WO2020210650A1 - Neutralization of tgf-beta or alpha-v-beta-8 integrin for s. aureus infections - Google Patents

Abstract

The present disclosure is directed to methods of using compounds that neutralize TGF-Beta or Alpha-V-Beta-8 (e.g., anti-TGF-Beta or anti-Alpha-V-Beta-8 antibodies and antigen-binding fragments thereof) for the treatment or prevention of S. aureus infections in patients in need thereof. Such patients include patients with an increased incidence of severe bacterial infections, e.g., diabetic patients.

Description

NEUTRALIZATION OF TGF-BETA OR ALPHA-V-BETA-8 INTEGRIN FOR S.

A UREUS INFECTIONS

BACKGROUND

[0001] Staphylococcus aureus is a major bacterial pathogen responsible for causing many types of human disease. Infections caused by antimicrobial resistant (AMR) bacterial pathogens are an increasing threat to public health. The ongoing AMR epidemic has been fueled, in part, by empiric broad spectrum antibiotic therapy. This has led to the exploration of pathogen specific methods, including monoclonal antibodies (mAbs), to prevent or treat serious bacterial infections. Several monoclonal antibodies that bind to targets on the bacteria are currently in development for the prevention or treatment of antibiotic resistant bacterial infections (see, e.g., DiGiandomenico, A., and B.R. Sellman, Curr. Opin.

Microbiol., 27: 78-85 (2015)). Such passive immunization strategies have the potential to provide an immediate and potent immunoglobulin response against the target pathogen.

[0002] Diseases associated with chronic inflammation, such as diabetes, are linked to an increased risk of infection with opportunistic bacteria such as S. aureus. However, the mechanisms of this increased risk are not understood. Therefore, methods of protecting patients against S. aureus infections, especially those at increased risk, are needed.

BRIEF SUMMARY OF THE INVENTION

[0003] As demonstrated herein, low density neutrophils (LDNs) are elevated during S. aureus infections in diabetic hosts, and the release of pro-inflammatory NETs by LDNs is correlated with increased mortality. However, blocking steps in the LDN maturation pathway, for example using antibodies or antigen-binding fragments thereof that bind to TGFp or the integrin responsible for its activation, anb8, can block NET induction and prevent mortality increases associated with infection in the diabetic host.

[0004] Provided herein are methods of treating or preventing a Staphylococcus aureus (S. aureus) infection in a subject with diabetes comprising administering to the subject an antibody or antigen-binding fragment thereof that binds to anb8 integrin.

[0005] Provided herein are antibodies or antigen-binding fragments thereof that bind to anb8 integrin for use in treating or preventing a S. aureus infection in a subject with diabetes. [0006] Provided herein are uses of an antibody or antigen-binding fragment thereof that binds to anb8 integrin in the preparation of a medicament for treating or preventing a S. aureus infection in a subject with diabetes.

[0007] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment thereof that binds to anb8 integrin is a neutralizing antibody.

[0008] Provided herein are methods of treating or preventing a S. aureus infection in a subject with diabetes comprising administering to the subject an antibody or antigen-binding fragment thereof that binds to transforming growth factor-beta (b) (TORb).

[0009] Provided herein are antibodies or antigen-binding fragments thereof that bind to

TQRb for use in treating or preventing a S. aureus infection in a subject with diabetes.

[0010] Provided herein are uses of an antibody or antigen-binding fragment thereof that binds to TQRb in the preparation of a medicament for treating or preventing a S. aureus infection in a subject with diabetes.

[0011] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment thereof that binds to TϋRb comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of 1D11.16.8.

[0012] In certain instances of a method, antibody, or use provided herein the antibody or antigen-binding fragment thereof that binds TQRb comprises a VH comprising the amino acid sequence of the VH of 1D11.16.8.

[0013] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment thereof that binds TORb comprises a VL comprising the amino acid sequence of the VL of 1D11.16.8.

[0014] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment thereof that binds TQRb binds to the same TORb epitope as 1D11.16.8.

[0015] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment thereof that binds TQRb competitively inhibits binding of iDi i.16.8 to tsrb.

[0016] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment thereof that binds to TQRb is a neutralizing antibody.

[0017] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment thereof that binds to TGFB is 1D11.16.8. [0018] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment that binds to anb8 integrin or the antibody or antigen-binding fragment thereof that binds to TGFp further comprises a heavy chain constant region. In certain instances, the heavy chain constant region is selected from the group consisting of human immunoglobulin IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2 heavy chain constant regions. In certain instances, the heavy chain constant region is a human IgGi constant region.

[0019] In certain instances of a method, antibody, or use provided herein, the antibody or antigen-binding fragment that binds to anb8 integrin or the antibody or antigen-binding fragment thereof that binds to TGI^ further comprises a light chain constant region. In certain instances, the light chain constant region is selected from the group consisting of human immunoglobulin IgGK and IgG light chain constant regions. In certain instances, the light chain constant region is a human IgGK light chain constant region.

[0020] In certain instances of a method, antibody or use provided herein, the antibody or antigen-binding fragment that binds to anb8 integrin or the antibody or antigen-binding fragment thereof that binds to TGI^ is an IgG antibody or antigen-binding fragment thereof.

[0021] In certain instances of a method, antibody or use provided herein, the antibody or antigen-binding fragment that binds to anb8 integrin or the antibody or antigen-binding fragment thereof that binds to TGI^ is a monoclonal antibody or antigen-binding fragment thereof.

[0022] In certain instances of a method, antibody or use provided herein, the antibody or antigen-binding fragment that binds to anb8 integrin or the antibody or antigen-binding fragment thereof that binds to TGI^ is a full-length antibody.

[0023] In certain instances of a method, antibody or use provided herein, the antibody or antigen-binding fragment that binds to anb8 integrin or the antibody or antigen-binding fragment thereof that binds to TGI^ is an antigen-binding fragment. In certain instances, the antigen-binding fragment comprises a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide linked Fv, intrabody, IgGACH2, minibody, F(ab')3, tetrabody, triabody, diabody, DVD-Ig, Fcab, mAh², (scFv)2, or scFv-Fc.

[0024] In certain instances of a method, antibody or use provided herein, the antibody or antigen-binding fragment that binds to anb8 integrin or the antibody or antigen-binding fragment thereof that binds to TORb decreases low density neutrophils (LDNs) in the subject or prevents the increase of LDNs in the subject. [0025] In certain instances of a method, antibody or use provided herein, the antibody or antigen-binding fragment that binds to anb8 integrin or the antibody or antigen-binding fragment thereof that binds to TGFp decreases neutrophil extracellular traps (NETs) in the subject or prevents the increase of NETs in the subject.

[0026] Provided herein are methods of treating or preventing a Staphylococcus aureus ( S . aureus) infection in a subject with diabetes comprising administering to the subject a compound that neutralizes anb8 integrin.

[0027] Provided herein are compounds that neutralize anb8 integrin for use in treating or preventing a S. aureus infection in a subject with diabetes.

[0028] Provided herein are uses of a compound that neutralizes anb8 integrin in the

preparation of a medicament for treating or preventing a S. aureus infection in a subject with diabetes.

[0029] In certain instances of a method, compound, or use provided herein, the

compound is a polypeptide, an antibody or antigen-binding fragment thereof, a small molecule, an siRNA, an antisense oligonucleotide, or an aptamer.

[0030] Provided herein are methods of treating or preventing a S. aureus infection in a subject with diabetes comprising administering to the subject a compound that neutralizes tsrb

[0031] Provided herein are compounds that neutralizes TQRb for use in treating or

preventing a S. aureus infection in a subject with diabetes.

[0032] Provided herein are uses of compound that neutralizes TQRb in the preparation of a medicament for treating or preventing a S. aureus infection in a subject with diabetes.

[0033] In certain instances of a method, compound, or use provided herein, the

compound is a polypeptide, an antibody or antigen-binding fragment thereof, a small molecule, an siRNA, an antisense oligonucleotide, or an aptamer.

[0034] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is sepsis.

[0035] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is bacteremia.

[0036] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is pneumonia. [0037] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is ICU pneumonia.

[0038] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is a skin or soft tissue infection (SSTI).

[0039] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is a diabetic infection of the lower limbs.

[0040] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is a diabetic foot ulcer (DFU).

[0041] In certain instances of a method, antibody, compound, or use provided herein, the

DFU is uninfected. In certain instances, the DFU is infected. In certain instances, the DFU is a grade 1, 2 or 3 DFU.

[0042] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is a bone or joint infection.

[0043] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection is a joint infection, a device infection, a wound infection, a surgical site infection, or osteomyelitis.

[0044] In certain instances of a method, antibody, compound, or use provided herein, the subject is a surgical subject.

