Antibodies against staphylococcus aureus agglutination factor (CLFA)

Abstract

The present application relates to antibodies against Staphylococcus aureus agglutination factor A (CLFA). The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to Staphylococcus aureus agglutination factor A protein (ClfA), and a composition comprising the monoclonal antibody. The present invention is also directed to methods of treating Staphylococcus aureus infection by administering to an individual an anti-ClfA monoclonal antibody alone or in combination with a monoclonal antibody that specifically binds to Staphylococcus aureus alpha toxin (AT) protein. The invention also provides bispecific monoclonal antibodies that specifically bind to both ClfA and AT and methods of using the monoclonal antibodies.

Description

[0001] This application is a divisional application of an invention patent application with an application date of July 24, 2019, application number “201980058857.X”, and name “Antibodies against Staphylococcus aureus Agglutinating Factor A (CLFA)”. Technical Field

[0002] The present application generally relates to antibodies against Staphylococcus aureus Agglutinating Factor A (CLFA). Background Art

[0003] Infections caused by antimicrobial resistant (AMR) bacterial pathogens are an increasing threat to public health. The ongoing AMR epidemic is partly fueled by empirical 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. Many monoclonal antibodies are currently being developed for the prevention or treatment of antibiotic-resistant bacterial infections (see, for example, DiGiandomenico, A. and BR Sellman, Curr. Opin. Microbiol., 27: 78-85 (2015)). This passive immunization strategy provides real-time and effective immunoglobulin responses against target pathogens. Ideally, monoclonal antibodies or monoclonal antibody mixtures provide multiple mechanisms of action to neutralize key bacterial virulence mechanisms and enhance host innate immune responses, thereby providing the greatest opportunity for clinical success.

[0004] Staphylococcus aureus is a bacterial pathogen that can cause a variety of diseases, including skin and soft tissue infections, endocarditis, osteomyelitis, pneumonia, and bacteremia (Lowy, FD, N. Engl. J. Med., 339(8):520-32 (1998)). Preclinical studies indicate that monoclonal antibody-based approaches have promise for the prevention and adjunctive treatment of S. aureus infections (see, e.g., Hazenbos et al., PLoS Pathog., 9(10):e1003653.doi:10.1371 / journal.ppat.10036532013 (2013); Rouha, H., MAbs, 7(1):243-254 (2015); Foletti et al., J. Mol. Biol., 425(10):1641-1654 (2013); Karauzum et al., J Biol Chem., 287(30):25203-15 (2012); and Hua et al., Antimicrob Agents Chemotherapy. Chemother., 58(2):1108-17 (2014)). A combination of multi-mechanistic monoclonal antibodies against S. aureus alpha toxin (AT) and clumping factor A (ClfA) showed enhanced protection and improved strain coverage relative to each monoclonal antibody alone in a lethal bacteremia model of S. aureus (Tkaczyk et al., MBio., 7(3).pii:e00528-16 (2016)); however, the ClfA monoclonal antibodies tested exhibited reduced binding affinity and functional activity to the ClfA founder sequence ClfA002 relative to two other founder sequences (ClfA001 and ClfA004).

[0005] Therefore, there remains a need for compositions and methods for treating S. aureus infections, particularly infections that are resistant to currently available antibiotics. The present invention provides such compositions and methods. Summary of the invention

[0006] Provided herein are antibodies or antigen-binding fragments that bind to Staphylococcus aureus agglutinating factor A (ClfA) proteins. In some cases, antibodies or antigen-binding fragments thereof that specifically bind to Staphylococcus aureus ClfA proteins include a variable heavy chain (VH) complementary determining region (CDR) 1 containing the amino acid sequence of SEQ ID NO: 1, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 2, a VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, a variable light chain (VL) CDR 1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, and the antibody or antigen-binding fragment includes a heavy chain constant domain containing the amino acid sequence of CSYHLC (SEQ ID NO: 21). In some cases, the antibody or its antigen-binding fragment includes a VH containing the amino acid sequence of SEQ ID NO: 13. In some cases, the antibody or its antigen-binding fragment includes a VL containing the amino acid sequence of SEQ ID NO: 14.

[0007] In some cases, an antibody or antigen-binding fragment thereof that specifically binds to a S. aureus ClfA protein comprises VH, VL, and a heavy chain constant domain comprising the amino acid sequence of CSYHLC (SEQ ID NO:21), wherein the VH comprises the amino acid sequence of SEQ ID NO:13.

[0008] In some cases, an antibody or antigen-binding fragment thereof that specifically binds to a S. aureus ClfA protein comprises VH, VL, and a heavy chain constant domain comprising the amino acid sequence of CSYHLC (SEQ ID NO:21), wherein the VL comprises the amino acid sequence of SEQ ID NO:14.

