitudinal BLI revealed significantly reduced in vivo S. aureus growth in humanized BLT mice treated with OPDUALAG™ (n = 3-4, ANOVA, *p < 0.05). On day 14 after surgery, terminal ex vivo CFU counts in the tibia and viscera (heart, liver, kidney, and spleen) revealed that OPDUALAG™-treated animals exhibited lower disease severity. (Figure 6C) Interestingly, histopathology revealed significantly reduced SAC formation in OPDUALAG™-treated animals compared with controls. (Figure 6D) μCT analysis revealed a reduced bone response in OPDUALAG™-treated animals, with rough, depressed areas around the implant holes (red arrows). The blue color indicates holes formed at the infection site due to infected implants. (Figure 6E) Immunofluorescence micrographs showing a significant reduction in LAG3+ and PD-1+ T cells in OPDUALAG™-treated animals. In addition, increased proliferating PCNA+ T cells were observed in OPDUALAG™-treated animals, potentially suggesting functional recovery of depleted T cells. [Figure 7A-D]Preliminary feasibility data demonstrate a trend toward efficacy of the immune checkpoint blocker sabatolimab (anti-TIM-3) in reducing S. aureus burden resulting from implant-associated osteomyelitis. (Figure 7A) Schematic of the experimental design. Eight-week-old C57BL / 6 mice were subjected to transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 LAC::lux) and treated with sabatolimab or a saline placebo control. The concentrations of anti-TIM-3 mAb used in the study are also outlined. (Figure 7B-C) Longitudinal BLI revealed significantly reduced in vivo S. aureus growth in humanized BLT mice treated with sabatolimab (n = 9-10, ANOVA, *p < 0.05). At day 14 after surgery, terminal ex vivo CFU in the tibia and viscera (heart, liver, kidney, and spleen) revealed that sabatolimab-treated animals exhibited lower disease severity (n=9-10, t-test, *p<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0039] The present disclosure provides methods for reducing the number of S. aureus bacterial cells in a subject, and methods for treating or preventing a disease or disorder caused by S. aureus. The present disclosure also provides methods for diagnosing or prognosing a disease or disorder caused by S. aureus.

[0040] Immune checkpoint blockade therapy for treating S. aureus infection Method for reducing the bacterial load of pathogenic bacteria or fungi, such as A. Saureus In one aspect, the invention provides methods for reducing the number of pathogenic bacterial or fungal cells, such as S. aureus bacterial cells, in a subject in need thereof. In some embodiments, the methods comprise reducing the bacterial load of S. aureus or eradicating the bacteria in the bloodstream or heart of a mammalian subject. In some embodiments, the methods comprise administering to the subject an effective amount of an inhibitor of an immune checkpoint molecule. In some embodiments, the methods comprise administering to the subject an additional therapeutic agent, such as an antimicrobial or antibacterial agent (e.g., an antibiotic).

[0041] The bacterial load in the bloodstream or heart of a mammalian subject can be routinely measured through methods known in the art to determine the amount of bacteria in the bloodstream or heart.For example, bacterial load can be measured by plating a sample from an organism onto an agar plate, incubating the plate, and then quantifying the number of colony-forming units (CFU) on the plate.Such methods are known in the art.Additional suitable methods for determining bacterial load can also be used.

[0042] Using the method, the bacterial burden (i.e., the amount of bacteria as measured by colony forming units) in a subject infected with S. aureus can be reduced by at least 30% (e.g., at least 40%, 50%, 60%, 70%, 80%, or 90%) in subjects treated with an inhibitor of an immune checkpoint molecule, compared to a subject infected with S. aureus but not administered the inhibitor. In some embodiments, the inhibitor of an immune checkpoint molecule can be administered as soon as possible after diagnosis of infection with S. aureus, e.g., within hours or days. The duration and amount of the inhibitor of an immune checkpoint molecule to be administered can be readily determined by one of skill in the art.

[0043] As used herein, the term "immune checkpoint molecule" refers to a molecule on an immune cell, such as a T cell, that is important under normal physiological conditions for maintaining self-tolerance (or preventing autoimmunity) and protecting host cells and tissues when the immune system responds to foreign pathogens. Certain immune checkpoint molecules are costimulatory molecules that amplify signals involved in the T cell response to an antigen, while certain immune checkpoint molecules are inhibitory molecules (e.g., CTLA-4 or PD-1) that reduce signals involved in the T cell response to an antigen.

[0044] As used herein, the term "inhibitory immune checkpoint molecule" includes, inter alia, inhibitory immune checkpoint receptors that are expressed on immune effector cells, such as T cells, and that can mediate downregulation or inhibition of immune responses upon engagement by their respective ligands. Examples of "inhibitory immune checkpoint molecules" include, but are not limited to, CTLA-4, PD-1, PD-L1, PD-L2, VISTA, TIM-1, TIM-3, TIM-4, LAG-3, TIGIT, galectin-1, galectin-9, CEACAM-1, CEACAM-5, CD69, CD113, GPR56, VISTA, B7-H3 (CD276), B7-H4 (VTCN1), 2B4 (CD244), SLAMF2 (CD48), GITR, BTLA, HVEM, KIR family receptors, GARP, and PD1H. Inhibitors (e.g., antagonistic (blocking) compositions) that bind to inhibitory immune checkpoint molecules may also be referred to herein as immune checkpoint inhibitors.

[0045] In some embodiments, the immune checkpoint molecule is an inhibitory molecule that reduces signals involved in T cell responses to antigens. For example, CTLA4 is expressed on T cells and plays a role in downregulating T cell activation by binding to CD80 (also known as B7.1) or CD86 (also known as B7.2) on antigen-presenting cells. PD-1 is another inhibitory immune checkpoint molecule expressed on T cells. PD-1 limits the activity of T cells in peripheral tissues during inflammatory responses. PD-L1 is widely expressed on antigen-presenting cells and other immune cells, and its binding to PD-1 on T cells drives them to apoptosis or a regulatory phenotype. In some embodiments, the inhibitory immune checkpoint molecule is lymphocyte-activation gene 3 (LAG-3) or T-cell membrane protein 3 (TIM-3). Non-limiting examples of immune checkpoint molecules include LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1.

[0046] As used herein, the term "inhibit" or "inhibitor" refers to a direct or indirect alteration, interference, reduction, downregulation, blocking, suppression, cessation, or degradation in the expression, amount, or activity of a target or signaling pathway compared to a control, endogenous, or reference target or pathway, or the absence of the target or pathway, where the alteration, interference, reduction, downregulation, blocking, suppression, cessation, or degradation is statistically, biologically, or clinically significant. For example, an "inhibitor" of an immune checkpoint molecule blocks, inactivates, reduces, or minimizes the activity of an immune checkpoint molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Non-limiting examples of inhibitors include antibodies or antigen-binding fragments thereof, small molecules, proteins, polypeptides, peptides, peptidomimetics, nucleic acids, antisense molecules, ribozymes, RNAi molecules, lipids, lipopeptides, carbohydrates, or combinations thereof.

[0047] Inhibitors targeting immune checkpoint molecules can be used to enhance T cell responses to S. aureus. Small molecule inhibitors of immune checkpoint molecules are described, for example, in Smith WM, et al. (Smith WM, et al. Therapeutic targeting of immune checkpoints with small molecule inhibitors. Am J Transl Res 11(2), 529-541(2019). PMID:30899360), the contents of which are incorporated herein by reference. For example, non-limiting examples of small molecule inhibitors of LAG-3 include BMS-986016 (BMS-ONO) and GSK2831781. Non-limiting examples of small molecule inhibitors of TIM-3 include EPZ005687 and DZNep. Non-limiting examples of small molecule inhibitors of CTLA-4 include entinostat, panobinostat, ACY-241, and azacitidine. Non-limiting examples of small molecule inhibitors of PD-1 / PD-L1 include BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, JQ1, and I-BET151.

[0048] In some embodiments, the inhibitor of an immune checkpoint molecule is an antibody, e.g., a monoclonal antibody. In some embodiments, the inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the inhibitor comprises an anti-LAG-3 antibody. In some embodiments, the inhibitor comprises a monoclonal anti-LAG-3 antibody. In some embodiments, the inhibitor comprises an anti-TIM-3 antibody. In some embodiments, the inhibitor comprises a monoclonal anti-TIM-3 antibody. In some embodiments, the inhibitor comprises an anti-CTLA-4 antibody. In some embodiments, the inhibitor comprises a monoclonal anti-CTLA-4 antibody. In some embodiments, the inhibitor comprises an anti-PD-1 antibody. In some embodiments, the inhibitor comprises a monoclonal anti-PD-1 antibody. In some embodiments, the inhibitor comprises an anti-PD-L1 antibody. In some embodiments, the inhibitor comprises a monoclonal anti-PD-L1 antibody. In some embodiments, the inhibitor is a combination of two or more of an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody. In some embodiments, the inhibitor is a combination of two or more of an anti-LAG-3 monoclonal antibody, an anti-TIM-3 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, an anti-PD-1 monoclonal antibody, and an anti-PD-L1 monoclonal antibody.

[0049] In some embodiments, the inhibitor comprises (i) an anti-LAG-3 antibody or antigen-binding fragment thereof, (ii) a combination of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-LAG-3 antibody or antigen-binding fragment thereof, or (iii) a bispecific antibody that binds to PD-1 and LAG-3. In some embodiments, the anti-PD-1 antibody is nivolumab or the anti-LAG-3 antibody is leratolimab.

[0050] In some embodiments, the administered LAG-3 inhibitor is an anti-LAG-3 antibody or antigen-binding fragment thereof. In some embodiments, the administered LAG-3 inhibitor is BMS986016, MK-4280 (28G-10), REGN3767, GSK2831781, IMP731 (H5L7BW), BAP050, IMP-701 (LAG-5250), TSR-033, LAG525, BI754111, Sym022, FS-118, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the administered anti-LAG-3 antibody is MK-4280, IMP-761, GSK2837781, MGD013, BAP050, or IBI110 IMP321, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the administered anti-LAG-3 antibody is leratolimab, or an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the administered anti-LAG-3 antibody is a antibody described in U.S. Patent Nos. 9,244,059, 10,266,591, 9,908,936, 10,358,495, or 10,188,730, or in WO 2008 / 132601, 2010 / 019570, 2014 / 008218, 2017 / 219995, 2014 / 008218, 2018 / 066054, 2019 / 066055, 2019 / 066056, 2019 / 066057, 2019 / 066058, 2019 / 066059 ... 019 / 141092, 2017 / 220555, 2019 / 129137, 2018 / 069500, 2018 / 066054, 2014 / 008218, 2015 / 138920, 2017 / 037203, or 2018 / 034227 (each of which is incorporated by reference in its entirety).

[0051] In some embodiments, the administered LAG-3 inhibitor is an antibody or antigen-binding fragment of an antibody that specifically binds to human LAG-3 and cross-competes with one or more of the antibodies listed above for binding to human LAG-3. For example, in some embodiments, the administered LAG-3 inhibitor is an antibody or antigen-binding fragment of an antibody that specifically binds to human LAG-3 and cross-competes with leratolimab for binding to human LAG-3. The ability of antibodies and antigen-binding fragments to cross-compete for binding to an antigen indicates that these antibodies / fragments bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies / fragments are expected to have functional properties very similar to the antibodies (e.g., leratolimab) with which they compete by binding to the same epitope region of LAG-3. Cross-competing antibodies can be readily identified based on their ability to cross-compete with a reference LAG-3 binding antibody (e.g., leratolimab) in standard LAG-3 binding assays such as surface plasmon resonance (BIACORE®) analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0052] In some embodiments, the administered TIM-3 inhibitor is an anti-TIM-3 antibody or antigen-binding fragment thereof. In some embodiments, the administered TIM-3 inhibitor is MBG-453, TSR-022, TRL-6061, BGBA425, LY-3321367, Sym023, INCAGN-2390, MBS-986258, RO-7121661, BC-3402, SHR-1702, or LY-3415244, or an antigen (TIM-3)-binding fragment thereof. In some embodiments, the administered TIM-3 inhibitor is antibody 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, or 8C4, or an antigen (TIM-3)-binding fragment thereof, as described in WO 2019 / 046321A1. WO 2019 / 046321A is incorporated herein in its entirety. In some embodiments, the administered anti-TIM-3 antibody is TSR-022 or LY3321367, or an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the administered anti-TIM-3 antibody is leratolimab, or an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the anti-TIM-3 antibody administered is any of the antibodies described in WO 2011 / 155607, WO 2011 / 159877, WO 2013 / 006490, WO 2015 / 109931, WO 2015 / 117002, WO 2016 / 068803, WO 2016 / 068802, WO 2016 / 071448, WO 2016 / 111947, WO 2016 / 144803, WO 2016 / 161270, WO 2017 / 019897, WO 2017 / 031242, WO 2017 / 055399, WO 2017 / 061448 ... or 2019 / 0046321 , U.S. Patent Nos. 8,552,156 , 8,841,418 , or 9,163,087 , or Chinese Patent Application No. 2010 / 6632675 , each of which is incorporated herein by reference in its entirety.

[0053] In some embodiments, the administered TIM-3 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to human TIM-3 and cross-competes with one or more of the antibodies listed above for binding to human TIM-3. For example, in some embodiments, the administered TIM-3 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to human TIM-3 and cross-competes with a reference TIM-3-binding antibody (e.g., 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, or 8C4) for binding to human TIM-3 in a standard TIM-3 binding assay, such as surface plasmon resonance (BIACORE®) analysis, ELISA assay, or flow cytometry (see, e.g., WO 2013 / 173223). In another example, in some embodiments, the administered TIM-3 inhibitor is an antibody or antigen-binding fragment of an antibody that specifically binds to human TIM-3 and cross-competes with a reference TIM-3-binding antibody disclosed in WO2019 / 0046321 for binding to human TIM-3 in a standard TIM-3 binding assay, such as surface plasmon resonance (BIACORE®) analysis, ELISA assay, or flow cytometry.

[0054] Additional TIM-3 inhibitors that can be used in the methods described herein include MBG-453, TSR-022, TRL-6061, BGBA425, and LY-3321367, and antigen-binding fragments thereof, as well as any other TIM-3 inhibitors, e.g., antibodies, peptides, small molecules, and bispecific molecules such as bispecific antibodies (e.g., anti-TIM-3 / anti-PD-1 bispecific molecules).

[0055] As used herein, TIM-3 inhibitors include, but are not limited to, anti-TIM-3 antibodies, and antigen-binding portions thereof, and soluble TIM-3 polypeptides (e.g., TIM-3-Fc fusion proteins capable of binding to TIM-3 ligands).

[0056] Bispecific antibodies that specifically bind to PD-1 and LAG-3 include those described in WO2018 / 185043A1 (Roche), the contents of which are incorporated herein by reference.

[0057] In some embodiments, the administered CTLA-4 inhibitor is an anti-CTLA-4 antibody or antigen-binding fragment thereof, hi some embodiments, the administered CTLA-4 inhibitor comprises ipilimumab (e.g., YERVOY®), tremelimumab (ticilimumab, CP-675,206), AGEN-1884, ATOR-1015, antigen-binding fragments thereof, or combinations thereof.

[0058] In some embodiments, the anti-CTLA-4 antibody administered is a CTLA-4 binding protein or antigen-binding fragment thereof disclosed in U.S. Patent Nos. 5,811,097, 6,682,736, or 7,605,238, WO 2000 / 32231, 2000 / 37504, or WO 1997 / 20574 (each of which is incorporated by reference in its entirety). In some embodiments, the CTLA-4 inhibitor administered is an antibody or antigen-binding fragment of an antibody that specifically binds to human CTLA-4 and cross-competes with one or more of the antibodies listed above for binding to human CTLA-4. For example, in some embodiments, the administered CTLA-4 inhibitor is an antibody or antigen-binding fragment of an antibody that specifically binds to human CTLA-4 and cross-competes with a reference CTLA-4 binding antibody (e.g., ipilimumab or tremelimumab) for binding to human CTLA-4 in a standard CTLA-4 binding assay such as surface plasmon resonance (BIACORE®) analysis, ELISA assay, or flow cytometry (see, e.g., WO 2013 / 173223).