[0045] In certain instances of a method, antibody, compound, or use provided herein, the

S. aureus infection comprises antibiotic-resistant S. aureus.

[0046] In certain instances of a method, antibody, compound, or use provided herein, the subject is human.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0047] FIGURES 1A-G show that elevated glucose levels correlate with more severe S. aureus infections. (A and B) After infection with S. aureus , diabetic db/db (A) and STZ (B) mice had increased mortality as compared to non-diabetic controls. (C) After infection with S. aureus , diabetic db/db mice had similar levels of S. aureus in their kidneys as non-diabetic controls. (D) After infection with S. aureus , diabetic STZ mice had similar levels of S.

aureus in their kidneys as non-diabetic controls. (E, F, and G) Treatment with Rosiglitazone for 1 week prior to infection with S. aureus reduced circulating glucose (E) and increased survival (F), but did not affect the bacterial burden in the kidney (G). (See Example 1.) [0048] FIGURES 2A-D show that systemic infection of the diabetic host lead to an AT- dependent increase in circulating NETs. (A) After infection with S. aureus , ELISA detected increased serum NETs in diabetic mice as compared to non-diabetic controls. (B)

Neutralization of S. aureus alpha toxin (AT) with the anti-alpha toxin monoclonal antibody MEDI4893* significantly reduced the number of NE-DNA complexes in the serum 48 hours post-infection in diabetic mice. (C) After infection with S. aureus , Western blot showed increased citrinulated Histone H3 (H3cit) in diabetic mice as compared to non-diabetic controls. (D) Neutralization of S. aureus AT increased survival of diabetic mice infected with S. aureus. (See Example 2.)

[0049] FIGURES 3A-D show that diabetic db/db mice have increased low density

neutrophils (LDNs). (A) After infection with S. aureus , the amount of LDNs in the blood of infected diabetic db/db mice was significantly increased as compared to uninfected db/db mice or non-diabetic controls. (B) Treatment with Rosiglitazone for 1 week prior to infection with S. aureus reduced LDNs 48 hours post-infection. (C and D) Neutralization of S.

aureus AT prior to infection reduced LDNs (C) but did not affect overall numbers of neutrophils (D) in diabetic db/db mice. (See Example 3.)

[0050] FIGURE 4 shows that, after infection with S. aureus , diabetic STZ mice had increased low density neutrophils LDNs. (See Example 3.)

[0051] FIGURES 5A-D shows that delivery of a TGFP neutralizing antibody prior to infection is protective in diabetic mice (A) TGFP significantly increased the number of LDNs in diabetic db/db blood, but not in non-diabetic control blood. (B and C) Delivery of a TGFp neutralizing antibody provided prior to S. aureus infection reduced LDNs in blood (B), but did not affect the amount of bacteria in the kidney (C). (D) Delivery of a TGFP neutralizing antibody provided prior to infection increased survival of diabetic db/db mice. (See Example 4.)

[0052] FIGURES 6A-E show that blocking the anb6/8 pathway prior to infection is protective in diabetic mice. (A) b8 positive inflammatory monocytes and dendritic cells (DCs) increased in the livers of diabetic db/db mice, not C57BKS mice, following infection. (B) Integrin expression increased on the surface of monocytes, and the overall number of DCs (not the density of b8 on DCs) increased. (C) Neutralizing anb6/8 prior to infection decreased LDNs in the blood stream as compared to administration of an anti-a.nbό antibody or a control antibody (c-IgG). (D) Neutralizing aUbό/8 prior to infection did not affect the amount of bacteria in the kidney. (E) Neutralizing anb6/8 prior to infection increased survival as compared to administration of a control antibody (c-IgG). (See Example 4.)

[0053] FIGURES 7A-C show that AT influences activation of TGFB independently of anb8 expression on innate immune cells. (A) pSMAD levels were higher in the livers of infected diabetic mice as compared with naive diabetic mice and infected non-diabetic mice. (B) Neutralizing AT significantly reduced pSMAD levels in the liver. (C) Neutralizing AT did not alter the numbers of anb8 expressing innate immune cells. (See Example 5.)

[0054] FIGURES 8A-C show altered gene and protein expression in LDNs. (A) RNA expression in LDN and HDN. (B) Protein expression in LDN and HDN. (C) Relative pathway activity in LDN and HDN. (See Example 6.)

[0055] FIGURES 9A-9B, FIGURES 9C-9D and FIGURE 9E show that excessive

NET production results from increased PTEN levels in LDN. (A) GPCRs are upregulated in LDNs as compared to HDNs. (B) Schematic of the PTEN pathway. (C) Reduced Akt activation in LDNs is demonstrated by membrane associated Akt and phosphorylated Akt.

(D) Exposure of purified HDN and LDN to fMLP ex vivo only increases Akt activation minimally. (E) The small molecule PTEN inhibitor VO-OHpic increases survival of mice infected with S. aureus. (See Example 6.)

DETAILED DESCRIPTION OF THE INVENTION

[0056] The present disclosure provides methods of using compounds that neutralize

TORb or anb8, such as anti-TGFb or anti-aV]38 antibodies and antigen-binding fragments thereof (e.g., monoclonal antibodies and antigen-binding fragments thereof), for the treatment or prevention of S. aureus infections in patients in need thereof, including, e.g., diabetic patients.

I. Definitions

[0057] The use of the terms“a” and“an” and“the” and“at least one” and similar

referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0058] The use of the term“at least one” followed by a list of one or more items (for example,“at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context.

[0059] The terms“comprising,”“having,”“including,” and“containing” are to be

construed as open-ended terms (i.e., meaning“including, but not limited to,”) unless otherwise noted.

[0060] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0061] As used herein, the term "transforming growth factor-beta (b)" or "TGFP" refers to TGFp polypeptides including, but not limited to, native TGFp polypeptides and isoforms of TGFp polypeptides. “TGFP” encompasses full-length, unprocessed TGFp polypeptides as well as forms of TGFp polypeptides that result from processing within the cell. As used herein, the term "human TGFP" refers to a polypeptide comprising the amino acid sequence of

ALDTNY CF SSTEKNCCVRQL YIDFRKDLGWKWIHEPKGYHANF CLGPCP YIW SLDT QYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

(SEQ ID NO:4).

[0062] A compound that "neutralizes" TGFP is a compound that reduces TGFP

expression or activity. For example, compounds that neutralize TGF include compounds that reduce TGFP-induced phosphorylation of SMADs and/or reduce TGFB-l inhibition of IL-4- dependent proliferation, e.g., in the HT-2 mouse T cell line. Tsang, M. et al. Lymphokine Res. 9:607 (1990). In certain instances, a compound that neutralizes TGFP binds to TGFP protein. In certain instances, a compound that neutralizes TGFP binds to a polynucleotide encoding TGFp. A compound that neutralizes TGFP can be any class of compound including, e.g., a polypeptide, an antibody, a small molecule, an siRNA, an antisense oligonucleotide, or an aptamer.

[0063] As used herein, the terms "anb8 integrin," "anb8," and "anb8 integrin receptor" refer to a complex containing the b8 integrin subunit associated with the alpha-V (aV) subunit. The term "human anb8 integrin" refers to a complex containing the human b8 integrin subunit associated with the human (aV) subunit.

[0064] A compound that "neutralizes" anb8 is a compound that reduces anb8 activity, optionally by blocking binding of a ligand to anb8. For example, compounds that neutralize anb8 include compounds that reduce anb8 activation of TGFB. In certain instances, a compound that neutralizes anb8 binds to anb8 protein. In certain instances, a compound that neutralizes anb8 binds to a polynucleotide encoding anb8. A compound that neutralizes anb8 can be any class of compound including, e.g., a polypeptide, an antibody, a small molecule, an siRNA, an antisense oligonucleotide, or an aptamer.

[0065] As used herein, the term "human aV integrin" refers to a polypeptide comprising the amino acid sequence of:

MAFPPRRRLRLGPRGLPLLL SGLLLPLCRAFNLD VD SP AEYSGPEGS YF GF AVDFF VP S AS SRMFLL V GAPK ANTT QPGIVEGGQ VLKCD W S S TRRC QPIEFD AT GNRD Y AKDDP LEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYAP CRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPN VYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYI YDGKNMS SLYNFTGEQMAAYFGF S VAATDINGDD YADVFIGAPLFMDRGSDGKLQ EVGQ V S V SLQRASGDF QTTKLNGFEVF ARFGS AIAPLGDLDQDGFNDIAIAAPY GGE DKKGI V YIFN GRS T GLN A VP S QILEGQ W A ARSMPP SF GY SMKGATDIDKN GYPDLI V GAF GVDRAIL YRARP VIT VNAGLEVYP SILNQDNKT C SLPGT ALK VSCFNVRF CLK A DGKGVLPRKLNF QVELLLDKLKQKGAIRRALFL Y SRSPSHSKNMTISRGGLMQCEEL IAYLRDESEFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDCGE DN V CKPKLE V S VD SD QKKI YIGDDNPLTLI VK AQN Q GEGA YE AELI V SIPLQ ADFIGV VRNNEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSV KFDLQIQ S SNLFDK V SP VV SHKVDL AVL AAVEIRGV S SPDHVFLPIPNWEHKENPETE ED V GP V V QHI YELRNN GP S SF SK AMLHLQ WP YK YNNNTLL YILH YDIDGPMNCT SD MEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIHTLGCGVAQCLKIV CQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIEFPYKNLPIEDI TN STL VTTNVTW GIQP APMPVP VW VIIL AVL AGLLLL AVLVF VMYRMGFFKRVRPP QEEQEREQLQPHENGEGN SET (SEQ ID NO:5).