[0009] In some cases, the antibody or antigen-binding fragment thereof that specifically binds to the ClfA protein of Staphylococcus aureus comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of SAR114. In some cases, the CDR is a CDR defined by Kabat, a CDR defined by Chothia or a CDR defined by AbM. In some cases, the antibody or antigen-binding fragment comprises a heavy chain constant domain comprising the amino acid sequence of CSYHLC (SEQ ID NO: 21).

[0010] In some cases, the heavy chain constant domain comprises the amino acid sequence of MHEACSYHLCQKSLSLS (SEQ ID NO: 23). In some cases, the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO: 24. In some cases, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 50. In some cases, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 26.

[0011] In some cases, the IC50 of the antibody or antigen-binding fragment thereof against ClfA001, ClfA002, and ClfA004 in a fibrinogen binding inhibition assay is within 2 μg / ml of each other. In some cases, the IC50 of the antibody or antigen-binding fragment thereof against ClfA001, ClfA002, and ClfA004 in a fibrinogen binding inhibition assay is within 2 μg / ml of each other. 50 In some cases, the binding affinity (K of the antibody or antigen-binding fragment thereof for ClfA001, ClfA002 and ClfA004 is between 1 μg / ml and 5 μg / ml. D ) are between 200pM and 350pM.

[0012] In some cases, the antibody or antigen-binding fragment thereof has a monomer purity that decreases by no more than 5% after exposing the antibody or antigen-binding fragment to 2 kLux / hr of conventional white light at 23°C for 14 days. In some cases, the antibody or antigen-binding fragment comprises a mutation that extends the half-life in human FcRn mice relative to the same antibody without the mutation. In some cases, the antibody or antigen-binding fragment comprises a mutation that extends the half-life relative to the same antibody without the mutation, and wherein the mutation does not inhibit OPK activity relative to the same antibody or antigen-binding fragment without the mutation.

[0013] Also provided herein are bispecific antibodies and antigen-binding fragments thereof that specifically bind to the ClfA protein of Staphylococcus aureus and the alpha toxin (AT) protein of Staphylococcus aureus. In some cases, the bispecific antibody or antigen-binding fragment thereof that specifically binds to the ClfA protein of Staphylococcus aureus and the alpha toxin (AT) protein of Staphylococcus aureus comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some cases, the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 13. In some cases, the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 14. In some cases, the antibody or antigen-binding fragment thereof further comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some cases, the antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 15. In some cases, the antibody or antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0014] In some cases, the antibody or antigen-binding fragment thereof that specifically binds to the ClfA protein of Staphylococcus aureus comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of SAR72, SAR80, SAR113, SAR132, SAR352, SAR372, SAR510, SAR547, SAS1, SAS19 or SAS203. In some cases, the CDRs are Kabat-defined CDRs, Chorthia-defined CDRs or AbM-defined CDRs. In some cases, the antibody or antigen-binding fragment comprises a variable heavy chain and a variable light chain sequence comprising an amino acid sequence as shown below: (a) SEQ ID NOs: 17 and 18, respectively, (b) SEQ ID NOs: 30 and 31, respectively, (c) SEQ ID NOs: 32 and 33, respectively, (d) SEQ ID NOs: 34 and 35, respectively, (e) SEQ ID NOs: 36 and 37, respectively, (f) SEQ ID NOs: 38 and 39, respectively, (g) SEQ ID NOs: 40 and 41, respectively, (h) SEQ ID NOs: 42 and 43, respectively, (i) SEQ ID NOs: 44 and 45, respectively, (j) SEQ ID NOs: 46 and 47, respectively, or (k) SEQ ID NOs: 48 and 49, respectively.

[0015] In some cases, the antibody or antigen-binding fragment thereof that specifically binds to the S. aureus ClfA protein comprises a VH and a VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 17, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48.

[0016] In some cases, the antibody or antigen-binding fragment thereof that specifically binds to the S. aureus ClfA protein comprises a VH and a VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 18, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49.

[0017] In some cases, the antibody or its antigen-binding fragment further comprises a heavy chain constant region. In some cases, the heavy chain constant region is selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 heavy chain constant regions. In some cases, the heavy chain constant region is a human IgG1 constant region. In some cases, the heavy chain constant region comprises N3, N3E or N3F mutations. In some cases, the heavy chain constant region comprises a YTE mutation.

[0018] In some cases, the antibody or its antigen-binding fragment further comprises a light chain constant region. In some cases, the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions. In some cases, the light chain constant region is a human IgGκ light chain constant region.

[0019] In certain instances, the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.

[0020] In some cases, the antibody or antigen-binding fragment is a full-length antibody. In some cases, the antibody or antigen-binding fragment is an antigen-binding fragment. In some cases, the antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, intrabody, IgGΔCH2, minibody, F(ab')3, tetrafunctional antibody, trifunctional antibody, bifunctional antibody, DVD-Ig, Fcab, mAb 2 , (scFv)2 or scFv-Fc.