[0059] In some embodiments, the PD-1 inhibitor administered is an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the administered PD-1 inhibitor comprises pembrolizumab (e.g., KEYTRUDA®, MK-3475), pidilizumab (e.g., CT-011), nivolumab (e.g., OPDIVO®, BMS-936558), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), B7-DC Fc fusion protein), PF-06801591, BGB-A317, BI754091, JNJ-63723283, tislelizumab, Sym021, MDX-1106, MDX-1106-04, ONO-4538, SHR-1210, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the PD-1 inhibitor administered is human antibody 17D8, 2D3, 4H1, 4A11, 7D3, or 5F4, or an antigen (PD-1) binding fragment thereof, as described in U.S. Pat. No. 8,008,449.

[0060] In some embodiments, the anti-PD-1 antibody administered is a PD-1 binding protein or antigen-binding fragment thereof disclosed in U.S. Pat. Nos. 8,008,449, 8,354,509, 8,609,089, or 8,747,847, WO 2004 / 056875, 2009 / 114335, 2006 / 121168, 2009 / 101611, 2010 / 027827, 201 / 0027423, or 2011 / 066342 (each of which is incorporated by reference in its entirety).

[0061] In some embodiments, the administered PD-1 inhibitor is an antibody or antigen-binding fragment of an antibody that specifically binds to human PD-1 and cross-competes with one or more of the antibodies listed above for binding to human PD-1. For example, in some embodiments, the administered PD-1 inhibitor is an antibody or antigen-binding fragment of an antibody that specifically binds to human PD-1 and cross-competes with nivolumab for binding to human PD-1 (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105 and WO 2013 / 173223). The ability of antibodies and antigen-binding fragments to cross-compete for binding to an antigen indicates that these antibodies / fragments bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies / fragments are expected to have functional properties very similar to the antibodies (e.g., nivolumab) with which they compete by binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with a reference PD-1 binding antibody (e.g., nivolumab) in standard PD-1 binding assays such as surface plasmon resonance (BIACORE®) analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0062] In some embodiments, the anti-PD-1 antibody administered is nivolumab (e.g., OPDIVO®). In some embodiments, the anti-PD-1 antibody administered is pembrolizumab (e.g., KEYTRUDA®).

[0063] In some embodiments, the administered PD-L1 inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof. In some embodiments, the administered PD-L1 inhibitor comprises atezolizumab (e.g., TECENTRIQ®), RG7446, MPDL3280A, ATEZO®, R05541267), durvalumab (e.g., MEDI4736, IMFINZI®), BMS-936559, avelumab (e.g., BAVENCIO®), LY3300054, CS1001, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105, W243.55.S70, STI-A1014, YW243.55.570, MSB-0010718C, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the anti-PD-L1 antibody administered is a PD-L1 binding protein or antigen-binding fragment thereof disclosed in U.S. Patent Nos. 7,943,743, 8,217,149, 8,779,108, or 9,324,298, or WO 2013 / 079174 or WO 2016 / 061142, which are incorporated by reference in their entireties.

[0064] In some embodiments, the PD-L1 inhibitor administered is an antibody or antigen-binding fragment of an antibody that specifically binds to human PD-L1 and cross-competes with one or more of the antibodies listed above for binding to human PD-L1. For example, in some embodiments, the PD-L1 inhibitor administered is an antibody or antigen-binding fragment of an antibody that specifically binds to human PD-L1 and cross-competes with a reference PD-L1-binding antibody (e.g., atezolizumab, durvalumab, or avelumab) for binding to human PD-L1 in a standard PD-L1 binding assay, such as surface plasmon resonance (BIACORE®) analysis, ELISA assay, or flow cytometry (see, e.g., WO 2013 / 173223).

[0065] In some embodiments, the administered TIGIT inhibitor is an anti-TIGIT antibody or an antigen-binding fragment thereof. In some embodiments, the administered TIGIT inhibitor is BMS-986207, AB154, COM902 (CGEN-15137), or etigilimab (e.g., OMP-313M32), or an antigen (TIGIT)-binding fragment thereof. In some embodiments, the administered TIGIT inhibitor is the anti-TIGIT antibody MBSA43, MK-7684, AB154, MTIG7192A, MTIG7192A, or an antigen (TIGIT)-binding fragment thereof. In some embodiments, the administered anti-TIGIT antibody is BMS-986207 or an antigen-binding fragment thereof. In some embodiments, the administered anti-TIGIT antibody is OMP-313M32 or an antigen-binding fragment thereof. In some embodiments, the administered anti-TIGIT antibody is MTIG7192A or an antigen-binding fragment thereof. In some embodiments, the anti-TIGIT antibody administered is a TIGIT binding protein, or an antigen-binding fragment thereof, disclosed in International Publication Nos. 2016 / 011264, 2016 / 106302, 2016 / 191643, or 2017 / 053748 (each of which is incorporated by reference in its entirety).

[0066] In some embodiments, the administered TIGIT inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and cross-competes with one or more of the antibodies listed above for binding to human TIGIT. For example, in some embodiments, the administered TIGIT inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and cross-competes with reference TIGIT-binding antibodies (e.g., BMS-986207, OMP-313M32, and MTIG7192A) for binding to human TIGIT in a standard TIGIT binding assay, such as surface plasmon resonance (BIACORE®) analysis, ELISA assay, or flow cytometry.

[0067] In some embodiments, the administered inhibitor is a bispecific antibody that binds to at least one inhibitory immune checkpoint molecule. In some embodiments, the administered bispecific antibody binds to two different inhibitory immune checkpoint inhibitor molecules. In further embodiments, the administered bispecific antibody binds to PD-1 and LAG-3. In further embodiments, the administered bispecific antibody comprises antigen-binding fragments of nivolumab and leratolimab.

[0068] antibody In some embodiments, antibodies targeting immune checkpoint molecules described herein can be derived from specific murine heavy and light chain germline sequences and / or contain specific structural features, such as CDR regions containing specific amino acid sequences. Further provided are methods of making such antibodies, or immunoconjugates comprising such antibodies or antigen-binding fragments thereof, as well as pharmaceutical compositions formulated to contain the antibodies or fragments. Also provided herein are methods of using the antibodies to prevent and treat S. aureus infections. Accordingly, the antibodies described herein can be used therapeutically in a wide variety of therapeutic applications, including, for example, treating bacteremia, osteomyelitis, and sepsis.

[0069] Unless otherwise indicated or clear from the context, the term "antibody," as used herein, includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibody" refers, in one embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (herein referred to as V H In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region is composed of three domains: CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (herein abbreviated as V L The light chain constant region consists of one domain, C LIt consists of: V H and V L The region can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four framework regions (FRs) arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0070] Antibodies are typically -7 ~10 -11 The following dissociation constants (K D ) specifically bind to their cognate antigen with high affinity, reflected by a .about.10 -6 Any K greater than M D As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds with high affinity to the antigen and a substantially identical antigen, and -7 M or less, preferably 10 -8 M or less, and even more preferably 5×10 -9 M or less, and most preferably 10 -8 M~10 -10 K below M D"Substantially identical" means that an antibody has a high degree of sequence identity with a given antigen, but does not bind with high affinity to unrelated antigens. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity with the given antigen, e.g., if it exhibits at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% sequence identity with the sequence of the given antigen. By way of example, an antibody that specifically binds to a human immune checkpoint molecule may also cross-react with immune checkpoint molecules from certain non-human primate species (e.g., cynomolgus monkeys), but may not cross-react with immune checkpoint molecules from other species or antigens other than immune checkpoint molecules.

[0071] Unless otherwise specified, immunoglobulins can be from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as human IgG1, exist in several allotypes, which differ from each other by up to a few amino acids. Unless otherwise specified, "antibody" can include, by way of example, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, fully synthetic antibodies, and single-chain antibodies.

[0072] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding portion / fragment" of an antibody include: (i) Fab fragment - V L , V H , C L (ii) a F(ab')2 fragment—a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody L and VH Fv fragments consisting of domains, and (v) V H Examples of such fragments include dAb fragments consisting of a domain (Ward et al. (1989) Nature 341:544-546). An isolated complementarity-determining region (CDR), or a combination of two or more isolated CDRs joined by a synthetic linker, may comprise an antigen-binding domain of an antibody capable of binding to an antigen.

[0073] Single chain antibody constructs are also included in the present invention. Furthermore, the two domains of the Fv fragment, V L and V H are encoded by separate genes, but they can be synthesized using recombinant methods. L and V H The regions can be linked by synthetic linkers that allow them to be produced as a single paired protein chain, forming monovalent molecules known as single-chain Fvs (scFvs); see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., Proc Natl Acad Sci. (USA) 85, 5879-5883 (1988). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion / fragment" of an antibody. These and other potential constructs are described by Chan & Carter, Nat Rev Immunol 10, 301 (2010). These antibody fragments are routinely obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions / fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0074] The term "monoclonal antibody," as used herein, refers to an antibody displaying a single binding specificity and affinity for a particular epitope, or a composition of antibodies in which all of the antibodies display a single binding specificity and affinity for a particular epitope. Typically, such monoclonal antibodies are derived from a single cell or nucleic acid encoding the antibody and are propagated without the intentional introduction of any sequence changes. Thus, the term "human monoclonal antibody" refers to a monoclonal antibody having variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas obtained, for example, by fusing B cells obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome containing human heavy chain and light chain transgenes) with immortalized cells.

[0075] The term "recombinant human antibody," as used herein, includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from recombinant, recombinant human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences and are encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, e.g., Lonberg, Nature Biotech 23(9, 1117-1125 (2005)), the variable regions comprise antigen-binding domains encoded by different genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangement, the variable regions can be further modified by multiple single amino acid changes (termed somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant regions change in further response to antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid sequences encoding light and heavy chain immunoglobulin polypeptides in response to an antigen are not identical to the original germline sequences, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0076] The term "human" antibody refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0077] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody, e.g., a murine antibody, are replaced with corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not abolish the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0078] A "chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, such as an antibody whose variable region sequences are derived from a murine antibody and whose constant region sequences are derived from a human antibody. A "hybrid" antibody refers to an antibody with different types of heavy and light chains, such as a murine (parental) heavy chain and a humanized light chain, or vice versa.

[0079] An "isolated antibody," as used herein, refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to an immune checkpoint molecule (e.g., PD-1) is substantially free of antibodies that specifically bind to antigens other than the immune checkpoint molecule). However, an isolated antibody that specifically binds to an epitope of an immune checkpoint molecule may have cross-reactivity to other immune checkpoint molecules from different species.

[0080] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., huPD-1) to which an immunoglobulin or antibody specifically binds. Epitopes within protein antigens can be formed both from contiguous amino acids (usually linear epitopes) or from noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. The term "epitope mapping" refers to the process of identifying molecular determinants on an antigen involved in antibody-antigen recognition. Methods for routinely determining which epitope is bound by a given antibody are known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or contiguous peptides are tested for reactivity with a given antibody (e.g., an anti-PD-1 antibody), X-ray crystallography, two-dimensional nuclear magnetic resonance, yeast display, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0081] Also provided herein are "conservative sequence modifications" to the antibody sequences provided herein, i.e., nucleotide and amino acid sequence modifications that do not interfere with the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. For example, modifications can be routinely introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in an antibody can be replaced with another amino acid residue from the same side chain family. Methods for routinely identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are known in the art. See, for example, Brummell et al. Biochem 32, 1180-1187 (1993), Kobayashi et al. Protein Eng 12(10), 879-884 (1999), and Burks et al. Pro. Natl Acad Sci (USA) 94, 412-417 (1997).

[0082] antibody generation The various antibodies of the present invention can be routinely produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256, 495-497 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can also be used, such as viral or oncogenic transformation of B lymphocytes, and phage display techniques using libraries of human antibody genes.

[0083] The preferred animal system for preparing hybridomas is the mouse system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for routine isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0084] To generate hybridomas producing the monoclonal antibodies described herein, splenocytes and / or lymph node cells from mice immunized with immune checkpoint molecules or immunogenic fragments can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, a single-cell suspension of splenic lymphocytes from immunized mice can be fused with 50% PEG to Sp2 / 0 non-secreting mouse myeloma cells (ATCC, CRL1581). Cells can be plated at approximately 2 x 10 in a flat-bottom microtiter plate. 5Cells can be seeded at 1000 x 1000 cells / well and subsequently incubated for 2 weeks in selection medium containing 10% fetal bovine serum, 18% "653" conditioned medium, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin, and 1x HAT (Sigma). After approximately 2 weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium can be observed, usually after 10-14 days. Antibody-secreting hybridomas can be replated and screened again, and if necessary, monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

[0085] To purify monoclonal antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography using Protein A-Sepharose. The eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0086] Chimeric or humanized antibodies can be prepared based on the sequences of the murine monoclonal antibodies prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the desired murine hybridoma and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be routinely linked to human constant regions using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody, murine CDR regions can be inserted into a human framework using routine methods known in the art (see, e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0087] Human monoclonal antibodies directed against immune checkpoint molecules can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include those referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice." See, e.g., Lonberg, et al., Nature 368, 6474 (1994): 856-859; Lonberg, N., Handbook of Experimental Pharmacology 113, 49-101 (1994); Lonberg, N. and Huszar, D., Intern Rev Immunol 13, 65-93 (1995); and Harding, F. and Lonberg, N., Ann NY Acad Sci 764, 536-546 (1995)). The preparation and use of HuMab mice and the genomic modifications carried by such mice are described in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., Proc Natl Acad Sci (USA) 90, 3720-3724 (1993), Choi et al., Nature Genetics 4, 117-123 (1993), Chen, J. et al., EMBO J 12, 821-830 (1993), Tuaillon et al., J Immunol 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), and Fishwild, D. et al. Nature Biotechnology 14, 845-851 (1996), the contents of all of which are specifically incorporated herein by reference in their entireties.See also U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, all to Lonberg and Kay; U.S. Patent No. 5,545,807 to Surani et al.; WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962, all to Lonberg and Kay; and Korman et al. See International Publication No. WO 01 / 14424 to Ishida et al. In some embodiments, the antibodies described herein are raised using mice carrying human immunoglobulin sequences in a transgene and transchromosome, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice," are described in detail in International Publication No. WO 02 / 43478 to Ishida et al. Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-immune checkpoint molecule (e.g., anti-PD-1) antibodies described herein. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used, and such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

[0088] Alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to raise the antibodies described herein. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice," can be used; such mice are described in Tomizuka et al., Proc Natl Acad Sci (USA) 97, 722-727 (2000). Furthermore, cattle carrying human heavy and light chain transchromosomes have been reported in the art (Kuroiwa et al., Nature Biotechnology 20, 889-894 (2002)) and can be used to raise the antibodies described herein.

[0089] Additional mouse systems described in the art for raising human antibodies include (i) the VELOCIMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavy and light chain variable regions have been replaced, via homologous recombination, with human heavy and light chain variable regions operably linked to endogenous mouse constant regions, such that chimeric antibodies (human V / mouse C) are raised in the mouse and then converted to fully human antibodies using standard recombinant DNA techniques, and (ii) the MeMo® mouse (Merus Biopharmaceuticals, Inc.), in which the mouse contains unrearranged human heavy chain variable regions but a single rearranged human common light chain variable region. Such mice, and their use to raise antibodies, are described, for example, in WO 2009 / 15777, WO 2011 / 072204, WO 2011 / 097603, WO 2011 / 163311, WO 2011 / 163314, and WO 2012 / 148873, and U.S. Publication No. 2010 / 0069614.