[0066] The polynucleotide sequence of human av integrin (CCDS 2292.1) can be found at accession number: NCBI Reference Sequence: NM_002210.4. [0067] As used herein, the term "human b8 integrin" refers to a polypeptide comprising the amino acid sequence of:

MCGS ALAFFT AAF VCLQNDRRGPASFLWAAWVF SLVLGLGQGEDNRC AS SNAASC ARCLALGPECGWCVQEDFISGGSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEIN T Q VTPGE V SIQLRPGAE ANFMLKVHPLKKYP VDL YYL VD V S ASME1NNIEKLN S VGN DLSRKMAFF SRDFRLGF GS YVDKTV SP YISIHPERIIdNQC SD YNLDCMPPHGYIHVLS LTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQ T SHL ALD SKL AGI VVPNDGN CHLKNN V Y VK S TTMEHP SLGQL SEKLIDNNFN1 VIF A V QGKQFFiWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQKLISEVKVQVENQVQGIY FNIT AICPDGSRKPGMEGCRNVT SNDEVLFNVT VTMKKCD VT GGKNY AIIKPIGFNET AKIHfflRNCSCQCEDNRGPKGKCVDETFLDSKCFQCDENKCFfFDEDQFSSESCKSFfK DQP V C SGRGV C VCGKC SCHKIKLGK VY GK Y CEKDDF SCP YHHGNLC AGHGECE AG RCQCFSGWEGDRCQCPSAAAQHCVNSKGQVCSGRGTCVCGRCECTDPRSIGRFCEH CPTCYTACKENWNCMQCLHPHNLSQAILDQCKTSCALMEQQHYVDQTSECFSSPSY LRIFFIIFIVTFLIGLLKVLIIRQ VILQWN SNKIKSS SDYRV S ASKKDKLILQ S VCTRAVT YRREKPEEIKMDISKLNAHETFRCNG (SEQ ID NO: 6)

[0068] The polynucleotide sequence of human b8 integrin can be found at accession number: NCBI Reference Sequence: NM_002214.2.

[0069] The term“antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term“antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three- dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc. [0070] The term“monoclonal antibodies,” as used herein, refers to antibodies that are produced by a single clone of B-cells and bind to the same epitope. In contrast, the term “polyclonal antibodies” refers to a population of antibodies that are produced by different B- cells and bind to different epitopes of the same antigen.

[0071] The term“antibody fragment” refers to a portion of an intact antibody. An

“antigen-binding fragment,”“antigen-binding domain,” or“antigen-binding region,” refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain the antigenic determining regions of an intact antibody (e.g., the complementarity determining regions (CDR)). Examples of antigen-binding fragments of antibodies include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies. An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0072] A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI , CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework regions, whose sequences are relatively conserved. The term“framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the hypervariable or complementary determining regions (CDRs). There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the b sheets that provide the structural framework of the variable region (see, e.g., C.A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). The three CDRs, known as CDR1, CDR2, and CDR3, form the“hypervariable region” of an antibody, which is responsible for antigen binding.

[0073] The terms“VL” and“VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0074] The terms“VH” and“VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody. [0075] The term“Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In certain aspects, CDRs can be determined according to the Kabat numbering system (see, e.g ., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0076] Chothia refers instead to the location of the structural loops (Chothia and Lesk, J.

Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35 A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

Loop Kahat AbM Chothia

LI L24-L34 1,24-1.34 L24-L34

1,2 L50-L56 1,50-1.56 I.,50-L56

L3 1, 89-1,97 L89-L97 L89-L97

HI H31-H35B H26-H35B H26-H32..34

(Kabat Numbering)

HI H31-H35 H26-H35 H26-H32

(Chothia Numbering)

[0077] As used herein, the term“constant region” or“constant domain” are

interchangeable and have its meaning common in the art. The constant region is an antibody portion, e.g ., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an

immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0078] As used herein, the term“heavy chain” when used in reference to an antibody can refer to any distinct type, e.g. , alpha (a), delta (d), epsilon (e), gamma (g), and mu (m), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGi, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In specific embodiments, the heavy chain is a human heavy chain.

[0079] As used herein, the term“light chain” when used in reference to an antibody can refer to any distinct type, e.g, kappa (K) or lambda (l) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

[0080] A“chimeric” antibody refers to an antibody or fragment thereof comprising both human and non-human regions. A“humanized” antibody is a antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat). A humanized antibody can comprise, one, two, or three CDRs obtained or derived from a non-human antibody. A fully human antibody does not contain any amino acid residues obtained or derived from a non-human animal. It will be appreciated that fully human and humanized antibodies carry a lower risk for inducing immune responses in humans than mouse or chimeric antibodies (see, e.g., Harding et al., mAbs , 2(3): 256-26 (2010)).

[0081] The anti-AT antibody "MEDI4893 " (also known as "suvratoxumab") is the half- life extended (YTE) version of MEDI4893 * or“LC10” described previously in International Patent Application Publications WO 2012/109285 and WO 2014/074540 (both of which are herein incorporated by reference in their entireties). MEDI4893 (or suvratoxumab) contains a heavy chain with the amino acid sequence set forth in SEQ ID NO: 1 and a light chain with the amino acid sequence set forth in SEQ ID NO:2. MED 14893 * contains a heavy chain with the amino acid sequence set forth in SEQ ID NO: 3 and a light chain with the amino acid sequence set forth in SEQ ID NO:2. The variable domains in each sequence are underlined, and the CDR sequences are shown in bold.

[0082] As used herein, an“epitope” is a term in the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non contiguous regions of a polypeptide or polypeptides (conformational, non-linear,

discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g ., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array -based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g, site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any of the known methods in the art (e.g, Giege R el al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251 : 6300-6303). Antibody/antigen-binding fragment thereof: antigen crystals can be studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see , e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,, · U.S.

2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al, (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art. See, e.g, Champe M et al, (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0083] An antibody that“binds to the same epitope” as a reference antibody refers to an antibody that binds to the same amino acid residues as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can determined by a hydrogen/deuterium exchange assay (see Coales et al. Rapid Commun. Mass Spectrom.

2009; 23: 639-647) or x-ray crystallography. [0084] As used herein, the terms“immunospecifically binds,”“immunospecifically recognizes,”“specifically binds,” and“specifically recognizes” are analogous terms in the context of antibodies or antigen-binding fragments thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain and that the binding entails some complementarity between the antigen binding domain and the epitope. Accordingly, for example, an antibody that“specifically binds” to a first integrin protein may also bind to other integrin proteins, but the extent of binding to an un-related, non-integrin protein is less than about 10% of the binding of the antibody to the first integrin as measured, e.g., by a radioimmunoassay (RIA), enzyme-linked

immunosorbent assay (ELISA), BiaCore or an octet binding assay.

[0085] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0086] The term“nucleic acid sequence” is intended to encompass a polymer of DNA or

RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms“nucleic acid” and“polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single- stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).

[0087] An S. aureus infection can occur, for example, as a skin or soft tissue infection

(SSTI) or bacteremia. S. aureus bacteria can travel through the bloodstream and infect a site in the body, resulting in pneumonia, ICU pneumonia, a diabetic infection of the lower limbs, diabetic foot ulcer (DFU), a bone or joint infection, a device infection, a wound infection, a surgical site infection, or osteomyelitis.

[0088] As used herein, the terms“treatment,”“treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. In one embodiment, the effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease.

[0089] A“therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., treat' ment of S. aureus infection). The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antigen-binding fragment to elicit a desired response in the individual.

[0090] A“prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of S. aureus infection or disease onset).

[0091] A subject "colonized" with S. aureus refers to a subject with S. aureus present in or on the body. Colonization can be determined, for example, by detecting S. aureus in a sample obtained from the subject. Infections that result from or that are attendant to the presence of S. aureus in or on the body of a subject show radiographic and/or clinical signs of the bacteria. Radiographic signs include, for example, X-rays showing infiltrates. Clinical signs include, for example, abnormal temperature, abnormal white blood cell count, cough, purulent sputum, bronchial breath sounds, dyspnea, tachypnea (respiratory rate > 30 breaths/minute), and/or hypoxemia. An S. aureus infection can occur, for example, as a skin or soft tissue infection (SSTI) or bacteremia. S. aureus bacteria can travel through the bloodstream and infect a site in the body, resulting in pneumonia, ICU pneumonia, a bone or joint infection, a device infection, a wound infection, a surgical site infection, or

osteomyelitis.

[0092] The terms“administer”,“administering”,“administration”, and the like, as used herein, refer to methods that may be used to enable delivery of a compound that neutralizes TGFp or anb8 to the desired site of biological action (e.g., intravenous administration). Administration techniques that can be employed with the agents and methods described herein are found in e.g. , Goodman and Gilman, The Pharmacological Basis of Therapeutics , current edition, Pergamon; and Remington’s, Pharmaceutical Sciences , current edition, Mack Publishing Co., Easton, Pa.