[0021] In some cases, an antibody that specifically binds to a S. aureus ClfA protein comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:50 and a light chain comprising the amino acid sequence shown in SEQ ID NO:26.

[0022] In some cases, the antibody or antigen-binding fragment thereof further comprises a detectable label.

[0023] Also provided herein are compositions comprising the antibodies provided herein. In some cases, the composition comprises an antibody provided herein and a pharmaceutically acceptable carrier.

[0024] In some cases, the composition comprises an antibody provided herein and an antibody or antigen-binding fragment that specifically binds to the AT protein of Staphylococcus aureus, and an optional pharmaceutically acceptable carrier. In some cases, the antibody or antigen-binding fragment that specifically binds to the AT protein of Staphylococcus aureus comprises a VHCDR1 containing the amino acid sequence of SEQ ID NO: 7, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 8, a VHCDR3 containing the amino acid sequence of SEQ ID NO: 9, a VL CDR1 containing the amino acid sequence of SEQ ID NO: 10, a VLCDR2 containing the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 12. In some cases, the antibody or antigen-binding fragment that specifically binds to the AT protein of Staphylococcus aureus comprises a VH containing the amino acid sequence of SEQ ID NO: 15. In some cases, the antibody or antigen-binding fragment that specifically binds to the AT protein of Staphylococcus aureus comprises a VL containing the amino acid sequence of SEQ ID NO: 16. In some cases, an antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus AT protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 27. In some cases, an antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus AT protein comprises a light chain comprising the amino acid sequence of SEQ ID NO:28.

[0025] Also provided herein are methods of using the antibodies provided herein. In some cases, a method of treating or preventing a Staphylococcus aureus infection in a subject comprises administering to the subject an antibody or antigen-binding fragment provided herein or a composition provided herein.

[0026] In some cases, a method for treating or preventing an individual's Staphylococcus aureus infection comprises administering to an individual an antibody or antigen-binding fragment provided herein and an antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha toxin (AT) protein. In some cases, an antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus AT protein comprises a VH CDR1 containing an amino acid sequence of SEQ ID NO: 7, a VHCDR2 containing an amino acid sequence of SEQ ID NO: 8, a VH CDR3 containing an amino acid sequence of SEQ ID NO: 9, a VLCDR1 containing an amino acid sequence of SEQ ID NO: 10, a VL CDR2 containing an amino acid sequence of SEQ ID NO: 11, and a VLCDR3 containing an amino acid sequence of SEQ ID NO: 12. In some cases, an antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus AT protein comprises a VH containing an amino acid sequence of SEQ ID NO: 15. In some cases, an antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus AT protein comprises a VL containing an amino acid sequence of SEQ ID NO: 16. In some cases, the antibody or antigen-binding fragment that specifically binds to the S. aureus AT protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 27. In some cases, the antibody or antigen-binding fragment that specifically binds to the S. aureus AT protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 28. In some cases, the anti-S. aureus CIfA antibody or antigen-binding fragment provided herein and the antibody or antigen-binding fragment that specifically binds to the S. aureus AT protein are administered simultaneously. In some cases, the anti-S. aureus CIfA antibody or antigen-binding fragment provided herein and the antibody or antigen-binding fragment that specifically binds to the S. aureus AT protein are administered sequentially.

[0027] In certain instances, treating or preventing a S. aureus infection in a subject comprises inhibiting S. aureus associated sepsis, inhibiting S. aureus agglutination, inhibiting thromboembolic lesion formation, toxin neutralization, inducing opsonophagocytosis, inhibiting S. aureus fibrinogen binding, or any combination of the foregoing.

[0028] In some cases, the subject has diabetes. In some cases, the subject is human.

[0029] Polynucleotides are also provided herein. In some cases, the isolated polynucleotides comprise nucleic acid molecules encoding the VH or heavy chains of the antibodies or antigen-binding fragments thereof provided herein. In some cases, the nucleic acid molecules encode the VH of SEQ ID NO: 13 or the heavy chains of SEQ ID NO: 25, 50 or 52.

[0030] In some cases, the isolated polynucleotide comprises a nucleic acid molecule encoding the VL or light chain of an antibody or antigen-binding fragment thereof provided herein. In some cases, the nucleic acid molecule encodes the VL of SEQ ID NO: 14 or the light chain of SEQ ID NO: 26.

[0031] In some cases, the isolated polynucleotide comprises a nucleic acid molecule encoding a VH or heavy chain of an antibody or antigen-binding fragment thereof provided herein and a VH or light chain of an antibody or antigen-binding fragment thereof.

[0032] Vectors are also provided herein. In some cases, an isolated vector comprises a polynucleotide provided herein.