[0090] Human monoclonal antibodies described herein can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. See, for example, U.S. Patent Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al., U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al., U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0091] The human monoclonal antibodies described herein can also be prepared using SCID mice into which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0092] Antibodies of the present invention can be routinely produced recombinantly in host cell transfections using a combination of recombinant DNA technology and gene transfection methods known in the art, for example.

[0093] For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). Using the light and heavy chain variable regions of the antibodies described herein, V H The segment is C in the vector H operably connected to the segment, V L The segment is C in the vector L Full-length antibody genes of any antibody isotype can be produced by inserting them operably linked to segments into an expression vector already encoding heavy and light chain constant regions of the desired isotype. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0094] In addition to the antibody chain genes, the recombinant expression vector can carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those skilled in the art will understand that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of the host cell to be transformed and the desired expression level of protein. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP) and polyoma). Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or β-globin promoter, can be used. Regulatory elements can also be composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al., Mol Cell Biol 8, 466-472 (1988)).

[0095] In addition to the antibody chain genes and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0096] For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, etc. While it is theoretically possible to express the antibodies described herein in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, most preferably mammalian host cells, is most preferred, as such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies. Prokaryotic expression of antibody genes has been reported to be ineffective for producing high yields of active antibody (Boss, MA and Wood, CR Immunology Today 6, 12-13 (1985)). The antibodies of the present invention can also be produced in glycoengineered strains of the yeast Pichia pastoris. Li et al. Nat Biotechnol 24, 210-215 (2006).

[0097] Preferred mammalian host cells for expressing the recombinant antibodies described herein include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells (described, for example, in Urlaub and Chasin, Proc Natl Acad Sci (USA) 77, 4216-4220 (1980)) using the DHFR selection marker (described, for example, in R.J. Kaufman and P.A. Sharp, Mol. Biol. 159, 601-621 (1982))), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 1987 / 04462, WO 1989 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells, or more preferably, to allow secretion of the antibody into the culture medium that the host cells are grown in. The antibody can be recovered from the culture medium using standard protein purification methods.

[0098] The N- and C-termini of antibody polypeptide chains of the present invention may differ from the expected sequences due to commonly observed post-translational modifications. For example, C-terminal lysine residues are often missing from antibody heavy chains. Dick et al. Biotechnol Bioeng 100, 1132 (2008). N-terminal glutamine residues, and to a lesser extent glutamic acid residues, are frequently converted to pyroglutamic acid residues in both the light and heavy chains of therapeutic antibodies. Dick et al. Biotechnol Bioeng 97:544 (2007); Liu et al. J Biol Chem 286, 11211 (2011).

[0099] composition In some embodiments, inhibitors of immune checkpoint molecules for use in the disclosed methods may be provided in a pharmaceutical composition, optionally formulated with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a therapeutic agent. The pharmaceutical composition may also be administered in combination therapy with, for example, an antibiotic or a vaccine. In some embodiments, the composition comprises the antibody at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 1-300 mg / ml, or 100-300 mg / ml.

[0100] In some embodiments, the therapeutic compositions disclosed herein can include other compounds, drugs, and / or agents used for the treatment of infections and / or associated conditions. Such compounds, drugs, and / or agents can include, for example, antibiotics.

[0101] Accordingly, any of the pharmaceutical compositions provided herein can further comprise one or more additional antimicrobial agents, non-limiting examples of which include linezolid, erythromycin, mupirocin, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalothin, cephalexin, cefracor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefotaxime ... Perazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telvancin, emifloxac, clindamycin, lincomycin, daptomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin Cillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin , temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamide chrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline.

[0102] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0103] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM, et al., J Pharm Sci 66, 1-19 (1977)). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphoric acid, as well as those derived from non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0104] In some embodiments, the pharmaceutical compositions can be in the form of a sterile aqueous solution or dispersion, or can be formulated in microemulsions, liposomes, or other ordered structures suitable to high drug concentration.

[0105] In some embodiments, inhibitors of inhibitory immune checkpoint molecules described herein can be administered as sustained-release formulations, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the inhibitor in the patient. For example, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over an extended period of time. In therapeutic applications, relatively high dosages at relatively short intervals may be required until the progression of the disease is reduced or terminated, or until the patient shows partial or complete remission of disease symptoms. A prophylactic regime can then be administered to the patient.

[0106] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration, but will generally be that amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of the active ingredient, in combination with a pharmaceutically acceptable carrier, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30%.

[0107] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. Alternatively, antibodies may be administered as sustained-release formulations, in which case less frequent administration is required. For administration of antibodies, the dosage ranges from about 0.0001 to 100 mg / kg, more usually 0.01 to 5 mg / kg, of the host body weight. For example, dosages may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or may be within the range of 1 to 10 mg / kg. Exemplary treatment regimes involve administration once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, or once every three to six months. In some embodiments, the dosage regimen of an antibody of the invention comprises 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration, with the antibody being administered using one of the following dosing schedules: (i) every four weeks for six doses, then every three months; (ii) every three weeks; or (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight every three weeks. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1 to 1000 μg / ml, and in some methods, about 25 to 300 μg / ml. A "therapeutically effective dosage" of an inhibitor may result in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or impairment due to the affliction of the disease.

[0108] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0109] Pharmaceutical compositions can be administered via medical devices such as needleless hypodermic infusion devices (e.g., U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556), microinfusion pumps (U.S. Pat. No. 4,487,603), transdermal devices (U.S. Pat. No. 4,486,194), infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224), and osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0110] In some embodiments, inhibitors of immune checkpoint molecules described herein can be formulated to ensure proper distribution in vivo, for example, they can be formulated in liposomes and can additionally contain targeting moieties that enhance selective delivery to particular cells or organs. For example, U.S. Patent Nos. 4,522,811, 5,374,548, 5,416,016, and 5,399,331; 153,1038-1044(1988), Bloeman et al.FEBS Lett 357,140-144(1995), M.Owais et al.Antimicrob Agents Chemother 39,180(1995), Briscoe et al.Am J Physiol 1233:134(1995), Schreier et al.J Biol Chem 269,9090-9098(1994), Keinanen and See Laukkanen FEBS Lett 346, 123-126 (1994), and Killion and Fidler Immunomethods 4, 273-279 (1994).

[0111] Methods for treating diseases or disorders caused by pathogenic bacteria or fungi, such as B. Saureus In another aspect, the disclosure also provides methods for preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or fungus, such as S. aureus, in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an inhibitor of an immune checkpoint molecule.

[0112] In some embodiments, the immune checkpoint molecule is selected from LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1.

[0113] In some embodiments, the inhibitor comprises an antibody or antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof.

[0114] In some embodiments, the inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof, hi some embodiments, the inhibitor comprises an anti-LAG-3 antibody.

[0115] In some embodiments, the inhibitor comprises (i) an anti-LAG-3 antibody or antigen-binding fragment thereof, (ii) a combination of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-LAG-3 antibody or antigen-binding fragment thereof, or (iii) a bispecific antibody that binds to PD-1 and LAG-3. In some embodiments, the anti-PD-1 antibody is nivolumab or the anti-LAG-3 antibody is leratolimab.

[0116] In some embodiments, the disease or disorder comprises a S. aureus infection. In some embodiments, the infection is a bone infection such as osteomyelitis. In some embodiments, the infection is a prosthetic joint infection, a fracture-related infection, a diabetic foot infection, hematogenous osteomyelitis, or a spinal infection.

[0117] In some embodiments, the infection is bacteremia, hi some embodiments, the disease or disorder comprises S. aureus-associated sepsis.

[0118] In some embodiments, the method further includes selecting a subject with a chronic or acute disease or disorder caused by S. aureus. In some embodiments, the subject is suffering from osteomyelitis. As used herein, the term "subject suffering from osteomyelitis" refers to a subject with one or more symptoms of osteomyelitis (e.g., including but not limited to, bone pain, bone tenderness, and swelling or warmth) or a positive diagnosis based on one or more diagnostic tests (e.g., including but not limited to, bone scan, blood culture, or culture of an infected lesion). As used herein, the term "subject suffering from osteomyelitis at a specific infection site" refers to a "subject suffering from osteomyelitis," where osteomyelitis is identified as being present in a specific bone or region of bone or several specific bones or regions of bone.

[0119] S. aureus infection In another aspect, the invention provides methods of treating a subject having or at risk of having an S. aureus infection and associated conditions (e.g., methicillin-resistant S. aureus (MRSA) infection, methicillin-susceptible S. aureus (MSSA), vancomycin-resistant S. aureus (VRSA), daptomycin-resistant S. aureus (DRSA), linezolid-resistant S. aureus (LRSA), and vancomycin-intermediate-susceptible S. aureus (VISA)), S. aureus bacteremia, S. aureus skin infection, S. aureus mastitis, S. aureus cellulitis or folliculitis, or an S. aureus-associated wound infection, abscess, osteomyelitis, endocarditis, pneumonia, septic shock, food poisoning, or toxic shock syndrome). The methods generally involve administering to a subject in need thereof (e.g., a human or another mammal, such as a cow, sheep, dog, cat, horse, goat, rabbit, pig, or bird), a therapeutically effective amount of a pharmaceutical composition described herein.

[0120] In some examples, the subject has been diagnosed or identified as having or at risk of having an S. aureus infection (e.g., an MRSA infection, an MSSA infection). Some embodiments further include (prior to the administering step) diagnosing, identifying, or selecting a subject as having or at risk of having an S. aureus infection (e.g., an MRSA infection, an MSSA infection). In some examples, the S. aureus infection is a hospital-acquired infection. In some examples, the subject has previously been treated with an antibacterial therapy, and the previous treatment was unsuccessful.

[0121] In some embodiments, prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, the subject: (i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) has been determined to have a serum titer of soluble TIM-3 (e.g., as determined by methods described herein or known in the art) of at least 2100, 2200, 2300, 2400, or 2500 pg / ml.

[0122] In some embodiments, prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, the subject: (a)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, or 2500 pg / ml; and (b)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0123] In some embodiments, prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, the subject: (i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) has been determined to have a serum titer of soluble LAG-3 (e.g., as determined by methods described herein or known in the art) of at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml.

[0124] In some embodiments, prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, the subject: (a)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml; and (b)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0125] In some embodiments, prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, the subject: (a)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, or 2500 pg / ml; and (b)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 of at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml; and / or (c)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0126] In some embodiments, the subject is diagnosed or identified as having or at risk of having a S. aureus infection or adverse outcome prior to surgery or receiving the implant, hi some embodiments, the subject is determined to have or be at risk of having a S. aureus infection or adverse outcome within 18 months, 12 months, 9 months, 6 months, 3 months, 1 month, 3 weeks, 2 weeks, or 1 week prior to surgery or receiving the implant.

[0127] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule and prior to surgery or receiving an implant, the subject: (iii) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (iv) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) has been determined to have a serum titer of soluble TIM-3 (e.g., as determined by methods described herein or known in the art) of at least 2100, 2200, 2300, 2400, or 2500 pg / ml.

[0128] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule and prior to surgery or receiving an implant, the subject: (a)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, or 2500 pg / ml; and (b)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0129] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule and prior to surgery or receiving an implant, the subject: (iii) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (iv) a serum titer of soluble LAG-3 that is higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of soluble LAG-3 (e.g., as determined by methods described herein or known in the art) of at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml.

[0130] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule and prior to surgery or receiving an implant, the subject: (a)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml; and (b)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0131] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule and prior to surgery or receiving an implant, the subject: (a)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, or 2500 pg / ml; and (b)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 of at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml; and / or (c)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0132] In some embodiments, the subject having or at risk of having an S. aureus infection has undergone surgery within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 2 years prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, hi some embodiments, the subject has undergone surgery within 6 months, 12 months, or 18 months prior to administration of the inhibitor of an inhibitory immune checkpoint molecule.

[0133] In some embodiments, the subject having or at risk of having an S. aureus infection receives the implant within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 2 years prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, hi some embodiments, the subject receives the implant within 6 months, 12 months, or 18 months prior to administration of the inhibitor of an inhibitory immune checkpoint molecule.

[0134] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule, and after surgery or receiving an implant, the subject: (i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) has been determined to have a serum titer of soluble TIM-3 (e.g., as determined by methods described herein or known in the art) of at least 2100, 2200, 2300, 2400, or 2500 pg / ml.

[0135] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule, and after surgery or receiving an implant, the subject: (a)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, or 2500 pg / ml; and (b)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0136] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule and after surgery or receiving an implant, the subject: (i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) has been determined to have a serum titer of soluble LAG-3 (e.g., as determined by methods described herein or known in the art) of at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml.

[0137] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule, and after surgery or receiving an implant, the subject: (a)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml; and (b)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0138] In some embodiments, prior to administration of an inhibitor of an inhibitory immune checkpoint molecule, and after surgery or receiving an implant, the subject: (a)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, or 2500 pg / ml; and (b)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 of at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml; and / or (c)(i) an elevated serum titer of CXCL13 compared to the subject's previously determined serum titer of CXCL13; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) has been determined to have a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

[0139] Bone infections, such as osteomyelitis, can be debilitating or even fatal and are notoriously difficult to treat. For example, osteomyelitis is an acute or chronic bone infection that can be caused by bacteria or fungi. The infection that causes osteomyelitis can begin elsewhere in the body and spread to the bone via the bloodstream. Infections can also spread to the bone from infected skin, muscle, or tendons next to the bone, as in osteomyelitis that occurs under chronic skin ulcers (sores). Bone infections can also begin after bone surgery, especially if the surgery is performed after an injury or if a metal rod or plate is placed in the bone. In children, long bones are usually affected. In adults, the feet, spine (vertebrae), and hip joints (pelvis) are most commonly affected.

[0140] As used herein, the term "osteomyelitis" refers to an infection or inflammation of bone. Osteomyelitis infections are generally caused by pathogenic microorganisms (e.g., bacteria or fungi). Osteomyelitis includes both acute and chronic (e.g., persistent) bone infections. It includes any inflammation of the bone marrow and adjacent bone. Often, the original site of infection is elsewhere in the body and spreads to the bone via the blood. Bones can become predisposed to infection due to recent minor trauma resulting in blood clots or hemostasis. In children, long bones are usually affected. In adults, the vertebrae, head, and pelvis are most commonly affected. Bacteria or fungi are common organisms, but either microorganism can contribute to the infection. Pus can form within the bone, leading to a bone abscess. The abscess then deprives the bone of its blood supply. Chronic osteomyelitis occurs when the causative microorganism becomes resistant to antimicrobial agents. This can occur due to the development of cellular mechanisms to evade antimicrobial agents, the formation of biofilms that prevent quiescent organisms from accessing antimicrobial agents, the death of bone tissue as a result of lost blood supply, and other mechanisms. Chronic infections can persist for several years with intermittent exacerbations. Risk factors for chronic infection are new trauma, diabetes, hemodialysis patients, IV drug abuse, and infections with organisms more adept at forming biofilms or developing antimicrobial resistance.

[0141] Bacteremia, also known as blood poisoning, occurs when S. aureus invades the bloodstream of mammals, including humans. Persistent fever is one symptom of bacteremia. The bacteria can migrate deep within the body, causing infections affecting internal organs such as the brain, heart, lungs, bones, and muscles, or surgically implanted devices such as artificial joints or cardiac pacemakers. One hallmark of S. aureus sepsis is bacterial aggregation and thromboembolic lesion formation, measured as bacterial colony-forming units (CFU) in the heart.

[0142] sepsis In some embodiments, methods are provided for treating or preventing pathogenic bacteria or fungi, such as S. aureus-associated sepsis, or reducing the severity of sepsis in a mammalian subject, suitably comprising administering to the subject an effective amount of an inhibitor of an immune checkpoint molecule as described herein.