II. Compounds that Neutralize TGFfi or anb8 integrin

[0093] As provided herein, compounds that neutralize TGFB or anb8 integrin can be used to treat or prevent S. aureus infections. Accordingly, antibodies and antigen-binding fragments thereof (e.g., monoclonal antibodies and fragments) that bind to TGFB or anb8 integrin can be used to treat or prevent S. aureus infections.

[0094] The cytokine, transforming growth factor-beta (b), (TORb), is a multifunctional regulator that modulates cell proliferation, differentiation, apoptosis, adhesion and migration of various cell types. TORb induces the production of extracellular matrix (ECM) proteins and almost all cell types, e.g., activated T and B cells, hematopoietic cells, macrophages, dendritic cells, produce TGF-b and/or are sensitive to its effects. (S. Dennler et ak, 2002, ./. Leukoc. Biol ., 71 :731-740). TORb is a member of a diverse superfamily that includes greater than 30 related members in mammals, viz, 3 TORb isoforms, 4 activins, and over 20 Bone Morphogenic proteins (BMPs). The 3 mammalian isoforms of TORb (TORb I , TORb2 and TORb3) share 70-82% homology at the amino acid level and have qualitatively similar activities in different systems. The active form of TORb is a dimer stabilized by hydrophobic interactions, which are further strengthened by an intersubunit disulfide bridge, in most cases. The TORbI isoform is the most abundant isoform in renal cells.

[0095] The mechanism by which TORb initiates intracellular signaling at the cell

membrane is generally well understood. See, e.g., I. Loeffler and G. Wolf, 2013, Nephrol. Dial. Transplant, 29:i37-i45). The intracellular mediators of TORb signaling are called Smads, which act downstream of the type 1 TORb receptor, Tb11- 1 , and which are

categorized into three classes. The receptor-regulated Smads (R-Smads), e.g., Smadl,

Smad2, Smad3, Smad5 and Smad8, which are directly phosphorylated and activated by TbR- 1 (which is a transmembrane receptor serine/threonine kinase), form hetero-oligomeric complexes with a second class of Smad, the common mediator Smads (Co-Smads), e.g., Smad4. These Smad complexes translocate into the nucleus where they interact with site- specific DNA transcription factors and participate in the regulation of target genes. Smad2 and Smad3 respond to signaling by the TGF-b subfamily. A third identified class of Smads includes the inhibitory Smads Smad6 and Smad7, which antagonize the activity of the receptor-regulated Smads by physically interacting with the activated TbRI- I receptor and can prevent the docking and phosphorylation of the R-Smads. {Ibid.). By virtue of its pleiotropic effects, TGFp can also directly activate other signal transduction cascades, including MAPK pathways, such as Ras, Raf, Erk, JNK and p38, in addition to Smad-mediated transcription. Moreover, TGFp can activate the phosphatidylinositol-3 -kinase (PI-3K) cascade by phosphorylation of its effector Akt, as well as Rho-like GTPases, including RhoA, Rac and cdc42. {Ibid.).

[0096] Compounds that neutralize TGFp can reduce any or all of these activities of

TGFp.

[0097] TGF-b is synthesized by a number of renal cell types and exerts its biological (and pathophysiological) effects through the above-noted signaling pathways. TGF-b is upregulated in renal diseases and induces renal cells to produce extracellular matrix proteins, which leads to glomerulosclerosis and tubule-interstitial (TI) fibrosis, which is characterized as a progressive, detrimental connective tissue deposition on the kidney parenchyma and is a damaging process, leading to the deterioration of renal function. Different types of renal cells undergo different pathophysiological changes induced by the activity of TGF-b, leading to apoptosis, tissue hypertrophy and podocyte foot processes abnormalities, ultimately causing renal dysfunction. {Ibid).

[0098] Integrins are cell-surface glycoproteins that are the principal receptors used by mammalian cells to bind to the extracellular matrix and mediate cell-cell and cell- extracellular matrix interactions. They are heterodimers (having a and b subunits bound noncovalently to each other) and function as transmembrane linkers between the extracellular matrix and the actin cytoskeleton of cells. Integrin proteins do not function as a passive glue, but rather are dynamic molecules that mediate the transfer of information across the cell membrane in both directions. Integrin-mediated adhesion can be regulated in response to signals by clustering and conformational changes triggered at integrins’ cytoplasmic tails, which function as signal transducers to activate various intracellular signaling pathways when activated by ligand binding. In addition, integrin signaling controls cell survival, cell cycle progression, and differentiation. The regulation of integrin-mediated adhesion structures is critical for many forms of cell migration. Integrins also contribute to the pathogenesis of a diverse array of acquired and hereditary diseases. [0099] There are several members of the integrin family of proteins, some of which have widespread tissue distribution. About twenty-four different integrins are present in vertebrates; a single cell may express multiple different types of integrin receptors on its surface. Human integrin b8 subunit, which is encoded by the ITGB8 gene, has ligands that include fibronectin and the TGF-bI and TGF^2 isoforms. In combination with the MT1 matrix metalloproteinase (MMP), anb8 integrin (a heterodimer comprising an alpha-V (av) subunit associated with a beta-8 (b8) subunit as further described infra ) is expressed on the cell surface and interacts with and mediates the activation of latent TORb in the cell matrix. The MT1 protease cleaves latent TORb to release the mature, active TORb polypeptide. Reactive oxygen species, other proteases, inflammation and pH change have also been demonstrated to be responsible for release of active THRb.

[0100] Compounds that neutralize THRb can reduce any or all of these activities of

TϋRb.

[0101] Exemplary compounds that neutralize THRb are known in the art. For example,

Gramont et al. Immunology 6: 31257453 (2017), which is herein incorporated by reference in its entirety discusses antibodies (e.g., fresolimumab) and antisense oligonucleotides

(Trabedersen) that neutralize TORb. In addition, U.S. Published Application No.

2018/0127246), which is herein incorporated by reference in its entirety discusses imidazole and thiazole small molecule compounds that inhibit TORb. Furthermore, antibodies that bind to TORb such as clone 1D11.16.8 are commercially available from BioXcell®.

[0102] Exemplary compounds that neutralize anb8 integrin and antibodies that bind to anb8 integrin are known in the art. For example, antibodies that bind to anb8 integrin are disclosed in WO 2013/026004 (which is herein incorporated by reference in its entirety).

[0103] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g, Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al, (1997) J Mol Biol 273: 927-948; Chothia C et al, (1992) J Mol Biol 227: 799-817; Tramontano A et al, (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Typically, when using the Rabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-H1 loop when numbered using the Rabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Rabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0104] In certain embodiments, antibodies or antigen-binding fragments thereof comprise one or more CDRs, in which the Chothia and Rabat CDRs have the same amino acid sequence. In certain embodiments, antibodies and antigen-binding fragments thereof for use in the methods provided herein comprise combinations of Rabat CDRs and Chothia CDRs.

[0105] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al, (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0106] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al. , (1996) J Mol Biol 262: 732-745. See also , e.g ., Martin A.“Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Rontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0107] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers AbM

hypervariable regions which represent a compromise between the Rabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).

[0108] In another aspect, the antibody or antigen-binding fragment thereof (e.g.,

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

[0109] An antibody or antigen-binding fragment (e.g. monoclonal antibody or fragment) described herein can be, or can be obtained from, a human antibody, a humanized antibody, a non-human antibody, or a chimeric antibody. In one aspect, an antibody described herein, or antigen-binding fragment thereof, is a fully human antibody.

[0110] A human antibody, a non-human antibody, a chimeric antibody, or a humanized antibody can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinantly) and in vivo sources (e.g., rodents, human tonsils). Methods for generating antibodies are known in the art and are described in, for example, Kohler and Milstein, Eur. J. Immunol ., 5: 511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)). In certain embodiments, a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples of transgenic mice wherein endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). A humanized antibody can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, N.J. (2009)), including, e.g., grafting of non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36( 1): 25-34 (2005); and Hou et al., J. Biochem., 144(1): 115- 120 (2008)). In one embodiment, a humanized antibody can be produced using the methods described in, e.g., U.S. Patent Application Publication 2011/0287485 AL

[0111] An anti-TGFp antibody or antigen-binding fragment thereof or an anti-avP8

integrin antibody or antigen-binding fragment thereof for use in the methods provided herein can be a neutralizing antibody.

[0112] A compound that neutralizes TGFP or anb8 integrin, an anti-TGFp antibody or antigen-binding fragment thereof, or an anti-avP8 integrin antibody or antigen-binding fragment thereof for use in the methods provided herein can decrease low density neutrophils (LDNs) or prevent an increase of LDNs.

[0113] A compound that neutralizes TGFp or anb8 integrin, an anti-TGFp antibody or antigen-binding fragment thereof, or an anti-av^8 integrin antibody or antigen-binding fragment thereof for use in the methods provided herein can decrease neutrophil extracellular traps (NETs) or prevent an increase of NETs.