[0033] Host cells are also provided herein. In some cases, host cells comprise polynucleotides provided herein, carriers provided herein, or first carriers comprising polynucleotides provided herein and second carriers comprising polynucleotides provided herein. In some cases, host cells are selected from the group consisting of CHO, NS0, PER-C6, HEK-293 and HeLa cells. In some cases, host cells are separated.

[0034] Also provided herein are methods for producing antibodies or antigen-binding fragments. In some cases, the method for producing an antibody or antigen-binding fragment thereof comprises culturing a host cell provided herein, thereby producing the antibody or antigen-binding fragment thereof.

[0035] Also provided herein are methods for detecting S. aureus or S. aureus CIfA. In some cases, methods for detecting S. aureus or S. aureus CIfA in a sample comprise contacting the sample with an antibody or antigen-binding fragment thereof provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results show that the anti-ClfA mAb 11H10 ( Figure 1 A) or SAR114( Figure 1 B) A series of graphs showing the inhibition of fibrinogen binding to the three major CIfA genotypes measured in the presence of .Data are representative of three independent experiments.

[0037] Figure 2 is a graph illustrating agglutination of S. aureus clinical isolates in the presence of human plasma and anti-CIfA mAb. Figure 2 Minimal concentrations of 11H10 and SAR114 mAbs required to inhibit bacterial agglutination are shown. Data are representative of two independent experiments. c-IgG was used as a negative control and did not show any inhibition at 200 μg / ml.

[0038] Figure 3 The results show that the anti-ClfA monoclonal antibody SAR114 is effective against the clinical isolate ARC635 (ST5) of Staphylococcus aureus ( Figure 3 A) SF8300(ST8)( Figure 3 B) NRS383(ST346)( Figure 3 C) NRS382(ST5)( Figure 3 D) NRS384(ST8)( Figure 3 E) and ARC2081(ST30)( Figure 3 F) A series of graphs showing the opsonophagocytic killing (OPK) activity of S. aureus strains were incubated with human HL-60 cells, human serum and serial dilutions of SAR114 (squares) or c-IgC (circles). The graphs represent the mean ± standard deviation (SD) of three independent experiments.

[0039] Figure 4 is a graph illustrating the inhibition of agglutination of multiple S. aureus types by the SAR114 monoclonal antibody.112 S. aureus clinical isolates representing 40 different sequence types (ST) and some not found (NF) were tested as described in Example 1 .

[0040] Figure 5 Shown is a graph illustrating competition between SAR114 and 11H10 for binding to ClfA001 genotypes. Figure 5 The graph in A shows the results of an ELISA competition binding assay as described in Example 1. The data represent the mean ± standard deviation (SD). Figure 5 The diagram in B shows the Combined analysis results.

[0041] Figure 6 Provided are graphs showing the effects of YTE and N3 mutations in anti-bacterial antibodies on opsonophagocytic killing (OPK). Cam004 (upper graph) is an anti-Pseudomonas antibody, and 2F4 (lower graph) is an anti-Staphylococcus aureus antibody. R347 antibody was used as a negative control. (See Example 2.)

[0042] Figure 7 A graph is provided showing that the N3 mutation does not reduce the ability of SAR114 to inhibit the binding of ClfA001, ClfA002 or ClfA1004 to fibrinogen. (See Example 2.)

[0043] Figure 8 A table is provided reporting the effects of N3F and N3Y mutations on SAR114 in coagulation and fibrinogen potency assays. (See Example 3.)

[0044] Fig. 9 Provided are graphs showing the effects of N3 (N3W), N3F and N3Y mutations on pharmacokinetics (PK) in human FcRn (upper graph) and OPK (lower graph) transgenic mice of SAR114. (See Example 3.)

[0045] Fig.10 AM is a series of graphs illustrating the strain coverage provided by the combination of SAR114 and MEDI4893* monoclonal antibodies in the lethal bacteremia mouse model described in Example 4. Different S. aureus clinical isolates from different sequence types (ST) and ClfA genotypes tested included: NRS123 (ST1, ClfA012), NRS387 (ST5, ClfA002), ARC635 (ST5, ClfA002), 3049043 (ST5, ClfA002), 3049057 (ST8, ClfA001), SF8300 (ST8, ClfA001), 3049088 (ST30, ClfA004), 3049114 (ST30, ClfA004), ARC2784 (ST188, ClfA019), 9043, 9057, 9157, and 2784. Data shown are representative of three independent experiments. The dashed line with a circle at the end represents mice treated with an IgG control antibody. The line with a square at the end represents mice treated with SAR114 (15 mpk). The line with an upward triangle at the end represents mice treated with MEDI4893* (15 mpk). The line with a downward triangle at the end represents mice treated with SAR114 and MEDI4893* (7.5 mpk each). (See Example 4.)