[0143] "Sepsis," as used herein, is a condition characterized by a systemic inflammatory state triggered by an infection. The infection can be caused by bacteria (such as S. aureus), viruses, or fungi. The body can develop this inflammatory response through the immune system to microorganisms in the blood, urine, lungs, skin, or other tissues. The slang term for sepsis is blood poisoning, which is also used to describe toxemia. Toxemia refers to the presence of pathogenic organisms in the bloodstream and is a related medical term that leads to sepsis.

[0144] A method for preventing S. aureus-associated sepsis in a mammalian subject includes administering to the subject an effective amount of an inhibitor of an immune checkpoint molecule described herein prior to an infectious event. As used herein, an "infectious event" refers to an event during which a subject is exposed or may be exposed to an S. aureus infection. Exemplary infectious events include, but are not limited to, surgery on any part of the body, including the head, mouth, hands, arms, legs, torso, internal organs (e.g., heart, brain, intestines, kidneys, stomach, lungs, liver, spleen, pancreas, etc.), bones, and skin. Surgery provides conditions such as open surgical wounds or organs that can be easily infected by S. aureus. Additional infectious events include trauma to any part of the body that provides an open wound or other access to the bloodstream through which an S. aureus infection may enter the body. Additional infectious events include blood transfusions, injection of medicines or illicit or legal drugs, needlesticks, tattoo needles, insertion and maintenance of intravenous (IV) lines, insertion and maintenance of surgical drains, and sites of skin breakdown, e.g., pressure sores (decubitus ulcers). In embodiments in which the method provides for prevention of S. aureus-associated sepsis, the antibody or antigen-binding fragment thereof can be administered at least 1 hour before the infectious event, e.g., at least 12, 18, 24, 30, 36, or 48 hours before the infectious event.

[0145] The antibodies described above can be used alone or in combination with other therapeutic agents to treat SA infections and related conditions. For example, prosthetic joint infections (PJIs) are a common complication of elective total joint replacement (TJR) surgery, the majority of which are caused by Staphylococcal species. The 1-5% incidence of PJIs is known to be a non-random event, primarily determined by patient-specific factors. Furthermore, approximately 13% of patients infected with S. aureus develop sepsis and die from multiple organ failure, while other patients recover with little or no intervention.

[0146] In some embodiments, the subject is undergoing or about to undergo surgery or an implant, hi some embodiments, the surgery is selected from the group consisting of orthopedic surgery, cardiothoracic surgery, plastic surgery, neurosurgery, oral surgery, total joint replacement, open reduction and internal fixation (ORIF), debridement for an open fracture, spinal surgery, median sternotomy, revision total joint surgery, revision ORIF, drainage of a soft tissue abscess, or organ transplant surgery.

[0147] In some embodiments, the subject has undergone surgery within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 2 years prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, hi some embodiments, the subject has undergone surgery within 6 months, 12 months, or 18 months prior to administration of the inhibitor of an inhibitory immune checkpoint molecule.

[0148] In some embodiments, the subject has received or is about to receive an implant. As used herein, the term "implant" refers to any medical device (object) that is not living tissue and is intended for placement within the body of a subject (e.g., a mammal such as a human). Implants have uses including orthopedic applications, dental applications, ear, nose, and throat ("ENT") applications, neurosurgical applications, and cardiovascular applications.

[0149] In some embodiments, the subject receives the implant within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, or 2 years prior to administration of the inhibitor of an inhibitory immune checkpoint molecule, hi some embodiments, the subject receives the implant within 6 months, 12 months, or 18 months prior to administration of the inhibitor of an inhibitory immune checkpoint molecule.

[0150] In some embodiments, the subject has received an implant associated with an infection, hi some embodiments, the implant comprises a biofilm.

[0151] In some embodiments, the subject has received an implant and the subject has a bone infection. In further embodiments, the subject has received an implant and the subject has osteomyelitis.

[0152] In some embodiments, the implant is an orthopedic implant. As used herein, the term "orthopedic implant" refers to an implant that replaces or provides fixation to bone, replaces an articular surface for articulation, provides an abutment for a prosthetic device, or a combination thereof.

[0153] Implants can include any combination of artificial materials selected for the specific properties of their components. For example, a hip implant can include a combination of a metal shaft for weight bearing, a ceramic prosthesis, and a polymer adhesive to secure the structure to the surrounding bone. Implants can be entirely within the body or partially within and partially outside the body. Implants can be intended for short-term or long-term residence in their location. Implants can be made from a variety of biocompatible materials, including metals, ceramics, polymers, gels, and fluids not normally found in the human body. Examples of polymers useful for manufacturing medical devices / implants include polymers such as silicone, rubber, latex, plastics, thermoplastics, polyanhydrides, polyesters, polyorthoesters, polyamides, polyacrylonitrile, polyurethanes, polyethylene, polytetrafluoroethylene, polyethylene tetraphthalate, polyphazenes, and fluoroplastics. Medical devices can also be manufactured using certain naturally occurring or processed naturally occurring materials.

[0154] To reduce the severity of S. aureus-associated sepsis in a mammalian subject, an effective amount of an inhibitor of an immune checkpoint molecule described herein can be administered to a subject exhibiting symptoms of S. aureus-associated sepsis, which may include, for example, chills, confusion or delirium, fever or low body temperature (hypothermia), lightheadedness due to low blood pressure, rapid heartbeat, tremors, skin rash, and warm skin.

[0155] As used herein, "reducing the severity" of sepsis refers to reducing the symptoms exhibited by a subject with S. aureus-associated sepsis. In some embodiments, the symptoms are reduced by at least 30% (e.g., at least 40%, 50%, 60%, 70%, 80%, or 90%) compared to a subject also with S. aureus-associated sepsis, but the subject has not been administered the antibody or antigen-binding fragment thereof.

[0156] The antibodies or antigen-binding fragments thereof described herein can be administered at a suitable dosage and dosage regimen, which can depend on the disease or condition. An effective dosage can be identified by determining whether the dosage and dosage regimen enhances a therapeutic effect or therapeutic endpoint (e.g., prevention). Administration of an inhibitor of an immune checkpoint molecule can be provided in a single dose or over multiple doses spaced apart according to the desired effect and other clinical considerations.

[0157] Exemplary ways in which an antibody or antigen-binding fragment thereof may be administered to a subject in any of the various methods described herein include, but are not limited to, intravenous (IV), intratumoral (IT), intralesional (IL), aerosol, transdermal, endoscopic, topical, intramuscular (IM), intradermal (ID), intraocular (TO), intraperitoneal (IP), transdermal (TD), intranasal (IN), intracerebral (IC), intraorgan (e.g., intrahepatic), sustained-release implant, or subcutaneous administration, or administration using an osmotic pump or mechanical pump.

[0158] Combination therapy The compositions and related methods disclosed herein can also be used in combination with additional therapeutic agents or therapies, non-limiting examples of which include antibiotics such as streptomycin, ciprofloxacin, doxycycline, gentamicin, chloramphenicol, trimethoprim, sulfamethoxazole, ampicillin, tetracycline, vancomycin, linezolid, teicoplanin, rifampin, or various combinations of antibiotics.

[0159] Alternatively, the therapy can precede or follow the other drug treatment by intervals ranging from minutes to weeks. In embodiments in which the other drug and / or protein or polynucleotide are administered separately, it is generally possible to ensure that a significant period of time does not elapse between each delivery so that the therapeutic compositions can still exert their beneficial combined effect on the subject. In some embodiments, both modalities may be administered within about 12-24 hours of each other, or within about 6-12 hours of each other. In some embodiments, the period for administration may be significantly extended, up to several days (2, 3, 4, 5, 6, or 7) or weeks (1, 2, 3, 4, 5, 6, 7, or 8) between each administration. Administration of the antibiotic typically can be via any common route, including, but not limited to, oral, nasal, or buccal administration. Alternatively, administration can be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection.

[0160] In some embodiments, the additional therapeutic agent is a second inhibitor of a second immune checkpoint molecule, an antibiotic, or a combination thereof. In some embodiments, the inhibitor and the additional therapeutic agent are contained in the same composition. In some embodiments, the second immune checkpoint molecule is selected from LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1. In some embodiments, the second inhibitor is a second inhibitory immune checkpoint inhibitor such as an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the second inhibitor is an inhibitory immune checkpoint inhibitor such as an anti-TIGIT antibody, or an antigen-binding fragment thereof, or a combination thereof.

[0161] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a killer cell immunoglobulin-like receptor (KIR) inhibitor. In some embodiments, the KIR inhibitor is an anti-KIR antibody or antigen-binding fragment thereof. In some embodiments, the anti-KIR antibody is lirilumab (1-7F9, BMS-986015, IPH2101) or IPH4102.

[0162] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a LAG-3 inhibitor. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-LAG-3 antibody or antigen-binding fragment thereof. In some embodiments, the administered LAG-3 antibody is BMS986016, MK-4280 (28G-10), REGN3767, GSK2831781, IMP731 (H5L7BW), BAP050, IMP-701 (LAG-5250), TSR-033, LAG525, BI754111, Sym022, FS-118, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the anti-LAG-3 antibody administered is MK-4280, IMP-761, GSK2837781, MGD013, BAP050, or IBI110 IMP321, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the anti-LAG-3 antibody administered is leratolimab, or an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the administered anti-LAG-3 antibody is a antibody described in U.S. Pat. Nos. 9,244,059, 10,266,591, 9,908,936, 10,358,495, or 10,188,730, or in WO 2008 / 132601, 2010 / 019570, 2014 / 008218, 2014 / 008218, 2019 / 141092, The LAG-3 binding protein or antigen-binding fragment thereof is disclosed in US Pat. Nos. 2017 / 220555, 2017 / 219995, 2019 / 129137, 2018 / 069500, 2018 / 034227, 2014 / 008218, 2015 / 138920, or 2017 / 037203 (each of which is incorporated by reference in its entirety).

[0163] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a TIGIT inhibitor. In one embodiment, the TIGIT antagonist is an anti-TIGIT antibody or an antigen-binding fragment thereof. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is the anti-TIGIT antibody BMS-986207, AB154, COM902 (CGEN-15137), or OMP-313M32, or an antigen-binding fragment thereof. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is the anti-TIGIT antibody BMS-986207, AB154, COM902 (CGEN-15137), or etigilimab (e.g., OMP-313M32), or an antigen (TIGIT)-binding fragment thereof. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-TIGIT antibody MBSA43, MK-7684, AB154, MTIG7192A, MTIG7192A, or an antigen (TIGIT)-binding fragment thereof. In some embodiments, the administered anti-TIGIT antibody is BMS-986207, or an antigen-binding fragment thereof. In some embodiments, the administered anti-TIGIT antibody is OMP-313M32, or an antigen-binding fragment thereof. In some embodiments, the second administered anti-TIGIT antibody is MTIG7192A, or an antigen-binding fragment thereof. In some embodiments, the second administered anti-TIGIT antibody is a TIGIT binding protein, or an antigen-binding fragment thereof, disclosed in International Publication Nos. 2016 / 011264, 2016 / 106302, 2016 / 191643, or 2017 / 053748 (each of which is incorporated by reference in its entirety).

[0164] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a TIM-3 inhibitor. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-TIM-3 antibody or an antigen-binding fragment thereof. In some embodiments, the administered anti-TIM-3 antibody is TSR-022 or LY3321367, or an antigen-binding fragment thereof. In some embodiments, the second administered anti-TIM-3 antibody is an antigen-binding fragment of TSR-022 or LY3321367. In some embodiments, the administered TIM-3 inhibitor is MBG-453, TRL-6061, BGBA425, Sym023, INCAGN-2390, MBS-986258, RO-7121661, BC-3402, SHR-1702, or LY-3415244, or an antigen (TIM-3)-binding fragment thereof. In some embodiments, the administered TIM-3 inhibitor is antibody 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, or 8C4, or an antigen (TIM-3)-binding fragment thereof, as described in WO 2019 / 046321A1. In some embodiments, the administered anti-TIM-3 antibody is TSR-022 or LY3321367, or an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the administered anti-TIM-3 antibody is leratolimab, or an antigen-binding fragment thereof, or a combination thereof.In some embodiments, the anti-TIM-3 antibody administered is any of the antibodies described in WO 2011 / 155607, WO 2011 / 159877, WO 2013 / 006490, WO 2015 / 109931, WO 2015 / 117002, WO 2016 / 068803, WO 2016 / 068802, WO 2016 / 071448, WO 2016 / 111947, WO 2016 / 144803, WO 2016 / 161270, WO 2017 / 019897, WO 2017 / 031242, WO 2017 / 055399, WO 2017 / 061448 ... or 2019 / 0046321 , U.S. Patent Nos. 8,552,156 , 8,841,418 , or 9,163,087 , or Chinese Patent Application No. 2010 / 6632675 , each of which is incorporated herein by reference in its entirety.

[0165] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-CTLA-4 antibody or antigen-binding fragment thereof. In some embodiments, the administered CTLA-4 antibody is ipilimumab (e.g., YERVOY®), tremelimumab (ticilimumab, CP-675,206), AGEN-1884, ATOR-1015, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a CTLA-4 binding protein, or an antigen-binding fragment thereof, disclosed in U.S. Patent Nos. 5,811,097, 6,682,736, 7,605,238, WO 2000 / 32231, WO 2000 / 37504, and WO 1997 / 20574 (each incorporated by reference in its entirety). In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody administered is pembrolizumab (e.g., KEYTRUDA®, MK-3475), pidilizumab (e.g., CT-011), nivolumab (e.g., OPDIVO®, BMS-936558), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), B7-DC Fc fusion protein), PF-06801591, BGB-A317, BI754091, JNJ-63723283, tislelizumab, Sym021, MDX-1106, MDX-1106-04, ONO-4538, SHR-1210, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the PD-1 inhibitor administered is human antibody 17D8, 2D3, 4H1, 4A11, 7D3, or 5F4, or an antigen (PD-1) binding fragment thereof, as described in U.S. Pat. No. 8,008,449.In some embodiments, the anti-PD-1 antibody administered is a PD-1 binding protein or antigen-binding fragment thereof disclosed in U.S. Pat. Nos. 8,008,449, 8,354,509, 8,609,089, or 8,747,847; WO 2004 / 056875; WO 2009 / 114335; WO 2006 / 121168; WO 2009 / 101611; WO 2010 / 027827; WO 2010 / 027423; WO 2011 / 066342; or WO 2015112900 (each of which is incorporated by reference in its entirety).

[0166] In some embodiments, the administered anti-PD-1 antibody is nivolumab (e.g., OPDIVO®). In some embodiments, the administered anti-PD-1 antibody is pembrolizumab (e.g., KEYTRUDA®). In some embodiments, the second administered inhibitory immune checkpoint inhibitor is nivolumab. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is pembrolizumab.

[0167] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a PD-L1 inhibitor, hi some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody administered is atezolizumab (e.g., TECENTRIQ®), RG7446, MPDL3280A, ATEZO®, R05541267), durvalumab (e.g., MEDI4736, IMFINZI®), BMS-936559, avelumab (e.g., BAVENCIO®), LY3300054, CS1001, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105, W243.55.S70, STI-A1014, YW243.55.570, MSB-0010718C, an antigen-binding fragment thereof, or a combination thereof. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof disclosed in U.S. Patent Nos. 7,943,743, 8,217,149, 8,779,108, or 9,324,298, or WO 2013 / 079174 or WO 2016061142A1, which are incorporated by reference in their entireties.

[0168] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5) inhibitor. In some embodiments, the administered CEACAM inhibitor is an anti-CEACAM antibody or antigen-binding fragment thereof.

[0169] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a CEACAM1 inhibitor. In some embodiments, the administered CEACAM1 inhibitor is an anti-CEACAM1 antibody or an antigen-binding fragment thereof. In some embodiments, the administered anti-CEACAM1 antibody is CM-24 (MK-6018), or an antigen-binding fragment thereof.