III. Pharmaceutical compositions and methods of using compounds that neutralize

TGFfi and anb8 integrin

[0114] This disclosure provides methods of administering compounds that neutralize

TGFB or anti-avP8 integrin, including e.g., anti-TGFB or anti-av^8 integrin antibodies and antigen-binding fragments thereof. The compound (e.g., antibody or antigen-binding fragment thereof) can be administered in a pharmaceutical composition.

[0115] A composition comprising a compound that neutralizes TGFB or anb8 integrin or an antibody or antigen-binding fragment thereof that binds to TGFB or anb8 integrin can be a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, such as a pharmaceutically acceptable (e.g., physiologically acceptable) carrier and the compound or antibody or antigen-binding fragment. Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition optionally can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0116] The composition desirably comprises the compound that neutralizes TGFB or anb8 integrin or antibody or antigen-binding fragment in an amount that is effective to treat and/or prevent a S. aureus infection, to decrease or prevent an increase in LDNs, and/or to decrease and/or prevent an increase in LDNs. To this end, the disclosed method comprises administering a therapeutically effective amount or prophylactically effective amount of compound that neutralizes TGFB or anb8 integrin or antibody or antigen-binding fragment thereof that binds to TGFB or anb8 integrin or a composition comprising the aforementioned compound or antibody or antigen-binding fragment thereof (including monoclonal antibodies or fragments).

[0117] The disclosure provides a method of treating or preventing a S. aureus infection in a subject (e.g., a human), which comprises administering the compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti-av^8 integrin antibody or antigen-binding fragment to a subject in need thereof, whereupon the S. aureus infection is treated or prevented in the subject. The disclosure also provides use of the compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti-av^8 integrin -binding antibody or antigen binding fragment, described herein, or the composition comprising the compounds or antibodies or fragments thereof described herein, in the manufacture of a medicament for treating or preventing a S. aureus infection.

[0118] As discussed herein, Staphylococcus aureus is a major human pathogen that

causes a wide range of clinical infections. S. aureus is a leading cause of bacteremia and infective endocarditis as well as osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections. Approximately 30% of the human population is colonized with S. aureus (Wertheim et al., Lancet Infect. Dis., 5: 751-762 (2005)). The symptoms of S. aureus skin infections include, for example, boils, cellulitis, and impetigo. S. aureus also may cause food poisoning, blood poisoning (also known as bacteremia), toxic shock syndrome, and septic arthritis. The epidemiology, pathophysiology, and clinical manifestations of S. aureus infections are described in detail in, e.g., Tong et al., Clin. Microbiol. Rev., 28(3): 603-661 (2015), and the genomes of several different S. aureus strains have been sequenced (see, e.g., GenB ank/EMBL Accession Nos. BX571856, BX571857, BX571858, FN433596,

FN433597, FN433598, HE681097, FR821777, FR821778, FR821779, and FR821780).

[0119] The disclosure also provides a method of treating a subject (e.g., a human)

colonized with S. aureus , which comprises administering the compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti-av^8 integrin antibody or antigen-binding fragment to the subject, wherein the administration decreases the incidence and/or severity of infection in the subject, decreases LDNs in the subject, prevents an increase of LDNs in the subject, decreases NETs in the subject, and/or prevents an increase of NETs in the subject.

[0120] The disclosure provides a method of decreasing LDNs in a subject (e.g., a

human), which comprises administering the compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti-av^8 integrin antibody or antigen-binding fragment to a subject in need thereof. The disclosure provides a method of preventing an increase of LDNs in a subject (e.g., a human), which comprises administering the compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti-av^8 integrin antibody or antigen-binding fragment to a subject in need thereof.

[0121] The disclosure provides a method of decreasing NETs in a subject (e.g., a human), which comprises administering the compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti-av^8 integrin antibody or antigen-binding fragment to a subject in need thereof. The disclosure provides a method of preventing an increase of NETs in a subject (e.g., a human), which comprises administering the compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti-av^8 integrin antibody or antigen-binding fragment to a subject in need thereof.

[0122] As discussed herein, the subject (e.g., human subject) can have diabetes.

[0123] Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients. For repeated administrations over several days or longer, depending on the condition, the treatment can be repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens can be useful and are within the scope of the present disclosure. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0124] The composition(s) comprising an effective amount of a compound that

neutralizes TGFB or anb8 integrin or an antibody or antigen-binding fragment thereof described herein can be administered to a subject, such as a human, using standard administration techniques, including intravenous, intraperitoneal, subcutaneous, and intramuscular administration routes. The composition may be suitable for parenteral administration. The term“parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In some embodiments, the composition is administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0125] The compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti- anb8 integrin antibody or antigen-binding fragment or composition comprising the same, can be administered alone or in combination with other drugs (e.g., as an adjuvant)

conventionally used for treating S. aureus infections. The composition comprising the compound that neutralizes TGFB or anb8 integrin or the anti-TGFB or anti-avp8 integrin antibody or antigen-binding fragment can be used in combination with, for example, one or more antibiotics, such as a penicillinase-resistant b-lactam antibiotic (e.g., oxacillin or flucloxacillin). Gentamicin can be used to treat serious infections, such as endocarditis. Most strains of S. aureus , however, are now resistant to penicillin, and two in 100 people carry methicillin-resistant strains of S. aureus (MRSA). MRSA infections typically are treated with vancomycin, and minor skin infections can be treated with triple antibiotic ointment.

[0126] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

Methods

In vivo Model of Systemic Infection

[0127] Frozen stock cultures of S. aureus USA300 strain SF8300 were thawed and

diluted to the appropriate inoculum in sterile PBS, pH 7.2 (Invitrogen) (Hua et al., Antimicrob Agents Chemother. 55:1108-17 (2014)). Specific-pathogen-free 7- to 8-week-old female BKS.Cg-Dok7<m>+/+Lepr,db>/J ( db/db ), C57BKS, C57BL/6J - STZ, and C57BL/6J mice (The Jackson Laboratory) were briefly anesthetized and maintained in 3% isoflurane (Butler Schein™ Animal Health) with oxygen at 3 L/min and infected intravenously. All bacterial suspensions were administered in 100 pL of PBS. In select experiments, neutralizing antibodies MEDI4893*, anti-aVp6/8, anti-a.nbό, c-IgG (Medlmmune antibodies), anti-TGFp (clone 1D11.16.8, BioXcell), or control mouse IgGl were administered (15 mg/kg) in 0.5 mL intraperitoneally (IP) 24 hours prior to infection. Rosiglitazone (Sigma- Aldrich) was administered (10 mg/kg) orally for 7 days. Mice were infected 24 hours following the final dose of rosiglitazone. Animals were euthanized with CO2 at the indicated time points, and blood, liver, or kidneys were collected for analysis. The bacterial load in kidneys was determined by plating serial dilutions on TSA.

NET ELISA

[0128] To measure NETs, a hybrid of 2 different ELISA kits were used. Plates were initially coated with anti-elastase capture antibody (R&D Systems). Fresh serum samples were added to the coated wells, then incubated, and washed. Next, anti-DNA-POD antibody (Roche) was used to detect DNA in the captured proteins in the wells. Plates were developed with ABTS solution and ABTS stop solution. Absorbances were measured at 405 nm on a plate reader using SoftMax Pro software.

HDN and LDN Purification

[0129] High and low density neutrophils (HDN and LDN) were isolated from whole

blood. Following sacrifice, blood was collected and layered over with histopaque 1077 (Sigma-Aldrich). Cells were separated by centrifugation (500g, 30 minutes). The lower fraction was treated with ACK lysis buffer (Thermo Fisher Scientific) to remove red blood cells from the high density neutrophils. The upper (PBMC) fraction was washed 2x with PBS, and low density neutrophils were isolated with the EasySep Mouse Neutrophil

Enrichment Kit (Stemcell Technologies). Purified cell populations were lysed for protein or RNA analysis.

Flow Cytometry

[0130] Either whole blood or purified low density cells, were washed twice in ice-cold

FACs buffer (PBS with 5% fetal bovine serum, and 0.1% sodium azide). Fc receptors were blocked with anti-mouse CD16/CD32 (eBioscience), and cells were stained with antibodies against mouse CD45 (PE conjugated, clone FA-11), CD1 lc (APC-Cy5.5 or FITC conjugated, clone N418), CD1 lb (BV605 conjugated, clone Ml/70), Ly6-G (BV421 or PE-Cy7 conjugated, clone 1 A8), and Ly6-C. Cells were imaged using the LSR II Flow Cytometer (BD Biosciences) and analyzed with FlowJo. A known concentration of counting beads (Bangs Laboratories) was added to each sample to calculate the number of cells.

Neutrophil Gene Expression

[0131] HDN and LDNs were purified as described above. Cells purified from 5 mice were pooled and treated as a single sample for analysis. Total RNA was extracted using RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol and treated with RNase- free DNase I to remove genomic DNA contamination. RNA was quantified using a

NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and the quality of RNA was assessed using the Agilent RNA ScreenTape assay in conjunction with a 4200 TapeStation system (Agilent Technologies). Only high-quality RNA samples with an RNA Integrity Number (RIN) greater than 9 were used for microarray hybridization.