[0046] Fig.11 Description BKS.Cg-Dock7 m + / +Lepr db A series of graphs showing that the combination of SAR114 and MEDI4893* monoclonal antibodies prevents CA-MRSA SF8300-induced IV lethal bacteremia in type 1 / J diabetic mice (db / db). The horizontal bars in the bottom two graphs represent the geometric mean CFU. The data represent three independent experiments. (See Example 5.)

[0047] Fig.12 Images are provided demonstrating the effect of the combination of SAR114 and MEDI4893* monoclonal antibodies on liver injury in db / db mice exposed to CA-MRSA SF8300-induced IV lethal bacteremia, either by gross pathology (left) or after hematoxylin and eosin staining of sections (right). (See Example 7.)

[0048] Fig.13is a series of graphs illustrating that the combination of SAR114 and MEDI4893* monoclonal antibodies provides strain-wide protection in a diabetic db / db mouse model of lethal bacteremia. (See Example 8.)

[0049] Fig.14 Provided using anti-ClfAmAb as scaffold ( Fig.14 A) or linked to the N-terminus of the heavy chain of the ClfA monoclonal antibody via a 10-amino acid linker (GGGGx2) Fig.14 B) or the heavy chain C-terminus ( Fig.14 C) Overview of the bispecific construct of the scFv of the anti-AT mAb MEDI4893*. (See Example 9.)

[0050] Fig.15 is a series of graphs illustrating the in vitro characterization of anti-CIfA SAR114 or 11H10 / MEDI4893* BiSAbs, as described in Example 9. Fig.15 A and 15D illustrate the activity of BiS2 and BiS3 compared to MEDI4893* in an AT-mediated rabbit RBC hemolysis assay. Serial dilutions of BiSAb and MEDI4893* were compared with AT alone ( Fig.15 A) or 10 M excess ClfA001 ( Fig.15 D) and RBCs were incubated together. The percentage of hemolysis inhibition was calculated as follows: 100*(100-(OD AT+mAb ) / (OD 单独的AT )). Data are representative of three independent experiments. Fig.15 B and 15C illustrate the results of the immobilized fibrinogen binding assay described in Example 9. Serial dilutions of BiSAb, SAR114, or 11H10 were mixed with ClfA alone ( Fig.15 B) or with 10M excess AT ( Fig.15 C) were cultured together. Data represent the mean standard deviation of three independent experiments. The percentage of binding inhibition was calculated as 100*(100-(OD ClfA+mAb ) / (OD 单独的ClfA )).

[0051] Fig.16 is a series of graphs illustrating the inhibition of fibrinogen binding to the three major ClfA genotypes described in Example 9. Fig.16 A) SAR114( Fig.16 B) or the respective bispecific antibodies ( Fig.16 C). A similar analysis was performed by incubating the cells with a 10 M excess of AT (6.6 mM) ( Fig.16 DF) to saturate the AT scFv.

[0052] Fig.17 is a series of graphs illustrating the opsonophagocytic killing (OPK) activity of anti-ClfA / AT bispecific antibodies (BiS). Staphylococcus aureus Newman isolates were incubated with human HL-60 cells, human serum, and 11H10 parental monoclonal antibodies or 11H10-BiS molecules ( Fig.17 A), or SAR114 parental monoclonal antibody or SAR114-BiS molecule ( Fig.17 B) were cultured together with serial dilutions. The graph represents the mean ± SD of two independent experiments. (See Example 9.)

[0053] Fig.18 is a series of graphs illustrating the efficacy of the anti-ClfA mAb / MEDI4893* bispecific antibody in a mouse model of bacteremia. Balb / c mice (n=10) were passively immunized IP with the indicated concentrations of SAR114 / MEDI4893*BiS2, BiS3, or a combination of SAR114+MEDI4893* and 24 hours later with Staphylococcus aureus isolate SF8300 (6e 7 cfu)( Fig.18 A) or 3049057(5e 7 cfu)( Fig.18 B) LD 90 IV infection. Fig.18 C) or 30419057( Fig.18 D) Challenge evaluation of the protective efficacy of 11H10 / MEDI4893*BiS2, BiS3 or 11H10+MEDI4893*mAb. Survival was monitored for 2 weeks. Results were analyzed using the log-rank (Mantel Cox) test. Statistical analysis relative to c-IgG was considered statistically different if p<0.05 and is indicated by an asterisk (*). Data represent three independent experiments. (See Example 10.)

[0054] Fig.19 is a series of graphs illustrating sequestration of SAR114 / MEDI4893* BiSAbs by ClfA in a mouse model of lethal pneumonia. C57 / B6 mice (n=10) were passively immunized IP with the indicated concentrations of BiS2, BiS3, MEDI4893*, or the SAR114+MEDI4893* mAb combination and 24 hours later with 1.5 e 6 cfu Staphylococcus aureus isolate SF8300 ( Fig.19 A) or SF8300ΔclfA isogenic mutant ( Fig.19B) Intranasal (IN) infection was performed. Survival was monitored for 6 days. Results were analyzed using the log-rank (Mantel-Cox) test. Statistical analysis relative to c-IgG was considered statistically different if p < 0.05. Data represent three independent experiments. (See Example 11.)