[0170] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a CEA inhibitor. In one embodiment, the administered CEA inhibitor is an anti-CEA antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CEA antibody is sergutuzumab amnaleukin (RG7813, RO-6895882), RG7802 (RO6958688), an antigen-binding fragment thereof, or a combination thereof.

[0171] In some embodiments, the second inhibitory immune checkpoint inhibitor is an indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor. In other embodiments, the IDO1 inhibitor is indoximod (NLG8189, 1-methyl-D-TRP), epacadostat (INCB-024360, INCB-24360), KHK2455, PF-06840003, navoximod (RG6078, GDC-0919, NLG919), BMS-986205 (F001287), or a pyrrolidine-2,5-dione derivative.

[0172] In some embodiments, the second administered inhibitory immune checkpoint inhibitor is a CD80 inhibitor. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-CD80 antibody or an antigen-binding fragment thereof. In some embodiments, the administered anti-CD80 antibody is galiximab, AV1142742, an antigen-binding fragment thereof, or a combination thereof.

[0173] In some embodiments, the second inhibitory immune checkpoint inhibitor is an A2aR inhibitor. In some embodiments, the A2aR inhibitor is a small molecule. In some embodiments, the A2aR inhibitor is CPI-444, PBF-509, istradefylline (KW-6002), preladenant (SCH420814), tozadenant (SYN115), bipadenant (BIIB014), HTL-1071, ST1535, SCH412348, SCH442416, SCH58261, ZM241385, or AZD4635.

[0174] In some embodiments, the additional administered therapeutic agent is a chemokine inhibitor. In some embodiments, the chemokine inhibitor is an antibody or antigen-binding fragment thereof. In some embodiments, the second administered therapeutic agent is an inhibitor of CXCL13. In embodiments, the agent is an antibody or antigen-binding fragment thereof that binds to CXCL13.

[0175] In some embodiments, the additional administered therapeutic agent is a CD20 inhibitor. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-CD20 antibody or antigen-binding fragment thereof. In some embodiments, the administered anti-CD20 antibody is rituximab (e.g., RITUXAN®, IDEC-102, IDEC-C2B8), ABP798, ofatumumab, or obinutuzumab, an antigen-binding fragment thereof, or a combination thereof.

[0176] In some embodiments, the additional administered therapeutic agent is a GARP inhibitor. In some embodiments, the second administered inhibitory immune checkpoint inhibitor is an anti-GARP antibody or an antigen-binding fragment thereof. In some embodiments, the administered anti-GARP antibody is ARGX-115, or an antigen-binding fragment thereof.

[0177] In some embodiments, the additional administered therapeutic agent is a CD40 inhibitor. In some embodiments, the CD40 inhibitor is an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the administered anti-CD40 antibody is BMS3h-56, lucatumumab (HCD122 and CHIR-12.12), CHIR-5.9, dacetuzumab (huS2C6, PRO64553, RG3636, SGN14, SGN-40), an antigen-binding fragment thereof, or a combination thereof. In another embodiment, the CD40 inhibitor is a soluble CD40 ligand (CD40-L). In one embodiment, the soluble CD40 ligand is a fusion polypeptide. In one embodiment, the soluble CD40 ligand is CD40-L / FC2 or monomeric CD40-L.

[0178] In some embodiments, the additional therapeutic agent administered is a CD47 inhibitor. In some embodiments, the administered CD47 inhibitor is an anti-CD47 antibody or antigen-binding fragment thereof. In some embodiments, the administered anti-CD47 antibody is HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231, Effi-DEM, an antigen-binding fragment thereof, or a combination thereof.

[0179] In some embodiments, the additional administered therapeutic agent is a poliovirus receptor-related immunoglobulin domain-containing protein (PVRIG) inhibitor. In some embodiments, the administered PVRIG inhibitor is an anti-PVRIG antibody or an antigen-binding fragment thereof. In one embodiment, the administered anti-PVRIG antibody is COM701 (CGEN-15029), or an antigen-binding fragment thereof.

[0180] In some embodiments, the additional administered therapeutic agent is a stimulator of interferon genes (STING) inhibitor. In some embodiments, the STING inhibitor is a 2' or 3'-monofluoro substituted cyclic dinucleotide, a 2'3'-difluoro substituted mixed linkage 2',5'-3',5' cyclic dinucleotide, a 2'-fluoro substituted bis-3',5' cyclic dinucleotide, a 2',2''-diF-Rp,Rp,bis-3',5' cyclic dinucleotide, or a fluorinated cyclic dinucleotide.

[0181] In some embodiments, the administered antibiotic has antibacterial activity against S. aureus. Non-limiting examples of antibiotics include linezolid, erythromycin, mupirocin, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalothin, cephalexin, cefracor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefopenem, cefotaxime ... razone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telvancin, clindamycin, lincomycin, daptomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, naf Cillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, tema These include floxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamide chrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline.

[0182] In some embodiments, the additional administered therapeutic agent is an antibiotic selected from the group consisting of vancomycin, tobramycin, cefazolin, erythromycin, clindamycin, rifampin, gentamicin, fusidic acid, minocycline, cotrimoxazole, clindamycin, linezolid, quinupristin-dalfopristin, daptomycin, tigecycline, dalbavancin, telavancin, oritavancin, ceftobiprole, ceftaroline, iclaprim, carbapenem CS-023 / RO-4908463, and combinations thereof.

[0183] In some embodiments, the additional administered therapeutic agent is an immunotherapeutic agent, hi some embodiments, the immunotherapeutic agent is tefibazumab, BSYX-A1 10, Aurexis™, or a combination thereof.

[0184] Diagnosis or prognosis of pathogenic bacterial or fungal S. aureus infections The present disclosure also provides a method for the diagnosis or prognosis of a disease or disorder caused by a pathogenic bacterium or fungus, such as S. aureus, in a subject in need thereof. In some embodiments, the method includes: (a) determining the level of each of a set of biomarkers in a sample from the subject, where the set of biomarkers may include an immune checkpoint molecule or cytokine; (b) determining a change in the level of each of the set of biomarkers compared to a reference level for each of the set of biomarkers; and (c) assessing the presence of the disease or disorder or the state of the disease or disorder based on the change in the level of each of the set of biomarkers compared to the reference level for each of the set of biomarkers. In some embodiments, the sample from the subject is a serum sample. In some embodiments, the sample from the subject contains bone marrow cells. In some embodiments, the sample contains blood cells (e.g., peripheral blood mononuclear cells (PBMCs), neutrophils, metamyelocytes, monocytes, or T cells). In further embodiments, the sample contains T cells, e.g., Th1 / Th17 cells.

[0185] In some embodiments, the change in the level of each of the one or more biomarkers is an increase in the expression level of each of the one or more biomarkers compared to a reference level, hi some embodiments, subjects with acute S. aureus infection have increased expression levels of each of the one or more biomarkers compared to subjects with chronic S. aureus infection.

[0186] In some embodiments, the set of biomarkers comprises one or more of TIM-3, LAG-3, PD-1, CTLA-4, IFNγ, IL-2, TNFα, and IL-17.

[0187] In some embodiments, the set of biomarkers includes TIM-3. In some embodiments, the set of biomarkers includes TIM-3 and LAG-3. In some embodiments, the set of biomarkers includes TIM-3 and CTLA-4. In some embodiments, the set of biomarkers includes TIM-3 and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and CTLA-4. In some embodiments, the set of biomarkers includes TIM-3, CTLA-4, and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, CTLA-4, and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and PD-1. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and CTLA-4. In some embodiments, the set of biomarkers includes TIM-3, LAG-3, and PD-1. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, and PD-1.

[0188] In some embodiments, the set of biomarkers comprises TIM-3 and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, CTLA-4, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, CTLA-4, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, CTLA-4, PD-1, and IL-17. In some embodiments, the set of biomarkers comprises TIM-3, LAG-3, and CXCL13.

[0189] As used herein, the terms "increase," "elevate," "elevated," "enhance," and "activate" all generally refer to an increase by a statically significant amount compared to a reference level (e.g., a reference expression level or serum titer level in a healthy control subject). For the avoidance of doubt, these terms refer to an increase of at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) compared to the reference level, such as an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the reference level. In some embodiments, these terms refer to an increase of 10 to 20%, 10 to 30%, 10 to 40%, 10 to 50%, 10 to 60%, 10 to 70%, 10 to 80%, 10 to 90%, 10 to 100%, 10 to 110%, 10 to 120%, 10 to 130%, 10 to 140%, 10 to 150%, 10 to 160%, 10 to 170%, 10 to 180%, 10 to 190%, 10 to 200%, 10 to 210%, 10 to 220%, 10 to 230%, 10 to 240%, 10 to 250%, 10 to 260%, 10 to 270%, 10 to 280%, 10 to 290%, or 10 to 300% compared to a reference level. In some embodiments, these terms refer to 10-300%, 20-300%, 30-300%, 40-300%, 50-300%, 60-300%, 70-300%, 80-300%, 90-300%, 100-300%, 110-300%, 120-300%, 130-300%, 140-300%, 150-300%, 160-300%, 170-300%, 180-300%, 190-300%, 200-300%, 210-300%, 220-300%, 230-300%, 240-300%, 250-300%, 260-300%, 270-300%, 280-300%, 290-300%, 300-300%, 310-300%, 320-300%, 330-300%, 340-300%, 350-300%, 360-300%, 370-300%, 380-300%, 390-300%, 410-400%, 420-420%, 430-430%, 440-440%, 450-450%, 460-460%, 470-470%, 480-480%, 490-490%, 500-510%, 510-510%, 520-520%, 530-530%, 540-540%, 550-550%, 560-570%, 570-580%, 580-590 This refers to an increase of 0%, 150-300%, 160-300%, 170-300%, 180-300%, 190-300%, 200-300%, 210-300%, 220-300%, 230-300%, 240-300%, 250-300%, 260-300%, 270-300%, 280-300%, or 290-300%.In some embodiments, these terms refer to an increase of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold or more compared to a reference level.

[0190] The terms "reference level," "reference value," "predetermined value," and "predetermined level" are used interchangeably herein. An increased level of a biomarker compared to a predetermined reference value (e.g., expression level or serum titer level compared to a healthy subject) can be an increase of at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) compared to the predetermined reference value, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or any increase from 10% to 100% compared to the predetermined reference value, or at least a 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold increase, or any increase from 2-fold to 10-fold or more compared to the predetermined reference value.

[0191] In some embodiments, an increased level of a biomarker compared to a predetermined reference value (e.g., a reference value based on a healthy control subject) can be 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-110%, 10-120%, or greater than the predetermined reference value. , 10 to 130%, 10 to 140%, 10 to 150%, 10 to 160%, 10 to 170%, 10 to 180%, 10 to 190%, 10 to 200%, 10 to 210%, 10 to 220%, 10 to 230%, 10 to 240%, 10 to 250%, 10 to 260%, 10 to 270%, 10 to 280%, 10 to 290%, or 10 to 300% increase. In some embodiments, an increased level of a biomarker compared to a predetermined reference value can be 10-300%, 20-300%, 30-300%, 40-300%, 50-300%, 60-300%, 70-300%, 80-300%, 90-300%, 100-300%, 110-300%, 120-300%, 130-300%, 140-300%, 150-300%, 160-300%, 170-300%, 180-300%, 190-300%, 200-300%, 210-300%, 220-300%, 230-300%, 240-300%, 250-300%, 260-300%, 270-300%, 280-300%, 290-300%, 300-300%, 310-300%, 320-300%, 330-300%, 340-300%, 350-300%, 360-300%, 370-300%, 380-300%, 390-300%, 400-400%, 410-400%, 420-400%, 430-400%, 440-400%, 450-400%, 460-400%, 470-400%, 480-400%, 490-400%, 500-500%, 510-500%, 520-500%, 530-500%, 540-500%, 550-500%, 560-5 The increase may be 0%, 140-300%, 150-300%, 160-300%, 170-300%, 180-300%, 190-300%, 200-300%, 210-300%, 220-300%, 230-300%, 240-300%, 250-300%, 260-300%, 270-300%, 280-300%, or 290-300%. In some embodiments, an increased level of a biomarker compared to a predetermined reference value can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170%, 170-180%, 180-190%, 190-200%, 200-210%, 210-220%, 220-230%, 230-240%, 240-250%, 250-260%, 260-270%, 270-280%, 280-290%, 300-310%, 310-320%, 320-330%, 330-340%, 340-350%, 350-360%, 360-370%, 370-380%, 380-390%, 390-400%, 400-410%, 410-420%, 420-430%, 430-440%, 440-450%, 450-460%, 460-470%, 470-480%, 480-490%, 490-500%, 500-510%, 510-520%, 520-530%, 530-540%, 540-550%, 550-560%, 560-570%, 570-580%, 580- The increase may be 40-150%, 150-160%, 160-170%, 170-180%, 180-190%, 190-200%, 200-210%, 210-220%, 220-230%, 230-240%, 240-250%, 250-260%, 260-270%, 270-280%, 280-290%, or 290-300%.

[0192] As used herein, the term "higher" in reference to the measurement of a biomarker or biomarker complex refers to a statistically significant, measurable difference in the level of the measurement of the biomarker or biomarker complex compared to the level of another biomarker or biomarker complex, or a control level (e.g., the level of the biomarker or biomarker complex in a healthy control subject), where the measurement of the biomarker or biomarker complex is greater than the level of the other biomarker or biomarker complex or the control level. The difference is preferably at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%.

[0193] As used herein, the terms "reference level," "control level," "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refer to a level, standard, sample, cell, or tissue used for comparison purposes. The terms "reference level" and "control level" are used interchangeably herein. The terms "reference sample," "reference cell," "reference tissue," "control sample," "control cell," and "control tissue" are used interchangeably herein. In some embodiments, a reference level refers to the expression level or titer level of a biomarker or biomarker complex in a healthy control subject.

[0194] The methods described herein include subject prognosis, which may involve determining the severity of an S. aureus infection (e.g., sepsis) or determining a subject's risk for subsequent mortality, and risk assessment of a subject with an S. aureus infection (e.g., sepsis). In some embodiments, the methods involve determining the level (e.g., expression level) of one or more biomarkers (e.g., TIM-3, LAG-3, PD-1, CTLA-4, IFNγ, IL-2, TNFα, IL-17). In some embodiments, the methods involve determining the level of TIM-3 (e.g., TIM-3 expression in a PBMC sample or soluble TIM-3 in serum). In some embodiments, the methods involve determining the level of LAG-3 (e.g., LAG-3 expression in a PBMC sample or soluble LAG-3 in serum). In some embodiments, the methods involve determining the levels of TIM-3 and CXCL13 (e.g., in a serum sample). In some embodiments, the methods involve determining the levels of LAG-3 and CXCL13 (e.g., in a serum sample). In some embodiments, the methods include determining the levels of TIM-3, LAG-3, and CXCL13 (eg, in a serum sample).

[0195] These levels provide information regarding the severity and extent of infection in a subject. For example, as disclosed herein, osteomyelitis is associated with significantly elevated soluble LAG-3 serum titers.

[0196] These levels also provide information regarding the likelihood of a subject experiencing an adverse outcome, e.g., death, within a specific time period, e.g., 30 days, 60 days, 90 days, 6 months, 1 year, 2 years, 3 years, or 5 years. For example, as disclosed herein, elevated levels of soluble TIM-3 in the serum of a subject prior to surgery or an implant are significantly associated with adverse events and adverse outcomes in the subject after surgery and an implant. These levels also provide information regarding the severity of disease in the subject. In some embodiments, biomarker levels can be determined once, e.g., prior to an event suspected of S. aureus infection (e.g., TJR surgery). In some embodiments, biomarker levels are determined 2, 4, 6, 8, 12, 18, and / or 24 hours and / or 1-7 days after the infection event. If more than one level is determined, one can calculate a biomarker level that quantifies whether and by how much the level of one or more biomarkers (e.g., TIM-3, LAG-3, PD-1, CTLA-4, IFNγ, IL-2, TNFα, IL-17) in the subject is increased or decreased.