[0132] RNA samples were analyzed as previously described (Cohen, T. S. et al ., Cell Rep

22 2431-2441 (2018)). Briefly, RNA was amplified and labeled using MessageAmp Premier RNA Amplification Kit (Thermo Fisher Scientific). Total RNA was reverse transcribed to first strand cDNA with T7-oligo(dT) primer using ArrayScript reverse transcriptase, followed by second strand cDNA synthesis to generate double-stranded cDNA (ds-cDNA).

Subsequently, the ds-DNA was used as a template for in vitro transcription to synthesize biotin-labeled antisense-RNA (aRNA) molecules. The biotin-labeled aRNA was purified with RNA binding beads and then fragmented at 94°C for 35 minutes in fragmentation buffer (40 mM Tris-acetate, pH 8.2, 100 mM Potassium Acetate and 30 mM Magnesium Acetate). Fragmented aRNA (10 pg) was hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Array (Thermo Fisher Scientific) at 45 °C for 18 hours. Affymetrix GeneChip Fluidics Station 450 was used for washing and staining of the arrays, and hybridized arrays were scanned using a GeneChip Scanner 300 7G (Thermo Fisher Scientific) according to the manufacturer’s user guide. Array files were analyzed using packages from R Bioconductor: - normalized using the RMA method implemented in affy package; - probe IDs were converted into Entrez gene IDs and filtered to IRQ=0.33 using method nsFilter in genefilter package; - analysis for differential gene expression was performed with limma package; - ranking of genes from limma analysis was used as input to gene set enrichment analysis method gsePathway implemented in ReactomePA; - per-sample pathway activities for the heatmap visualization were estimated with GSVA package; heatmaps were shown with

ComplexHeatmap package; - MGSAT R software was used implement the entire analysis pipeline.

Western Blotting

[0133] Cells were lysed with Ripa buffer (ThermoFisher Scientific) containing complete protease inhibitor (Sigma) and frozen. In select experiments, IP3R was immunoprecipitated using anti-IP3R (Abeam cat#ab5804) and the Dynabeads protein G immunoprecipitation kit (ThermoFisher Scientific). Equal amounts of protein were separated on 4-12% bis-Tris NuPage gels and transferred to PVDF membranes (ThermoFisher Scientific).

Immunodetection was performed using anti-H3Cit (Abeam cat# ab5103), anti-lactoferrin (Abeam cat# ab77705), anti-MMP9 (Abeam cat#ab38898), anti-IP3R (Abeam cat#ab5804), anti-P-Ser/Thr (Abeam cat#ab 17464), and anti-actin (Sigma cat# A3854). Proteins were visualized with the Odyssey imaging system (Li-COR).

Example 1: Elevated glucose levels correlate with more severe S. aureus infections

[0134] Two models of murine diabetes, STZ induced and db/db , were used to study the effect of diabetes on the systemic response to systemic infection with S. aureus. In each model, the diabetic mice had a non-fasting glucose level greater than 450 dg/mL, while non diabetic control levels were less than 200 dg/mL. Mice were infected with 5e7 CFU S.

aureus (USA300, SF8300). CFU were collected from the kidney 48 hours post infection, and mortality was monitored for 14 days. Increased mortality was observed in both STZ (P = 0.0011) and db/db (P = 0.0241) models as compared with non-diabetic control (FIGs. 1A and IB). Of note, this did not correlate with a difference in bacterial CFU recovered from the kidneys 48 hours post-infection (FIGs. 1C and ID). To confirm that increased mortality was a consequence of elevated glucose in the diabetic host, mice were treated with

Rosiglitazone for 1 week prior to infection to reduce circulating glucose levels (FIG. IE). Rosiglitazone significantly reduced mortality (P = 0.0041) following infection with S. aureus , however the bacterial burden in the kidney was unaffected (FIGs. IF and 1G).

[0135] It is notable that no clearance defect was observed in the diabetic mice as

compared with non-diabetic controls. This highlights the contribution of excessive inflammation or exaggerated host response to the increase in mortality.

Example 2: Enhanced NETosis in Diabetic Mice

[0136] Neutrophils in a diabetic host, or in the presence of elevated glucose levels, are increasingly prone to NETosis. In the diabetic population, NET release has been shown to impair wound healing in mice, and the presence of NETs in the serum correlates with non healing wounds in patients (Fadini, G. P. et al. , Diabetes 65: 1061-1071 (2016) and Wong, S. L. et al. , Nat Med 21: 815-819 (2015)). Neutrophils also release NETs in response to bacterial infection, therefore it was hypothesized that S. aureus infection would result in increased systemic NET release in diabetic mice. Complexes of neutrophil elastase and double stranded DNA are used as a measurement of NET formation and quantified by ELISA (Fadini, G. P. et al. , Diabetes 65: 1061-1071 (2016)). Significant increases (P = 0.0003) in serum NETs were observed in diabetic mice intravenously infected with S. aureus for 24 hours, while significant increases were not observed in non-diabetic control mice (FIG. 2A). Levels of circulating NETs were not different in uninfected diabetic and non-diabetic mice.

[0137] Alpha toxin (AT), once released by S. aureus , binds to the receptor ADAMIO on the surface of platelets. (Neutrophils do not express ADAMIO.) In response to AT, platelets aggregate and bind to circulating neutrophils, resulting in activation of caspase-1 mediated signaling and NET production (Powers, M. E. et al. , Cell Host Microbe 17: 775-787 (2015) and Surewaard, B. G. J. et al. Cell Host Microbe 24: 271-284 (2018)). Consistent with these findings, neutralization of AT with monoclonal antibody MEDI4893* significantly reduced the number of NE-DNA complexes in the serum 48 hours post-infection in diabetic animals (FIG. 2B). Increased AT-dependent NET production was confirmed 48 hours post-infection by increased citrinulated Histone H3 (H3cit) in the liver as detected by western blot (FIG.

2C) Visualization of liver sections immunohistochemically stained with anti-Ly6G to mark neutrophils and anti-H3cit also showed increased AT-dependent NET (i.e., less anti-H3 cit staining in the livers of mice that received MEDI4893*) (Cohen TS, et al. Staphylococcus aureus drives expansion of low density neutrophils in diabetic mice. JCI 2019 IN PRESS). Neutralization of AT significantly increased survival (P = 0.0255) of diabetic mice infected with S. aureus (FIG 2D). These data indicate that systemic infection of the diabetic host lead to an AT-dependent increase in circulating NETs.

Example 3: Low Density Neutrophils Correlate with Increased NETosis

[0138] Similar to macrophages, neutrophils can be separated into different classes based on functional characteristics. Severe burns have been shown to alter the phenotype of circulating neutrophils and to alter TLR expression, cytokine production, and their ability to drive macrophage polarization (Tsuda, Y. et al. Immunity 21: 215-226 (2004)). Neutrophils are unique in that they can also be separated by cell density. High density neutrophils are anti-tumor, phagocytic cells, while low density neutrophils are considered pro-tumor phagocytic defective cells (Sagiv, J. Y. et al. Cell Rep 10: 562-573 (2015)). While Tsuda et. al. did not measure the density of neutrophils isolated from mice susceptible to S. aureus infection, the shape of the nuclei in these neutrophils was similar to the shape of nuclei in low density cells (Sagiv, J. Y. et al. Cell Rep 10: 562-573 (2015) and Fridlender, Z. G. et al. Cancer Cell 16: 183-194 (2009)). The shapes of the nuclei in neutrophils taken from non diabetic mice and diabetic mice also had striking differences. The nucleus in cells isolated from non-diabetic mice were multilobular or round, while large numbers of cells with ringed nuclei were observed in the blood of diabetic mice (Cohen TS, et al. Staphylococcus aureus drives expansion of low density neutrophils in diabetic mice. JCI 2019 IN PRESS). These structures were similar to those reported by Tsuda et. al to be found in the cells isolated from S. aureus susceptible mice, indicating that diabetic mice could have an increased number of low density, or immune impaired neutrophils.

[0139] Hyper NET production is a characteristic of low density neutrophils (LDN), and it was hypothesized that higher numbers of LDNs in infected diabetic mice were responsible for the increases in NETs (Villanueva, E. et al. J Immunol 187 : 538-552 (2011)). Blood was collected from C57BKS and db/db mice 48 hours post-IV infection and was analyzed for presence of LDNs. The amount of LDNs in the blood of infected db/db mice was

significantly increased compared to uninfected db/db mice (P < 0.0001) as well as infected C57BKS control mice (P = 0.0003) (FIG. 3A). Increases in LDNs were not observed in C57BKS mice (FIG. 3A). Similar increases were observed in STZ induced diabetic mice and not in C57BL/6 controls (FIG. 4). Lowering glucose levels with Rosiglitazone prior to infection significantly (P = 0.0116) reduced LDNs 48 hours post-infection (FIG. 3B).