[0055] Fig. 20 Concentrations of SAR114, SAR114 N3F, and SAR114 N3Y are shown in cynomolgus monkeys over 60 days following administration of 5 mg / kg antibody.

[0056] Fig.21 is a graph illustrating the immunogenicity of wild-type and N3Y Fc regions using an ex vivo PBMC stimulation assay. DETAILED DESCRIPTION

[0057] The present invention provides antibodies and antigen-binding fragments thereof (e.g., monoclonal antibodies and antigen-binding fragments thereof) that bind to Staphylococcus aureus clumping factor A (ClfA) protein (and optionally also to Staphylococcus aureus alpha toxin (AT) protein). The present invention also provides compositions comprising such antibodies or fragments thereof, and methods of using such antibodies, fragments thereof or compositions.

[0058] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing) through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are referred to as α, δ, ε, γ, and μ, respectively, according to the identity of their heavy chain constant domains. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated with other molecules (such as toxins, radioisotopes, etc.).

[0059] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single pure line of B cells and bound to the same antigenic determinant. In contrast, the term "polyclonal antibody" refers to an antibody population produced by different B cells and bound to different antigenic determinants of the same antigen.

[0060] 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 may include an antigen-determining region (e.g., a complementary determining region (CDR)) of an intact antibody. 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. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially produced.

[0061] A complete antibody is usually composed of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain comprises an N-terminal variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain comprises an N-terminal variable (VL) region and a C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of the antibody. The VH and VL regions have the same general structure, each comprising four framework regions, whose sequences are relatively conservative. As used herein, the term "framework region" refers to a relatively conservative amino acid sequence within the variable region, which is located between the hypervariable region or the complementary determining region (CDR). There are four framework regions in each variable domain, which are named FR1, FR2, FR3 and FR4. The framework regions form beta sheets that provide the structural framework for the variable regions (see, e.g., CA Janeway et al. (Eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY (2001)). The three CDRs (called CDR1, CDR2, and CDR3) form the "hypervariable region" of the antibody, which is responsible for antigen binding.

[0062] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain aspects, CDRs may 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 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, the CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (which may optionally include one or two additional amino acids, 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the 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.

[0063] In contrast, Chthia refers to the location of a structural loop (Chthia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Depending on the length of the loop, the end of the Chthia CDR-H1 loop varies between H32 and H34 when numbered using the Kabat numbering convention (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, 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). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chthia structural loops and are used by the AbM antibody modeling software from Oxford Molecular.

[0064]

[0065] In one embodiment, the composition comprises a first antibody or an antigen-binding fragment thereof (e.g., a monoclonal antibody or fragment) that specifically binds to Staphylococcus aureus clumping factor A protein (ClfA) and a second antibody or an antigen-binding fragment thereof (e.g., a monoclonal antibody or fragment) that binds to Staphylococcus aureus alpha toxin (AT) protein. Among many Staphylococcus aureus surface adhesins, clumping factor A (ClfA) has been shown to play an important role in severe bloodstream infections (Foster et al., Nat. Rev. Microbiol., 12: 49-62 (2014); and Murphy et al., Hum. Vaccin., 7 (suppl.): 51-59 (2011)). CIfA binds fibrinogen and promotes bacterial adhesion to fibrinogen and bacterial agglutination, both of which are key attributes for the development of S. aureus bloodstream infections (Vaudaux et al., Infect. Immun., 63:585-590 (1995); McDevitt et al., Mol. Microbiol., 11:237-248 (1994); and McDevitt et al., Eur. J. Biochem., 247:416-424 (1997)). ClfA bound to fibrin or fibrinogen bound or coated on indwelling devices at the site of injury can promote bacterial colonization (Foster et al., supra) and bacterial agglutination, which is thought to enhance bacterial invasiveness (McDevitt et al., Eur. J. Biochem., 247:416-424 (1997); McAdow et al., PLoS Pathogens, 7:e1002307 (2011); Flick et al., Blood, 121:1783-1794 (2013); and Rothfork et al., J. Immunol., 171:5389-5395 (2003)). ClfA has also been reported to impair complement deposition required for opsonophagocytic bacterial killing (OPK) (Hair et al., Infection and Immunity, 78:1717-1727 (2010)). Consistent with these observations, the isogenic ΔclfA mutant exhibited reduced virulence in infection models (Mercado et al., supra; Josefsson et al., PLoS One, 3:e2206 (2008); and Josefsson et al., J Infect. Dis., 184:1572-1580 (2001)).Furthermore, passive immunization with human anti-ClfA-enriched intravenous (iv) immunoglobulin (Ig) (INH-A21 or Veronate) or monoclonal antibodies (tefibazumab or Aurexis) improves disease outcome in patients with S. aureus bloodstream infections (Vernachio et al., Antimicrobial Agents Chemother., 47:3400-3406 (2003); and Vernachio et al., Antimicrobial Agents Chemother., 50:511-518 (2006)). However, these antibody preparations failed to improve the results of clinical studies of prophylaxis or adjunctive therapy with vancomycin to prevent or treat S. aureus bacteremia in very low birth weight infants (DeJonge et al., J. Pediatr., 151:260-265 (2007); Capparelli et al., Antimicrobial Agents and Chemotherapy, 49:4121-4127 (2005); and Bloom et al., Pediatr. Infect. Dis., 24:858-866 (2005)). ClfA structure and function are described in detail in, for example, McDevitt et al., Mol. Microbiol., 11:237-248 (1994)).