[0197] Evaluating the circulating levels of one or more biomarkers in a subject typically involves obtaining a tissue or biological sample, such as serum, plasma, or blood, from the subject. The tissue or biological sample from the subject can be obtained by any known means, including needle prick, needle biopsy, swab, etc. In one aspect, the biological sample is a blood sample, preferably a plasma or serum sample, obtained, for example, by venipuncture. In a further aspect, the biological sample is a serum sample. The biological sample may be, or may have been, stored or banked under suitable tissue preservation conditions.

[0198] The level of one or more biomarkers in a sample can be routinely determined by measuring the level of a polypeptide in the sample using methods known in the art and / or described herein, e.g., an immunoassay such as ELISA. The antibody being measured can include its antigen-binding fragment, its degradation product, and / or its enzymatic cleavage product. Alternatively, the level of mRNA encoding the corresponding antibody chain can be routinely measured using methods known in the art and / or described herein, e.g., by quantitative PCR or Northern blotting analysis.

[0199] In some embodiments, the method comprises determining the presence, concentration, or amount of one or more biomarkers. The presence, concentration, or amount of one or more biomarkers, or ratios thereof, in a biological sample can be routinely determined using any suitable assay known in the art. Examples include sandwich immunoassays (e.g., radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA)) or ELISA (e.g., Quantikine ELISA assay, R&D These immunoassays include, but are not limited to, immunoassays such as monoclonal sandwich immunoassays (including monoclonal sandwich immunoassays, including those from BioSystems, Minneapolis, Minn.), competitive inhibition immunoassays (e.g., forward and reverse), fluorescence polarization immunoassays (FPIA), enzyme-linked immunosorbent assay (EMIT), bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescence assays. In SELDI-based immunoassays, a capture reagent that specifically binds to an antibody (or fragment thereof) of interest is attached to the surface of a mass spectrometry probe, such as a preactivated ProteinChip array. Other suitable methods include, for example, mass spectrometry and immunoassays for angiopoietins. and immunohistochemistry (e.g., using sections from tissue biopsies) using antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) or fragments thereof. Other detection methods include those described in, for example, U.S. Patent Nos. 6,143,576, 6,113,855, 6,019,944, 5,985,579, 5,947,124, 5,939,272, 5,922,615, 5,885,527, 5,851,776, 5,824,799, 5,679,526, 5,525,524, and 5,480,792, each of which is incorporated herein by reference in its entirety.

[0200] Immobilized capture proteins (e.g., antibodies specific for immune checkpoint molecules) or fragments thereof can be incorporated into immunoassays. Capture proteins can be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as a microtiter well), or a piece of solid substrate material. Assay strips can be prepared by coating the capture proteins in an array onto a solid support. The strip can then be immersed in a biological test sample and then rapidly processed through washing and detection steps to generate a measurable signal, such as a color spot. Any solid support known in the art can be used, including, but not limited to, solid supports made from polymeric materials in the form of wells, tubes, or beads. Capture proteins or antibodies can be attached to the solid support by adsorption, covalent attachment using chemical coupling agents, or other means known in the art, provided that such attachment does not interfere with the binding capacity of the capture protein. Furthermore, if necessary, the solid support can be derivatized to allow reactivity with various functional groups on proteins. Such derivatization requires the use of specific coupling agents such as, but not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0201] Additional tests may be performed to determine the subject's actual condition. More aggressive treatment may be administered either before or after the additional tests. For example, in suspected cases of sepsis, the subject may be referred for more extensive imaging tests.

[0202] Relative to a predetermined value or level used to monitor disease progression and / or treatment, the amount / concentration of one or more biomarkers or ratios thereof can be "unchanged," "favorable" (or "favorably altered"), or "unfavorable" (or "unfavorably altered"). "Elevated" or "increased" refers to an amount or concentration or ratio in a test sample that is higher than a typical or normal level or range (e.g., a predetermined value / level) or higher than another reference level or range (e.g., an initial or baseline sample). "Decreased" or "decreased" refers to an amount or concentration or ratio in a test sample that is lower than a typical or normal level or range (e.g., a predetermined value / level) or lower than another reference level or range (e.g., an initial or baseline sample). The term "altered" refers to an amount, concentration, or ratio in a sample that has changed (increased or decreased) over a typical or normal level or range (e.g., a predetermined value / level) or over another reference value / level or range (e.g., an initial or baseline sample).

[0203] Typical or normal values, levels, or ranges for ratios are defined according to standard practice. A so-called altered level or change can be considered to occur when there is any net change compared to a typical or normal level or range, or a reference level or range, that cannot be explained by experimental error or sample variation. Thus, the level measured in a particular sample is compared to the level or range of levels determined in similar samples from so-called normal subjects. In this context, a "normal subject" is an individual without a detectable disease or disorder, and a "normal" (sometimes called "control") patient or population is, for example, one that does not exhibit a detectable disease or disorder, respectively. An analyte level is said to be "elevated" when the analyte is normally undetectable (e.g., normal levels are zero or within about the 25th to about 75th percentile of a normal population) but is detectable in a test sample, as well as when the analyte is present in a test sample at levels higher than normal. Accordingly, among other things, the present disclosure provides methods for screening for subjects with or at risk of having S. aureus infection (e.g., sepsis).

[0204] The information obtained from the above methods is useful for the prognosis, identification of progression, and clinical management of diseases and other adverse conditions affecting the health status of an individual subject. In some embodiments, the information is useful for the prognosis, identification of progression, and management of total joint replacement surgery and post-operative care. More specifically, the information assists clinicians in designing appropriate therapeutic regimens for treating or preventing conditions such as S. aureus osteomyelitis and associated sepsis or septic death in affected subjects.

[0205] As used herein, the term "prognosis" refers to the prediction of the likely course and outcome of a clinical condition or disease, including relapse and drug resistance, such as the likelihood of S. aureus osteomyelitis and associated sepsis or septic death or progression thereof. Prognosis is usually made by assessing disease factors or symptoms that indicate a favorable or unfavorable course or outcome of the disease.

[0206] The term "prediction" is used herein to refer to the likelihood that a patient will respond, either favorably or unfavorably, to a treatment, or the degree of such response, or the likelihood that a patient will survive after a particular period of treatment (such as TJR surgery). The predictive methods of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment modality for any particular patient. The predictive methods described herein are valuable tools for predicting whether a patient is likely to respond favorably to a treatment regimen (such as surgical intervention) or whether the patient is likely to survive long-term after surgery and / or completion of other treatment modalities.

[0207] The phrase "determining a prognosis," as used herein, refers to a process by which one of skill in the art can predict the course or outcome of a condition in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy. Instead, one of skill in the art will understand that the term "prognosis" refers to an increased likelihood that a particular course or outcome will occur, i.e., that the course or outcome is more likely to occur in patients who exhibit a given condition compared to those individuals who do not exhibit that condition.

[0208] As used herein, the terms "favorable prognosis" and "positive prognosis," or "unfavorable prognosis" and "negative prognosis," are relative terms for predicting the likely course and / or likely outcome of a condition or disease. A favorable or positive prognosis predicts a better outcome for a condition than an unfavorable or negative prognosis. In a general sense, a "favorable prognosis" is a relatively better outcome than many other possible prognoses that may be associated with a particular condition, while an unfavorable prognosis predicts a relatively worse outcome than many other possible prognoses that may be associated with a particular condition. Typical examples of a favorable or positive prognosis include a better-than-average cure rate and a lower tendency for osteomyelitis and associated sepsis or septic death. For example, a positive prognosis would indicate that a patient has a 50% chance of recovering from osteomyelitis and associated sepsis after treatment, while the average patient with the same condition has only a 25% chance of recovery.

[0209] Additional definitions To aid in understanding the detailed description of the compositions and methods according to the present disclosure, some explicit definitions are provided to facilitate a clear disclosure of the various aspects of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0210] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0211] The term "biomarker," as used herein, refers to an indicator, e.g., a predictive, diagnostic, and / or prognostic indicator, that can be detected in a sample. A biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by specific molecular, pathological, histological, and / or clinical characteristics, and / or may serve as an indicator of a particular cell type or state (e.g., epithelial, mesenchymal, etc.) and / or response to therapy. Biomarkers include, but are not limited to, polynucleotide (e.g., DNA and / or RNA), polynucleotide copy number changes (e.g., DNA copy number), polypeptide, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrate, and / or glycolipid-based molecular markers. A biomarker may be present in a sample obtained from a subject prior to the onset of a physiological or pathophysiological condition (e.g., primary cancer, metastatic cancer, etc.), including symptoms thereof (e.g., response to therapy). Thus, the presence of a biomarker in a sample obtained from a subject can indicate an increased risk of the subject developing a physiological or pathophysiological condition or symptom thereof. Alternatively and / or additionally, a biomarker may be normally expressed in an individual, but its expression may be altered (i.e., it is increased (upregulated, overexpressed) or decreased (downregulated, overexpressed)) prior to the onset of a physiological or pathophysiological condition, including symptoms thereof. Thus, changes in the level of a biomarker can indicate an increased risk of the subject developing a physiological or pathophysiological condition or symptom thereof. Alternatively, or in addition, changes in the level of a biomarker can reflect a change in a particular physiological or pathophysiological condition, or symptom thereof, in a subject, thereby allowing the nature (e.g., severity) of a physiological or pathophysiological condition, or symptom thereof, to be tracked over time.

[0212] In some embodiments, the level of a biomarker comprises the concentration of the biomarker, the expression level of the biomarker, or the activity of the biomarker.

[0213] The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a sample. "Expression" generally refers to the process by which information (e.g., genetically encoded and / or epigenetic) is converted into structures present and operating in a cell. Thus, as used herein, "expression" refers to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, a translated polypeptide, or a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide) are also considered to be expressed, regardless of whether they arise from a transcript produced by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide, e.g., proteolysis.

[0214] As used herein, the terms "reduce," "lowering," and "inhibiting" generally refer to a reduction by a statistically significant amount. However, for the avoidance of doubt, the terms "reduced," "reducing," "reducing," or "inhibiting" refer to a reduction of at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) compared to a reference level, including, for example, a reduction of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% reduction (e.g., a non-existent level compared to a reference sample), or any reduction between 10% and 100%. In some embodiments, these terms refer to a decrease of 10 to 20%, 10 to 30%, 10 to 40%, 10 to 50%, 10 to 60%, 10 to 70%, 10 to 80%, 10 to 90%, 10 to 100%, 10 to 110%, 10 to 120%, 10 to 130%, 10 to 140%, 10 to 150%, 10 to 160%, 10 to 170%, 10 to 180%, 10 to 190%, 10 to 200%, 10 to 210%, 10 to 220%, 10 to 230%, 10 to 240%, 10 to 250%, 10 to 260%, 10 to 270%, 10 to 280%, 10 to 290%, or 10 to 300% compared to a reference level. In some embodiments, these terms refer to 10-300%, 20-300%, 30-300%, 40-300%, 50-300%, 60-300%, 70-300%, 80-300%, 90-300%, 100-300%, 110-300%, 120-300%, 130-300%, 140-300%, 150-300%, 160-300%, 170-300%, 180-300%, 190-300%, 200-300%, 210-300%, 220-300%, 230-300%, 240-300%, 250-300%, 260-300%, 270-300%, 280-300%, 290-300%, 300-300%, 310-300%, 320-300%, 330-300%, 340-300%, 350-300%, 360-300%, 370-300%, 380-300%, 390-300%, 410-400%, 420-420%, 430-430%, 440-440%, 450-450%, 460-460%, 470-470%, 480-480%, 490-490%, 500-510%, 510-510%, 520-520%, 530-530%, 540-540%, 550-550%, 560-570%, 570-580%, 580-590 This refers to a decrease of 0%, 150-300%, 160-300%, 170-300%, 180-300%, 190-300%, 200-300%, 210-300%, 220-300%, 230-300%, 240-300%, 250-300%, 260-300%, 270-300%, 280-300%, or 290-300%.In some embodiments, these terms refer to 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170%, 170-180%, 180-190%, 190-200%, 200-210%, 210-220%, 220-230%, 230-240%, 240-250%, 250-260%, 260-270%, 270-280%, 280-290%, 290-300%, 30 This refers to a reduction of 50-160%, 160-170%, 170-180%, 180-190%, 190-200%, 200-210%, 210-220%, 220-230%, 230-240%, 240-250%, 250-260%, 260-270%, 270-280%, 280-290%, or 290-300%.

[0215] "Antigen" refers to a substance that elicits an immunological response or binds to the products of that response. The term "epitope" refers to the region of an antigen to which an antibody or T cell binds.

[0216] A "patient" or "subject" includes animals such as humans, cows, horses, sheep, lambs, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. The animals can be mammals, such as non-primates and primates (e.g., monkeys and humans). In one embodiment, the patient is a human, such as a human infant, child, adolescent, or adult. A "subject in need thereof" refers to a subject at risk for or suffering from a disease, disorder, or condition (e.g., a hyperproliferative disorder such as cancer, a chronic infection) that can be treated or ameliorated by a compound provided herein or a composition thereof. Subjects in need of administration of a therapeutic agent as described herein include subjects suspected of having cancer, subjects exhibiting existing cancer, subjects receiving a cancer vaccine, subjects suspected of being infected with an infectious pathogen, subjects exhibiting an infection or infectious disease, or subjects receiving a vaccine against an infectious pathogen. The subject can be any organism that can develop or be infected with cancer, such as a human, pet, livestock, show animal, zoo specimen, or other animal. For example, the subject can be a human, non-human primate, dog, cat, rabbit, horse, etc. In some embodiments, the subject in need is a human. In certain embodiments, the subject in need has a disease, such as cancer or a chronic infection, that is associated with immune resistance.

[0217] A "control subject" is a healthy subject, i.e., a subject without clinical signs or symptoms of sepsis. Preferably, the control subject is clinically evaluated for otherwise undetectable signs or symptoms of sepsis, which evaluation includes periodic laboratory testing. Preferably, the healthy control subject has a mean or average titer of soluble TIM-3 of at least 10, 50, or 100 subjects.

[0218] A "reference subject" or "reference population" defines the source of a reference standard. In one embodiment, the reference is a human subject or a population of subjects who have one or more clinical indicators of S. aureus infection but who did not develop S. aureus osteomyelitis or sepsis. In another embodiment, the reference is a human subject or a population of subjects who had S. aureus infection, S. aureus osteomyelitis, or sepsis but survived without treatment. In another embodiment, the reference is a human subject or a population of subjects who had S. aureus infection, S. aureus osteomyelitis, or sepsis but survived with treatment. The above reference subject or population can be used to obtain the first predetermined reference referred to above.

[0219] In another embodiment, the reference is a human subject or a population of subjects who had an S. aureus infection, S. aureus osteomyelitis, or sepsis, but did not survive without treatment. In another embodiment, the reference is a human subject or a population of subjects who had an S. aureus infection, S. aureus osteomyelitis, or sepsis, but did not survive even with treatment. These reference subjects or populations can be used to derive the second predetermined reference referred to above.

[0220] The terms "effective amount," "effective dose," or "effective dosage" are defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of functional impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dosage" of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of the disease, inhibits the onset or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the onset or recurrence of a disease can be routinely evaluated using a variety of methods known to skilled physicians, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0221] As used herein, "administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. In some embodiments, routes of administration for therapeutic agents (e.g., antibodies) described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, therapeutic agents described herein can be administered via parenteral routes, e.g., topical, epidermal, or mucosal routes of administration, e.g., intranasal, oral, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0222] The term "drug" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, antibody, protein, or portion thereof, e.g., a peptide), or an extract made from biological material such as a bacterial, plant, fungal, or animal (e.g., mammalian) cell or tissue. The activity of such drugs may make them suitable as "therapeutic agents," which are biologically, physiologically, or pharmacologically active substances or substances that act locally or systemically in a subject.