[0140] To ensure that the observations were not based on degranulated neutrophils, LDNs and high density neutrophils (HDNs) were isolated from the blood of infected db/db mice, and the amounts of lactoferrin (secondary granules) and MMP9 (tertiary granules) were measured by western blot. Equivalent amounts of both were observed, indicating that LDNs have similar granular content as compared to HDNs (Cohen TS, et al. Staphylococcus aureus drives expansion of low density neutrophils in diabetic mice. JCI 2019 IN PRESS).

Neutralizing AT prevented systemic NET release, therefore the influence of AT on the number of LDNs was assessed. LDNs in the blood of db/db mice treated 24 hours prior to infection with c-IgG or MEDI4893* and infected with S. aureus for 48 hours were measured. A significant reduction in LDNs in mice prophylactically treated with MEDI4893* (FIG.

3C) was observed, while overall numbers of neutrophils were not affected (FIG. 3D), indicating that AT contributes to the increase in LDNs.

[0141] These data indicate that LDNs contribute to the pathology associated with diabetic

S. aureus infection and that these LDNs are associated with excessive NET release in both the liver, a key target organ of systemic infections, and systemically in the blood. Example 4: TGFB Drives Expansion of LDN Population

[0142] TGFp has been implicated as a central regulator of neutrophil phenotype, and in tumor models it can drive a phenotypic switch from high to low density neutrophil (Sagiv, J. Y. et al. Cell Rep 10: 562-573 (2015) and Fridlender, Z. G. et al. Cancer Cell 16: 183-194 (2009). Sagiv et. al. demonstrated that the addition of TGFp to blood taken from tumor bearing mice, not naive mice, will increase numbers of LDNs in vitro {id). This study was repeated with blood from non-diabetic and diabetic mice. The addition of TGFp to diabetic blood significantly increased (P = 0.0021) the number of LDNs (FIG. 5A). The same was not observed in non-diabetic blood. Based on this in vitro evidence demonstrating that TGFp can increase numbers of LDNs, its necessity for their induction by blocking in vivo was tested. Diabetic mice were prophylactically treated with neutralizing TGFP antibody 24 hours prior to infection with S. aureus. The numbers of LDNs in the bloodstream was significantly reduced (P = 0.0003) by inhibition of TGFP, while numbers of bacteria in the kidneys were similar between groups (FIGs. 5B and 5C). Survival was significantly improved (P = 0.0072) by neutralizing TGFP (FIG. 5D). Visualization of NETs in the liver demonstrated a loss of NETs when TGFP was neutralized (Cohen TS, et al. Staphylococcus aureus drives expansion of low density neutrophils in diabetic mice. JCI 2019 IN PRESS). These data suggest that reducing LDNs by blocking TGFp could promote survival.

[0143] TGFp is secreted as a pro-form protein (pro-TGFB) and requires cleavage to be activated. Binding of pro-TGFp by anb8 integrin has been linked to its activation and prevention of colitis, and its expression on dendritic cell and monocyte subsets is increased in response to inflammation (Travis, M. A. et al. Nature 449: 361-365 (2007) and Kelly, A. et al. J Exp Med, doi: 10.1084/jem.20171491 (2018)). To determine if S. aureus infection influences expression of anb8 integrin, innate immune cells were isolated from the liver and spleen of C57BKS and db/db mice 24 hours post-infection, and the expression of anb8 was analyzed by flow cytometry. Numbers of b8 positive inflammatory monocytes and dendritic cells increased in the livers of db/db mice, not C57BKS mice, following infection (FIG. 6A). Interestingly, while integrin expression increased on the surface of monocytes, it was the overall number of DCs that increased, not the density of b8 (FIG. 6B). To demonstrate the functional relevance of anb8 in this model, mice were prophylactically treated with antibodies neutralizing anb6/8, anb6 or c-IgG and infected with S. aureus. Forty-eight hours post infection LDNs were significantly decreased (P = 0.0090) in the bloodstream in the mice treated with anb6/8 neutralizing antibody compared with c-IgG (FIG. 6C).

Neutralization of anbό alone did not reduce the numbers of these cells. Integrin inhibition did not affect the numbers of bacteria in the kidneys 48 hours post-infection (FIG. 6D). Survival was significantly improved in mice treated with anti-aVB6/8 antibody as compared with c-IgG treated mice (FIG. 6E). Therefore, consistent with directly neutralizing TORb, blocking the integrin responsible for activating this pathway was protective in diabetic mice.

[0144] These data show that neutralization of either aVB6/8 or TORb prevents LDN

increases and reduces mortality. These data also show that dendritic cells play a central role in the pathogenesis of diabetic infection due to their ability to activate TORb and promote expansion of LDNs.

Example 5: AT Drives TGFB Activation

[0145] It was hypothesized that AT was influencing LDN numbers by affecting the TORb pathway. Following its activation, TGFB binds to its receptor complex, activates SMAD transcription factors, and drives expression of downstream genes. Therefore, activation of SMAD signaling is commonly used as a surrogate measurement of TORb activation.

pSMAD levels were analyzed in the livers of diabetic and non-diabetic mice that were infected (24 hours) with S. aureus. Significantly increased pSMAD was observed in the livers of infected diabetic mice as compared to naive diabetic mice (P < 0.0001) and infected non-diabetic mice (P = 0.0338) (FIG. 7A). In diabetic mice, MEDI4893* significantly reduced (P < 0.0001) pSMAD levels in the liver, indicating that AT was contributing to activation of TORb signaling (FIG. 7B). Neutralizing AT did not alter the numbers of anb8 expressing innate immune cells (FIG. 7C). These data indicate that AT influences activation oίTϋRb through a mechanism that is independent of anb8 expression on innate immune cells. Accordingly, neutralization of AT, which is a key S. aureus virulence factor, limits activation of TORb signaling, and subsequently reduces LDN numbers and NET release.

[0146] These data indicate that, in addition to binding to ADAMIO on platelets, AT can act through a second pathway that alters the neutrophil phenotype and subsequent response to S. aureus infection. In the diabetic host, AT-dependent activation of TGFB signaling drives expansion of LDNs. Thus, AT is both promoting the expansion of the LDN population which spontaneously release NETs and activating platelets, which can bind and further activate neutrophils. Example 6: Increased PTEN in LDNs Results in Excessive NET Production

[0147] To further study the increased NET release by LDNs, LDNs and HDNs were

purified from infected mice. Gene expression analysis and mass spectrometry (MS) analyses were performed on isolated cell membranes. A large number of genes and proteins were significantly up or down regulated in LDNs as compared to HDNs (FIGs. 8A and 8B).

Pathway analysis revealed LDN enrichment in innate immune pathways including

phagocytotic and toll-like signaling, transendothelial migration, and inositol phosphate metabolism. In addition, significant upregulation in pathways such as platelet activation (platelet binding proteins) and G-protein-coupled receptor (GPCR) signaling were observed (FIG. 8C). Platelet activation in response to AT has previously been demonstrated to induce NET release by neutrophils. Therefore, the mechanism of GPCR activation resulting in enhanced NET release by LDNs was studied (Powers, M. E., et al. Cell Host Microbe 17: 775-787 (2015).)

[0148] Ligation of GPCRs activates phospholipase C (PLC), which leads to the

degradation of PIP2 into DAG and IP3. IP3 induces calcium release from the endoplasmic reticulum following binding to its receptor, IP3R. (Cohen TS, et al. Staphylococcus aureus drives expansion of low density neutrophils in diabetic mice. JCI 2019 IN PRESS.) Calcium release is required for the induction of NET release. (Douda, D. N., et al. Proc Natl Acad Sci USA 112: 2817-2822 (2015); Zhou, Y. et al. Sci Rep 8: 15228 (2018); and Gupta, A. K., et al. PLoS One 9 : e97088 (2014).) Therefore activation of this pathway in LDNs could explain why they are so prone for NET secretion. Four GPCRs were found to be upregulated in the LDNs as compared to HDNs by MS analysis; however only three were also significantly different (P < 0.05) in the RNA dataset (FIG. 9A). PIP2 is converted from PIP3 by the phosphatase PTEN, and loss of PIP3 can be monitored by a drop in Akt phosphorylation. (Marte, B. M. & Downward, J. Trends Biochem Sci 22: 355-358 (1997); and Stephens, L. et al. Science 279: 710-714 (1998).) (FIG. 9B.)

[0149] Upregulation of PTEN shunts signaling away from Akt, which is involved in

chemotaxis and sensing of bacteria via TLR signaling (Barati, M. T., et al. Cell Signal 27: 1178-1185 (2015); Kumar, S. et al. J Exp Med 211: 1741-1758 (2014); and Strassheim, D. et al. J Immunol 172: 5727-5733 (2004)). By degrading PIP3 into PIP2, PTEN decreases Akt recruitment to the cell membrane, preventing its phosphorylation, as well as enabling GPCR degradation of PIP2 into IP3 and activating Ca2+ signaling from the ER. When active, Akt blocks IP3 signaling through its interaction with the IP3R on the ER membrane. PTEN therefore not only increases the substrate for IP3R signaling, but also limits the ability of the cell to block signaling through this receptor.