[0066] Alpha toxin (AT) is a key virulence factor for several S. aureus diseases, including pneumonia, skin and soft tissue infections (SSTIs), and bacteremia (Bubeck Wardenburg, J. and Schneewind, O., J. Exp. Med., 205:287-294 (2008); Inoshima et al., J. Invest. Dermatol., 132:1513-1516 (2012); and Foretti et al., supra). Passive immunization with anti-AT monoclonal antibodies reduces disease severity in models of pneumonia and skin necrosis (Hua et al., Antimicrobial Agents and Chemotherapy, 58: 1108-1117 (2014); Tkachik et al., Clin. Vaccine Immunol., 19: 377-385 (2012); and Ragle, BE and Bubeck-Waldenburg, Infection and Immunity, 77: 2712-2718 (2009)), and vaccination with an AT toxoid containing the H35L mutation (ATH35L) prevents mortality in a lethal bacteremia and pneumonia model in mice (Bubeck-Waldenburg, supra, Foretti et al., supra, Hua et al., supra, Ragle, supra, Menzies, BE and Knodel, DS Kernodle, Infection and Immunity, 77:2712-2718 (2009); and Adhikari et al., PLoS One, 7:e38567 (2012). AT contributes to multiple aspects of S. aureus pathology during bacteremia and sepsis, including stimulation of the exaggerated inflammatory response characteristic of sepsis and activation of ADAM10-mediated cleavage of endothelial tight junctions, leading to loss of vascular integrity (Powers et al., J Infect. Dis., 206:352-356 (2012); Wilke, GA and Bubeck-Waldenburg, Proc. Natl. Acad. Sci. USA, 107:13473-13478 (2010); and Becker et al., J Innate Immun., 6:619-631 (2014)). AT has also been shown to target platelets, which may prevent repair of the damaged endothelial barrier and promote organ dysfunction through platelet-neutrophil aggregate formation (Bowers et al., Cell Host Microbe, 17:775-787 (2015)).The alpha toxin structure and function are described in detail in, for example, Bhakdi, S. and J. Tranum-Jensen, Microbiol. Mol. Biol. Rev., 55(4): 733-751 (1991).

[0067] Monoclonal and polyclonal antibodies that bind ClfA are known in the art (see, e.g., U.S. Pat. No. 7,364,738; Hall et al., Infection and Immunity, 71(12):6864-6870 (2003); and Farnejo et al., Antimicrobial Agents and Chemotherapy, 47(11):3400-3406 (2003)) and can be purchased from sources such as, for example, Creative Biolabs (Shirley, NY). As described above, while some anti-ClfA monoclonal antibodies (e.g., the 11H10 monoclonal antibody described in Tkachik et al., MBio., 7(3).pii:e00528-16 (2016)) have shown efficacy against S. aureus infection in a bacteremia model, such antibodies have been found to exhibit reduced affinity for ClfA and impaired inhibition of fibrinogen binding to the ClfA founder sequence (ClfA002) expressed by certain methicillin-resistant S. aureus (MRSA) strains. Accordingly, the present invention provides an antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody or fragment) that specifically binds to ClfA, has an affinity for three significant ClfA variants (including ClfA002) increased by more than 100-fold, and effectively inhibits bacterial agglutination of 112 different clinical isolates. In this regard, in one embodiment, the first antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody or fragment) of the composition described herein specifically binds to ClfA and comprises (i) a heavy chain polypeptide comprising a complementarity determining region 1 (CDR) amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, and (ii) a light chain polypeptide comprising, consisting essentially of, or consisting of a CDR1 amino acid sequence of SEQ ID NO: 4, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6. In another embodiment, the heavy chain polypeptide of the first antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) comprises, consists essentially of, or consists of a variable region amino acid sequence of SEQ ID NO: 13, and the light chain polypeptide of the first antibody or antigen-binding fragment comprises, consists essentially of, or consists of a variable region amino acid sequence of SEQ ID NO: 14. In certain instances, the antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) comprises a heavy chain constant domain comprising the amino acid sequence of CSYHLC (SEQ ID NO:21), MHEACSYHLCQKSLSLS (SEQ ID NO:23), or SEQ ID NO:24.