[0223] The terms "therapeutic agent," "therapeutic agent," or "therapeutic drug" are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. Beneficial effects include enabling a diagnostic determination; alleviating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or pathological condition.

[0224] The term "disease" as used herein is intended to be generally synonymous with, and used interchangeably with, the terms "disorder" and "condition" (in medical conditions), in that both reflect an abnormal condition of the human or animal body or parts thereof that impairs normal function, is typically manifested by clear signs and symptoms, and reduces the duration or quality of human or animal life.

[0225] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, or slowing the progression, onset, severity, or recurrence of symptoms, complications, conditions, or biochemical indicators associated with a disease, or preventing it. Prevention refers to administration to a subject not having a disease to prevent the disease from occurring or to minimize its effects if it does occur.

[0226] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the chance of developing a disorder or condition in a subject who does not have the disorder or condition but is at risk of or susceptible to developing the disorder or condition. The term includes preventing the spread of infection in a subject.

[0227] As used herein, the term "ameliorating" refers to alleviating the symptoms of a disease, disorder, or condition in a subject who already exhibits symptoms of the disease, disorder, and / or condition, i.e., causing regression of a disease, disorder, or condition that has already affected the subject.

[0228] "Combination" therapy, as used herein, unless otherwise clear from the context, is meant to encompass the administration of two or more therapeutic agents in a coordinated manner, including, but not limited to, simultaneous administration. Specifically, combination therapy encompasses both coadministration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent is in some way conditioned on the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a predetermined period of time. See, e.g., Kohrt et al., Blood 117, 2423 (2011).

[0229] The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of a composition calculated to produce the desired response discussed above in connection with its administration, i.e., the appropriate route and regimen. The amount administered, both according to number of treatments and unit dose, depends on the protection desired.

[0230] The terms "sample," "test sample," and "patient sample" may be used interchangeably herein. A sample can be a sample of serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells (e.g., antibody-producing cells), or tissue. Such samples can be used directly when obtained from a patient or can be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc. to alter the characteristics of the sample in some manner, as discussed herein or otherwise known in the art. The terms "sample" and "biological sample," as used herein, generally refer to biological material that is tested for and / or suspected of containing an analyte of interest, such as an antibody. A sample can be any tissue sample from a subject. A sample can include proteins from a subject. Any cell type, tissue, or bodily fluid can be utilized to obtain a sample. Such cell types, tissues, and fluids may include sections of tissue such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood (such as whole blood), plasma, serum, sputum, stool, tears, mucus, saliva, hair, skin, red blood cells, platelets, interstitial fluid, ocular lens fluid, cerebrospinal fluid, sweat, nasal fluid, synovial fluid, menstrual periods, amniotic fluid, semen, and the like. Cell types and tissues may also include lymph, ascites, gynecological fluid, urine, peritoneal fluid, cerebrospinal fluid, fluid collected by vaginal lavage, or fluid collected by vaginal flushing. While tissue or cell types may be provided by removing a sample of cells from an animal, it can also be achieved by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissue, such as that with a treatment history or outcome history, may also be used. Protein purification may not be required.

[0231] In some embodiments, the sample is serum. In some embodiments, the sample contains bone marrow cells. In some embodiments, the sample contains blood cells (e.g., peripheral blood mononuclear cells (PBMCs), neutrophils, metamyelocytes, monocytes, or T cells). In further embodiments, the sample contains T cells, e.g., Th1 / Th17 cells.

[0232] Doses are often expressed relative to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg, etc.) body weight," even if the term "body weight" is not explicitly mentioned.

[0233] As used herein, "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, such as in a test tube or reaction vessel, or in cell culture.

[0234] As used herein, "in vivo" refers to events that take place within a multicellular organism, such as a non-human animal.

[0235] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0236] As used herein, the words "including," "comprising," "containing," or "having," and their variations, are intended to encompass the subsequently listed items and equivalents thereof, as well as additional subject matter, unless otherwise stated.

[0237] As used herein, phrases such as "in one embodiment," "in various embodiments," and "in some embodiments" do not necessarily refer to the same embodiment, but may, unless the context indicates otherwise.

[0238] As used herein, the term "and / or" or " / " means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0239] The term "substantially" as used herein does not exclude "completely", e.g., a composition that is "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definitions of the present disclosure.

[0240] As used herein, the term "approximately" or "about," when applied to one or more subject values, refers to a value similar to the stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value in either direction (greater or less), unless otherwise stated or clear from the context (except when such number exceeds 100% of the possible values). Unless otherwise indicated herein, the term "about" is intended to include values, e.g., weight percent, that are close to the stated range and are equivalent in terms of the functionality of the individual component, composition, or embodiment.

[0241] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur where express disclosure or context clearly dictates otherwise.

[0242] As disclosed herein, several ranges of values ​​are provided. Unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value within that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the disclosure, subject to any specifically excluded limits in the specified range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0243] Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illustrate the invention and do not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0244] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. For any of the methods provided, the method steps may occur simultaneously or sequentially. When method steps occur sequentially, the steps may occur in either order unless otherwise stated. In cases where a method includes a combination of steps, each and every combination or subcombination of steps is encompassed within the scope of the present disclosure unless otherwise stated herein.

[0245] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety, to the extent not inconsistent with this disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present disclosure. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the publication dates of publications provided may be different from the actual publication dates, which may need to be independently confirmed.

[0246] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. [Example]

[0247] Example 1 S. aureus osteomyelitis is widely considered incurable due to the persistent biofilm formation and colonization of the osteocyte-lacunocanalicular network (OLCN) of cortical bone, and cannot be eradicated by standard treatments other than amputation (Masters, E.A. et al. Nat Rev Microbiol 20, 385-400 (2022)). However, it is also known that patients can recover from acute infection and live their entire lives with asymptomatic S. aureus osteomyelitis (Masters, E.A. et al. Bone Res 7, 20 (2019)). Unfortunately, the diagnostic methods currently available to guide this important decision are very limited, and the 2018 International Consensus Meeting on Musculoskeletal Infection concluded that the development of a functional definition of acute versus chronic osteomyelitis is a major priority in the field (Schwarz, EM et al. J Orthop Res 37, 997-1006 (2019)).

[0248] To this end, a preclinical natural history study evaluated transitions in host immunity and found an early, robust proinflammatory response during the acute phase of infection, transitioning from Th1 and Th17 to a suppressive Treg adaptive immune response over time (Sokhi, UK et al. J Bone Miner Res (2021)). Because S. aureus is a human-specific pathogen and the immune response to this pathogen differs between humans and animal models, a novel model (huNSG) was recently developed in which non-obese diabetic (NOD)-SCID IL2R gamma (null) (NSG) mice were engrafted with human hematopoietic stem cells (huNSG), and the fate of human immune cells in response to S. aureus implant-associated infection was examined over time (Muthukrishnan, G. et al. Front Immunol 12, 651-515 (2021)).

[0249] As demonstrated in this example, we developed a humanized mouse model to examine the entire T cell repertoire during S. aureus bone infection. Humanized BLT (bone marrow, liver, thymus, BLT model) mice were generated by engrafting human hematopoietic stem cells (CD34+), autologous fetal liver, and thymus into transgenic NSG mice (NSG-SGM3) encoding human stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3), and subjected them to S. aureus transtibial osteomyelitis. The following results were observed:

[0250] First, S. aureus infection was much more severe in huNSG-SGM3 BLT mice compared with murinized NSG-SGM3 and C57BL6 (WT) mice (Fig. 1). Furthermore, huNSG-SGM3 BLT mice showed enhanced MRSA dissemination to distant organs, suggesting that these mice were septic to S. aureus osteomyelitis.

[0251] To this end, humanized NSG-SGM3 BLT mice were generated by engrafting CD34+ human hematopoietic cells, autologous human fetal liver, and thymus from three different human donors. Murinized NSG-SGM3 BLT mice were generated using CD34+ murine hematopoietic cells derived from three different C57BL / 6 WT mice. Twenty-week-old humanized NSG-SGM3 BLT mice, murinized NSG-SGM3 mice, and C57BL6 (WT) mice were subjected to transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 LAC::lux). As shown in Figures 1C and 1D, longitudinal assessment of in vivo S. aureus growth via bioluminescence imaging revealed increased in vivo S. aureus growth in humanized NSG-SGM3 BLT mice. Furthermore, as shown in Figure 1E, MRSA spread from the infection site to distal internal organs was observed. On postoperative day 14, huNSG-SGM3 BLT mice and control animals were euthanized, and ex vivo CFU quantification was performed on the implant, tibia, soft tissue surrounding the tibia, and internal organs (heart, liver, kidney, and spleen). As shown in Figures 1F-1I, CFU quantification revealed that huNSG-SGM3 BLT mice exhibited increased susceptibility and sepsis due to S. aureus osteomyelitis compared with murinized NSG-SGM3 and C57BL6 (WT) animals (n = 25; ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001).

[0252] After euthanasia, MRSA-infected and sterile implant-inserted tibiae from a subset of huNSG-SGM3 BLT, NSG-SGM3, and C57BL / 6 animals were demineralized, sectioned, and processed for histopathological analysis. Braun staining to identify bacteria revealed numerous SAC formations and increased SAC area across the tibia region in humanized BLT mice compared to control animals (n = 4-5, ANOVA, *p < 0.05). See Figures 1J-1K.

[0253] Second, single-cell RNA-seq analysis of human T (CD3+) cell populations in the bone marrow of huNSG-SGM3 BLT mice revealed significant heterogeneity in gene expression (Figure 2A-B) and T cell population numbers (Figure 2C) between the sterile and infected surgery groups.

[0254] More specifically, bone marrow cells were isolated from the tibiae of humanized NSG-SGM3 BLT mice that underwent surgery with or without bioluminescent MRSA-contaminated transtibial implants on day 14 after surgery. Isolated bone marrow cells were FACS-sorted into human CD45+CD19+ B cells and CD45+CD3+ T cells. Equal proportions of B and T cells were subjected to sc-RNAseq and sc-TCR / BCR repertoire analysis. As shown in Figure 2B, UMAP plots of single-cell gene expression of T and B cells were generated for all approximately 30,000 BM cells from the tibiae of humanized NSG-SGM3 BLT mice. Figure 2C shows feature plots of the pan-T cell marker CD3E and the B cell marker CD19 in all integrated BM cells. As shown in Figures 2D and 2E, UMAP and DEG clustering analysis of hCD45+CD3+ T cells identified 24 T cell clusters. A bar plot showing the percentage of cell numbers in each cluster between the sterile sham-operated and infected implant groups was generated and is shown in Figure 2F. The number of Th1 / Th17 cells (clusters 8, 20) was found to be significantly increased in infected animals compared to sham-operated (sterile) animals.

[0255] Example 2 In this example, an assay was performed to examine immune checkpoint protein levels in mice, and immune checkpoint proteins were found to be elevated in CD4+ Th1 / Th17 cells in the tibiae of S. aureus-infected humanized BLT mice.

[0256] Briefly, Th1 / Th17 cells (clusters 8 and 20 from Figure 2D) were subjected to UMAP and differential gene expression (DEG) subclustering analysis. The analysis revealed seven clusters, as shown in Figure 3A. Bar plot analysis demonstrated that these cells were of the Th1 / Th17 phenotype (see Figure 3B). Some Th1 / Th17 clusters showed significantly increased expression of immune checkpoint molecules LAG3, TIM-3 (HAVCR2), and, to a lesser extent, other immunosuppressive genes such as CTLA-4 and TIGIT.

[0257] In Figure 3C, DEG analysis of transcription factors (TCF7, TOX1-2, EOMES, NR4A1), cytokines, and chemokines (IL-1, IL-17, CXCL13, CXCR5) associated with functional T cell exhaustion, long-term antigen stimulation (CD40L), and proliferation (MKi67) is shown. Lower expression of TCF7, MKi67, IL-1, and IL-17 genes, and higher expression of CXCL13 and TOX2, indicate transcriptional reprogramming of these cells to a terminally functionally exhausted state (*p<0.05).

[0258] Immunofluorescence analysis of tibia sections from sham-operated controls and humanized BLT mice demonstrated high accumulation of LAG3+, TIM-3+, and / or PD-1+ T cells near the site of infection in MRSA-infected BLT mice (see Figures 3D and 3E). In contrast, human T cell accumulation was poor in uninfected BLT mice, with minimal evidence of depletion observed near the site of infection.

[0259] A multicolor spectroscopic flow cytometry assay was developed, optimized, and performed on tibia bone marrow (BM) cells from uninfected and MRSA-infected BLT mice. Results are shown in Figure 3E. Live human CD45+ / CD3+ / T cells and their subpopulations (CD4+, CD8+, Tregs) were analyzed for immune checkpoint expression (LAG3, TIM-3, and PD-1) and proliferation (Ki67). The frequency of human CD3+CD4+ T cells expressing TIM-3, LAG3, and PD-1 in bone marrow cells from MRSA-infected BLT mice was found to be significantly higher than in controls (n = 4–8 mice, *p < 0.05, t-test).

[0260] Differentially expressed gene (DEG) analysis revealed that the immunosuppressive checkpoint signaling protein LAG-3 was significantly elevated in Th17 and proliferative Th17 cells in humanized mice infected with MRSA (Figure 3). LAG-3, along with PD-1, TIM-3, and CTLA-4, is also a well-known T cell depletion protein. These results suggest that human Th17 cell depletion may be responsible for increased susceptibility to osteomyelitis during the chronic phase of S. aureus infection.

[0261] We found that splenic and bone marrow CD4+ T cells expressing the TIM-3 and LAG3 checkpoint proteins exhibited reduced proliferative capacity due to S. aureus infection. Briefly, multicolor spectroscopic flow cytometry was performed on uninfected and MRSA-infected BLT mice (Figure 4A) spleen cells and (Figure 4B) tibial bone marrow cells using the protocol described herein. Subsequently, CD4+ TIM-3+ and CD4+ LAG3+ cells had significantly lower frequencies of proliferative Ki67+ cells in the spleen and tended to have lower amounts of proliferative Ki67+ cells in the bone marrow of infected BLT mice, suggesting functional exhaustion and dysfunction (n = 4–9 mice, *p < 0.05, ANOVA).

[0262] Most interestingly, serum LAG-3 levels were significantly elevated in patients with S. aureus osteomyelitis (Figure 5). Moderate trends toward elevated TIM-3 and CTLA-4 were also observed in infected patients. Multivariate logistic regression analysis, with risk characterized by odds ratios (ORs calculated per 10-fold increase in serum protein levels), revealed that TIM-3 levels were significantly associated with adverse outcomes, such as arthrodesis, reinfection, amputation, and septic death (OR = 485.1, 95% CI 2.49-94511.09, p = 0.02). Serum TIM-3, in combination with LAG-3, PD-1, and CTLA-4, highly predicted adverse outcomes in patients with osteomyelitis (AUC = 0.89, p < 0.00001).

[0263] Briefly, serum samples were collected from individuals undergoing total hip / knee arthroplasty (n = 15) and orthopedic patients with culture-confirmed S. aureus osteomyelitis at 1 year (n = 37; 12 were adverse events (AD), 11 were infection-controlled (IC), and 14 were inconclusive). Immune checkpoint proteins LAG3, TIM-3, CTLA-4, and PD-1, and cytokines (IFN-γ, IL-2, TNFα, IL-17A, and IL-17F) were assessed by multiplex Luminex assay. Results are shown in Figure 5, and data are expressed as mean + / - SEM for each experimental group. Receiver operating characteristic (ROC) curve analysis was performed using individual protein levels, alone or in combination, to distinguish between acute and chronic S. aureus infection (Figure 5B) and to generate area under the curve (AUC) for prognostic prediction of outcome (Figure 5C). Interestingly, no correlation was observed between immune checkpoint protein levels and clinical duration-based case classification of acute vs. chronic disease, whereas immune checkpoint proteins, especially TIM-3, were highly predictive of adverse events in these patients (*p<0.05, **p<0.01, ****p<0.00001).