[0150] Analysis of membrane associated Akt and phosphorylated Akt in LDNs as

compared with HDNs revealed significant (P < 0.0001) reduction of Akt activation in LDNs, consistent with a loss of PIP3 (FIG. 9C). To determine if reduced levels of pAkt were simply due to LDNs not being activated in vivo, purified HDN and LDN were exposed to N- Formylmethionine-leucyl-phenylalanine (fMLP), a neutrophil stimulator, ex vivo. Minimal additional activation of Akt was observed in either HDN or LDN, suggesting that both were maximally activated in vivo (FIG. 9D). PTEN expression (both RNA and protein) was significantly (P < 0.05) increased in LDNs, correlating with the loss of Akt activation (Cohen TS, et al. Staphylococcus aureus drives expansion of low density neutrophils in diabetic mice. JCI 2019 IN PRESS). The small molecule VO-OHpic inhibits PTEN. Therefore, the effect of VO-OHpic on survival of diabetic mice was tested. VO-OHpic treated mice survived at a significantly higher rate than control mice infected with S. aureus (P = 0.0491) (Fig. 9E). These data indicate that, due to increased PTEN levels in LDNs, the PIP2/PIP3 balance is shifted towards increased PIP2, resulting in excessive NET production, and inhibition of PTEN can protect diabetic mice against infection with S. aureus.

* * *

[0151] All references, including publications, patent applications, and patents, cited

herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0152] All methods described herein can be performed in any suitable order unless

otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0153] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method of treating or preventing a Staphylococcus aureus ( S . aureus) infection in a subject with diabetes comprising administering to the subject an antibody or antigen binding fragment thereof that binds to anb8 integrin.

2. An antibody or antigen-binding fragment thereof that binds to anb8 integrin for use in treating or preventing a S. aureus infection in a subject with diabetes.

3. Use of an antibody or antigen-binding fragment thereof that binds to anb8 integrin in the preparation of a medicament for treating or preventing a S. aureus infection in a subject with diabetes.

4. The method, antibody, or use of any one of claims 1-3, wherein the antibody or antigen binding fragment thereof that binds to anb8 integrin is a neutralizing antibody.

5. A method of treating or preventing a S. aureus infection in a subject with diabetes

comprising administering to the subject an antibody or antigen-binding fragment thereof that binds to transforming growth factor-beta (b) (TϋRb).

6. An antibody or antigen-binding fragment thereof that binds to TORb for use in treating or preventing a S. aureus infection in a subject with diabetes.

7. Use of an antibody or antigen-binding fragment thereof that binds to TQRb in the

preparation of a medicament for treating or preventing a S. aureus infection in a subject with diabetes.

8. The method, antibody, or use of any one of claims 5-7, wherein the antibody or antigen binding fragment thereof that binds to TQRb comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of 1D11.16.8.

9. The method, antibody, or use of any one of claims 5-8, wherein the antibody or antigen binding fragment thereof that binds TQRb comprises a VH comprising the amino acid sequence of the VH of 1D11.16.8.

10. The method, antibody, or use of any one of claims 5-9, wherein the antibody or antigen binding fragment thereof that binds TGFp comprises a VL comprising the amino acid sequence of the VL of 1D11.16.8.

11. The method, antibody, or use of any one of claims 5-10, wherein the antibody or antigen binding fragment thereof that binds TGFp binds to the same TGFp epitope as 1D11.16.8.

12. The method, antibody, or use of any one of claims 5-11, wherein the antibody or antigen binding fragment thereof that binds TGFP competitively inhibits binding of 1D11.16.8 to TGFp.

13. The method, antibody, or use of any one of claims 5-12, wherein the antibody or antigen binding fragment thereof that binds to TGFP is a neutralizing antibody.

14. The method, antibody, or use of any one of claims 5-13, wherein the antibody or antigen binding fragment thereof that binds to TGFB is 1D11.16.8.

15. The method, antibody, or use of any one of claims 1-14, wherein the antibody or antigen binding fragment that binds to anb8 integrin or wherein the antibody or antigen-binding fragment thereof that binds to TGFP further comprises a heavy chain constant region.

16. The method, antibody, or use of claim 15, wherein the heavy chain constant region is selected from the group consisting of human immunoglobulin IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2 heavy chain constant regions.

17. The method, antibody, or use of claim 16, wherein the heavy chain constant region is a human IgGi constant region.

18. The method, antibody, or use of any one of claims 1-17, wherein the antibody or antigen binding fragment that binds to anb8 integrin or wherein the antibody or antigen-binding fragment thereof that binds to TORb further comprises a light chain constant region.

19. The method, antibody, or use of 18, wherein the light chain constant region is selected from the group consisting of human immunoglobulin IgGK and IgG light chain constant regions.

20. The method, antibody, or use of claim 19, wherein the light chain constant region is a human IgGK light chain constant region.

21. The method, antibody or use of any one of claims 1-20, wherein the antibody or antigen binding fragment that binds to anb8 integrin or wherein the antibody or antigen-binding fragment thereof that binds to TGFp is an IgG antibody or antigen-binding fragment thereof.

22. The method, antibody or use of any one of claims 1-21, wherein the antibody or antigen binding fragment that binds to anb8 integrin or wherein the antibody or antigen-binding fragment thereof that binds to TGE^ is a monoclonal antibody or antigen-binding fragment thereof.

23. The method, antibody or use of any one of claims 1-22, wherein the antibody or antigen binding fragment that binds to anb8 integrin or wherein the antibody or antigen-binding fragment thereof that binds to TGE^ is a full-length antibody.

24. The method, antibody or use of any one of claims 1-23, wherein the antibody or antigen binding fragment that binds to anb8 integrin or wherein the antibody or antigen-binding fragment thereof that binds to TGE^ is an antigen-binding fragment.

25. The method, antibody or use of claim 24, wherein the antigen-binding fragment

comprises a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide linked Fv, intrabody, IgGACFLZ, minibody, F(ab')3, tetrabody, triabody, diabody, DVD-Ig, Fcab, mAh², (SCFV)2, or scFv-Fc.

26. The method, antibody or use of any one of claims 1-25, wherein the antibody or antigen binding fragment that binds to anb8 integrin or wherein the antibody or antigen-binding fragment thereof that binds to TORb decreases low density neutrophils (LDNs) in the subject or prevents the increase of LDNs in the subject.

27. The method, antibody or use of any one of claims 1-25, wherein the antibody or antigen binding fragment that binds to anb8 integrin or wherein the antibody or antigen-binding fragment thereof that binds to TORb decreases neutrophil extracellular traps (NETs) in the subject or prevents the increase of NETs in the subject.

28. A method of treating or preventing a Staphylococcus aureus ( S . aureus) infection in a subject with diabetes comprising administering to the subject a compound that neutralizes anb8 integrin.

29. A compound that neutralizes anb8 integrin for use in treating or preventing a S. aureus infection in a subject with diabetes.

30. Use of a compound that neutralizes anb8 integrin in the preparation of a medicament for treating or preventing a S. aureus infection in a subject with diabetes.

31. The method, compound, or use of any one of claims 28-30, wherein the compound is a polypeptide, an antibody or antigen-binding fragment thereof, a small molecule, an siRNA, an antisense oligonucleotide, or an aptamer.

32. A method of treating or preventing a S. aureus infection in a subject with diabetes

comprising administering to the subject a compound that neutralizes TOTb.

33. A compound that neutralizes TORb for use in treating or preventing a S. aureus infection in a subject with diabetes.

34. Use of compound that neutralizes TQRb in the preparation of a medicament for treating or preventing a S. aureus infection in a subject with diabetes.

35. The method, compound, or use of any one of claims 32-34, wherein the compound is a polypeptide, an antibody or antigen-binding fragment thereof, a small molecule, an siRNA, an antisense oligonucleotide, or an aptamer.

36. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is sepsis.

37. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is bacteremia.

38. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is pneumonia.

39. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is ICU pneumonia.

40. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is a skin or soft tissue infection (SSTI).

41. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is a diabetic infection of the lower limbs.

42. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is a diabetic foot ulcer (DFU).

43. The method, antibody, compound, or use of any one of claim 42, wherein the DFU is uninfected.

44. The method, antibody, compound, or use of claim 42, wherein the DFU is infected.

45. The method, antibody, compound, or use of claim 42, wherein the DFU is a grade 1, 2 or 3 DFU.

46. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is a bone or joint infection.

47. The method, antibody, compound, or use of any one of claims 1-35, wherein the S. aureus infection is a joint infection, a device infection, a wound infection, a surgical site infection, or osteomyelitis.

48. The method, antibody, compound, or use of any one of claims 1-47, wherein the subject is a surgical subject.

49. The method, antibody, compound, or use of any one of claims 1-48, wherein the S. aureus infection comprises antibiotic-resistant S. aureus.

50. The method, antibody, compound, or use of any one of claims 1-49, wherein the subject is human.