[0264] These results suggest that blocking T cell depletion or suppressing immune checkpoint proteins can be used to treat patients with bone infections. For example, (1) anti-LAG-3 monoclonal antibody (40 mg / kg) or LAG-3 chemical inhibitor, and (2) anti-TIM-3 mAb (40 mg / kg) as monotherapy or in combination with other immune checkpoint blockade therapies, such as anti-PD-1 and anti-CTLA-4, may be useful in combating debilitating and incurable bone infections in patients. These immunotherapies may be prescribed as adjunctive therapy in conjunction with standard-of-care antibiotic treatment.

[0265] Example 3 In this example, an assay was performed to examine the efficacy of the immune checkpoint blockade drug OPDUALAG™, a cocktail of anti-PD-1 (nivolumab) and anti-LAG-3 (leratolimab) mAbs, in reducing S. aureus burden in humanized BLT mice.

[0266] Briefly, 20-week-old humanized NSG-SGM3 BLT mice were subjected to transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 LAC::lux). Mice were then treated with OPDUALAG™ or saline placebo control. Longitudinal weight measurements as a measure of morbidity and longitudinal bioluminescence imaging were performed on days 0, 1, 3, 5, 7, 10, and 14 to assess planktonic growth of MRSA. See Figure 6A. Mice were sacrificed on day 14, and infection severity as a measure of (1) SAC formation (via histology) and (2) ex vivo CFU quantification of infected tibia, blood, liver, kidney, heart, and spleen was assessed between OPDUALAG™ and saline placebo control-treated animals. Results are shown in Figures 6B and 6C.

[0267] As shown in Figure 6B, longitudinal BLI revealed significantly reduced in vivo S. aureus growth in humanized BLT mice treated with OPDUALAG™ (n = 3-4, ANOVA, *p < 0.05). On day 14 after surgery, terminal ex vivo CFU in the tibia and viscera (heart, liver, kidney, and spleen) revealed that OPDUALAG™-treated animals exhibited lower disease severity. As shown in Figure 6C, histopathology revealed significantly reduced SAC formation in OPDUALAG™-treated animals compared to controls.

[0268] Example 4 Similar assays were performed to examine the efficacy trend of the immune checkpoint blocker sabatolimab (anti-TIM-3) in reducing S. aureus burden resulting from implant-associated osteomyelitis, and the results are shown in Figures 7A, 7B, 7C, and 7D.

[0269] Briefly, 8-week-old C57BL / 6 mice were subjected to transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 Lac::lux). Mice were then treated with sabatolimab or a saline placebo control. The concentrations of anti-TIM-3 mAb used in the study are also outlined in Figure 7A. As shown in Figures 7B and 7C, longitudinal BLI revealed significantly reduced in vivo S. aureus growth in humanized BLT mice treated with sabatolimab (n = 9-10, ANOVA, *p < 0.05). In addition, on day 14 after surgery, terminal ex vivo CFU counts in the tibia and viscera (heart, liver, kidney, and spleen) revealed that sabatolimab-treated animals exhibited lower disease severity (n = 9-10, t-test, *p < 0.05).

[0270] The present disclosure is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such variations are intended to be included within the scope of the appended claims.

Claims

1. 1. A method for reducing the number of pathogenic bacterial or fungal cells in a subject in need thereof, the method comprising administering to the subject an effective amount of an inhibitor of an inhibitory immune checkpoint molecule.

2. 2. The method of claim 1, wherein the administered inhibitor inhibits an inhibitory immune checkpoint molecule selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1.

3. 3. The method of claim 1 or 2, wherein the administered inhibitor comprises an antibody or antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof.

4. 4. The method of any one of claims 1 to 3, wherein the administered inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an antigen-binding fragment thereof, or a combination thereof.

5. 5. The method of any one of claims 1-4, wherein the administered inhibitor comprises (i) an anti-LAG-3 antibody or antigen-binding fragment thereof, (ii) a combination of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-LAG-3 antibody or antigen-binding fragment thereof, or (iii) a bispecific antibody that binds to PD-1 and LAG-3.

6. 6. The method of claim 5, wherein the administered anti-PD-1 antibody is nivolumab and the anti-LAG-3 antibody is leratolimab, or the bispecific antibody comprises an antigen-binding fragment of nivolumab and an antigen-binding fragment of leratolimab.

7. 1. A method for diagnosing or prognosing a disease or disorder caused by a pathogenic bacterium or a pathogenic fungus in a subject in need thereof, comprising: determining a level of each of a set of biomarkers in a sample from the subject, wherein the set of biomarkers comprises an inhibitory immune checkpoint molecule or a cytokine; determining a change in the level of each of the set of biomarkers compared to a reference level for each of the set of biomarkers; assessing the presence of the disease or disorder or the status of the disease or disorder based on the change in the level of each of the set of biomarkers compared to the reference level for each of the set of biomarkers.

8. the set of biomarkers is (a) LAG-3, PD-1, CTLA-4, TIM-3, IFNγ, IL-2, TNFα, and IL-17, or (b) one or more of LAG-3, TIM-3, and CXCL13.

9. The set of biomarkers is (a) TIM-3, (b) TIM-3 and LAG-3, (c) TIM-3 and CTLA-4, (d) TIM-3 and PD-1, (e) TIM-3 and IL-17, (f) TIM-3, LAG-3, and PD-1, (g) TIM-3, LAG-3, and CTLA-4, (h) TIM-3, CTLA-4, and PD-1, (i) TIM-3, LAG-3, CTLA-4, and PD-1, (j) TIM-3, LAG-3, CTLA-4, and PD-1, 9. The method of claim 7 or 8, comprising (a) TIM-3, CTLA-4, and IL-17, (b) TIM-3, CTLA-4, and IL-17, (c) TIM-3, CTLA-4, and IL-17, (d) TIM-3, CTLA-4, and IL-17, (e) TIM-3, CTLA-4, PD-1, and IL-17, (f) TIM-3, CTLA-4, PD-1, and IL-17, or (g) TIM-3, LAG-3, CTLA-4, PD-1, and IL-17.

10. The method of any one of claims 7 to 9, wherein the set of biomarkers comprises TIM-3, LAG-3, and CXCL13.

11. The method of any one of claims 7 to 10, wherein the sample is a bone marrow or PBMC sample.

12. The method according to any one of claims 7 to 11, wherein the sample is a serum sample.

13. The method of any one of claims 7 to 12, wherein the change in the level of a set of biomarkers is an increase in the expression level of each of the biomarkers in the set.

14. 1. A method of preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or pathogenic fungus in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an inhibitor of an inhibitory immune checkpoint molecule.

15. 11. A method of preventing, treating, or ameliorating a disease or disorder caused by a pathogenic bacterium or pathogenic fungus in a subject in need thereof, said method comprising administering a therapeutically effective amount of an inhibitor of an inhibitory immune checkpoint molecule, wherein said subject has been determined to have a disease or disorder caused by said pathogenic bacterium or pathogenic fungus according to the method of any one of claims 7 to 10.

16. The method of any one of claims 7 to 15, wherein the disease or disorder comprises an infectious disease.

17. 17. The method of claim 16, wherein the infection is bacteremia, a bone infection, bone loss, osteomyelitis, biofilm formation, an antimicrobial-resistant infection, or sepsis.

18. 17. The method of claim 16, wherein the infection is a prosthetic joint infection, a fracture-related infection, a diabetic foot infection, hematogenous osteomyelitis, or a spinal infection.

19. 19. The method of any one of claims 14 to 18, wherein the administered inhibitor inhibits an inhibitory immune checkpoint molecule selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1.

20. 20. The method of any one of claims 14-19, wherein the administered inhibitor comprises an antibody or antigen-binding fragment thereof, a small molecule, a protein, a polypeptide, a peptide, a peptidomimetic, a nucleic acid, an antisense molecule, a ribozyme, an RNAi molecule, a lipid, a lipopeptide, a carbohydrate, or a combination thereof.

21. 21. The method of any one of claims 14-20, wherein the administered inhibitor comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an antigen-binding fragment thereof, or a combination thereof.

22. 22. The method of any one of claims 14-21, wherein the administered inhibitor comprises (i) an anti-LAG-3 antibody or antigen-binding fragment thereof, (ii) a combination of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-LAG-3 antibody or antigen-binding fragment thereof, or (iii) a bispecific antibody that binds to PD-1 and LAG-3.

23. 23. The method of claim 22, wherein the anti-PD-1 antibody is nivolumab and the anti-LAG-3 antibody is leratolimab, or the bispecific antibody is an anti-PD-1 and anti-LAG-3 bispecific antibody (e.g., a bispecific antibody comprising an antigen-binding fragment of nivolumab and an antigen-binding fragment of leratolimab).

24. The method of any one of claims 1 to 6 and 14 to 23, further comprising administering to the subject an additional therapeutic agent.

25. 25. The method of claim 24, wherein the additional therapeutic agent comprises a second inhibitor of a second inhibitory immune checkpoint molecule, an antibiotic, an anti-pathogen antibody specific for the pathogenic bacterium or pathogenic fungus, or a combination thereof.

26. 26. The method of claim 25, wherein the additionally administered therapeutic agent is one selected from the group consisting of: (1) an anti-pathogen antibody that specifically binds to Staphylococcus aureus; (2) a second inhibitory immune checkpoint molecule selected from the group consisting of LAG-3, TIM-3, CTLA-4, PD-1, and PD-L1; and (3) an antibiotic with antibacterial activity against Staphylococcus aureus, or a combination thereof.

27. 27. The method of claim 26, wherein the additional administered therapeutic agent comprises an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or a combination thereof.

28. 28. The method of any one of claims 24 to 27, wherein the additional therapeutic agent is administered simultaneously with, prior to, or after the inhibitor.

29. 30. The method of claim 24 or 28, wherein the inhibitor and the additional therapeutic agent are contained in the same composition.

30. 30. The method of any one of claims 24-29, wherein the inhibitor and / or the additional therapeutic agent is administered to the subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, sustained release, controlled release, delayed release, as a suppository, or sublingually.

31. The method of any one of claims 7 to 30, wherein the subject has undergone or is about to undergo surgery or an implant.

32. The method of any one of claims 7 to 31, wherein the subject is undergoing surgery.

33. The method of any one of claims 7 to 31, wherein the subject is about to undergo surgery.

34. 34. The method of any one of claims 31 to 33, wherein the surgery is selected from the group consisting of orthopedic surgery, cardiothoracic surgery, plastic surgery, neurosurgery, oral surgery, total joint replacement, open reduction and internal fixation (ORIF), debridement for an open fracture, spinal surgery, median sternotomy, revision total joint surgery, revision ORIF, drainage of a soft tissue abscess, or organ transplant surgery.

35. 35. The method of any one of claims 31, 32, or 34, wherein the subject has received an implant.

36. 34. The method of claim 31 or 33, wherein the subject is about to receive an implant.

37. 37. The method of any one of claims 31 to 36, wherein the subject has received or is about to receive an orthopedic implant.

38. 38. The method of any one of claims 1 to 37, wherein the bacterium is selected from the group consisting of Staphylococcus aureus, S. epidermidis, S. lugdunensis, Cutibacterium acnes, Group B Streptococcus, and Enterobacteria.

39. 39. The method of claim 38, wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA).

40. The method of any one of claims 1 to 39, wherein the subject is a mammal.

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

42. Prior to said administration of said inhibitor, said subject: (i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined TIM-3 serum titer; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, 2500, or 3000 pg / ml.

43. 43. The method of claim 42, wherein the subject is administered the inhibitor prior to surgery or prior to receiving an implant.

44. 43. The method of claim 42, wherein the subject is administered the inhibitor after surgery or after receiving an implant.

45. 45. The method of claim 43 or 44, wherein the subject has undergone surgery or an implant within 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 12 months, or 18 months after the administration of the inhibitor of the inhibitory immune checkpoint molecule.

46. 46. ​​The method of any one of claims 43 to 45, wherein the surgery is selected from the group consisting of orthopedic surgery, cardiothoracic surgery, plastic surgery, neurosurgery, oral surgery, total joint replacement, open reduction and internal fixation (ORIF), debridement for an open fracture, spinal surgery, median sternotomy, revision total joint surgery, revision ORIF, drainage of a soft tissue abscess, or organ transplant surgery.

47. The method of any one of claims 42-46, wherein the serum titer of soluble TIM-3 in the subject is determined to be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (2-fold) higher in the subject than in healthy control subjects.

48. Prior to said administration of said inhibitor, said subject: (i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml.

49. 48. The method of claim 47, wherein the serum titer of soluble LAG-3 in the subject is determined to be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (2-fold) higher in the subject than in healthy control subjects.

50. Prior to said administration of said inhibitor, said subject: (i) an elevated serum titer of CXCL13 compared to a previously determined serum titer of CXCL13 in said subject; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) a serum titer of CXCL13 that is at least 50, 55, 60, 65, 70, or 75 pg / ml.

51. Prior to said administration of said inhibitor, said subject: (a)(i) an elevated serum titer of CXCL13 compared to a previously determined serum titer of CXCL13 in said subject; (ii) a serum titer of CXCL13 higher than that in a healthy control subject; or (iii) a serum titer of CXCL13 of at least 50, 55, 60, 65, 70, or 75 pg / ml; and / or (b)(i) an elevated serum titer of soluble TIM-3 compared to the subject's previously determined serum titer of TIM-3; (ii) a serum titer of soluble TIM-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble TIM-3 that is at least 2100, 2200, 2300, 2400, 2500, or 3000 pg / ml, and / or (c)(i) an elevated serum titer of soluble LAG-3 compared to the subject's previously determined LAG-3 serum titer; (ii) a serum titer of soluble LAG-3 that is higher than that in healthy control subjects; or (iii) a serum titer of soluble LAG-3 that is at least 80,000, 85,000, 90,000, 95,000, or 100,000 pg / ml; or any combination of (a) to (c).

52. 52. The method of any one of claims 48 to 51, wherein the subject is administered the inhibitor before surgery or before receiving an implant.

53. 10. The method of any one of the preceding claims, wherein the administered LAG-3 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment of an antibody selected from the group consisting of BMS986016, MK-4280 (28G-10), REGN3767, GSK2831781, IMP731 (H5L7BW), BAP050, IMP-701 (LAG-5250), TSR-033, LAG525, BI754111, and FS-118.

54. 10. The method of any one of the preceding claims, wherein the administered TIM inhibitor is an antibody or antigen-binding fragment of an antibody selected from the group consisting of TSR-022, LY3321367, EPZ005687, and DZNep.

55. 10. The method of any one of the preceding claims, wherein the administered inhibitor further comprises a TIGIT inhibitor.

56. 56. The method of claim 55, wherein the administered inhibitor is a TIGIT antibody or an antigen-binding fragment thereof.

57. 57. The method of claim 56, wherein the administered TIGIT inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of BMS-986207, AB154, COM902 (CGEN-15137), or OMP-313M32.

58. 10. The method of any one of the preceding claims, wherein the administered PD-1 inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, nivolumab, PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS00I, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI754091, and SHR-1210.

59. 10. The method of any one of the preceding claims, wherein the administered PD-L1 inhibitor is an antibody or an antigen-binding fragment of an antibody selected from the group consisting of atezolizumab, durvalumab, BMS-936559, avelumab, LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MPDL3280A, MEDI4736, MSB0010718C, and MDX-1105.

60. 10. The method of any one of the preceding claims, wherein the administered inhibitor is a small molecule selected from the group consisting of BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, JQI, and I-BET151.

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