[0068] Monoclonal and polyclonal antibodies that bind to AT are also known in the art (see, e.g., Hua et al., Antimicrobial Agents and Chemotherapy, 58(2): 1108-1117 (2014); and Oganesyan et al., J. Biol. Chem., 289: 29874-29880 (2014)) and can be purchased from sources such as, for example, Sigma Aldrich (St. Louis, MO) and AbCam (Cambridge, MA). In one embodiment, the second antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) of the composition described herein specifically binds to the Staphylococcus aureus alpha toxin (AT) protein and comprises (i) a heavy chain polypeptide comprising the CDR1 amino acid sequence of SEQ ID NO: 7, the CDR2 amino acid sequence of SEQ ID NO: 8, and the CDR3 amino acid sequence of SEQ ID NO: 9 and (ii) a light chain polypeptide comprising the CDR1 amino acid sequence of SEQ ID NO: 10, the CDR2 amino acid sequence of SEQ ID NO: 11, and the CDR3 amino acid sequence of SEQ ID NO: 12. In another embodiment, the heavy chain polypeptide of the second antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) comprises, consists essentially of, or consists of the variable region amino acid sequence of SEQ ID NO: 15, and / or the light chain polypeptide of the second monoclonal antibody comprises, consists essentially of, or consists of the variable region amino acid sequence of SEQ ID NO: 16.

[0069] Sequences of exemplary anti-ClfA and anti-AT antibodies are provided below. In some cases, the antibodies or antigen-binding fragments thereof described herein bind to ClfA and / or AT and comprise the 6 CDRs of the antibodies listed in the following two tables (that is, the 3 VH CDRs of the antibodies listed in the first table and the three VL CDRs of the same antibodies listed in the second table). The anti-AT antibody MEDI4893 is a half-life extended (YTE) form of "LC10" previously described in International Patent Application Publications WO 2012 / 109285 and WO 2014 / 074540 (both of which are incorporated herein by reference in their entirety). MEDI4893* does not contain a YTE mutation.

[0070] VH CDR amino acid sequences

[0071]

[0072] VL CDR amino acid sequences

[0073]

[0074] In certain instances, an antibody or antigen-binding fragment thereof described herein binds to CIfA and / or AT and comprises a VH of an antibody listed in the table below, e.g., in combination with a VL.

[0075] Variable heavy chain (VH) amino acid sequence

[0076]

[0077]

[0078] In some cases, an antibody or antigen-binding fragment thereof described herein binds to CIfA and / or AT and comprises a VL of an antibody listed in the table below, e.g., in combination with a VH (optionally, a VH of the same antibody listed in the table above).

[0079] Variable light chain (VL) amino acid sequence

[0080]

[0081]

[0082] In certain instances, an antibody or antigen-binding fragment thereof described herein binds to CIfA and / or AT and comprises a heavy chain of an antibody listed in the table below, e.g., in combination with a light chain.

[0083] Full-length heavy chain amino acid sequence

[0084]

[0085]

[0086] In certain cases, an antibody or antigen-binding fragment thereof described herein binds to CIfA and / or AT and comprises a light chain of an antibody listed in the table below, e.g., in combination with a heavy chain (optionally, a heavy chain of the same antibody listed in the table above).

[0087] Full length light chain amino acid sequence

[0088]

[0089]

[0090] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be identified according to the Chothia numbering scheme, which refers to the positions 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 et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226). Typically, when using the Kabat numbering convention, the Chthia CDR-H1 loop occurs at heavy chain amino acids 26 to 32, 33, or 34, the Chthia CDR-H2 loop occurs at heavy chain amino acids 52 to 56, and the Chthia CDR-H3 loop occurs at heavy chain amino acids 95 to 102, while the Chthia CDR-L1 loop occurs at light chain amino acids 24 to 34, the Chthia CDR-L2 loop occurs at light chain amino acids 50 to 56, and the Chthia CDR-L3 loop occurs at light chain amino acids 89 to 97. Depending on the length of the loop, the end of the Chthia CDR-H1 loop varies between H32 and H34 when numbered using the Kabat numbering convention (this is because the Kabat numbering scheme places insertions at H35A and H35B; if both 35A and 35B are absent, 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).

[0091] In certain aspects, provided herein are antibodies and antigen-binding fragments thereof comprising the Jyotiai VH and VL CDRs of the SAR114 and / or MEDI4893* antibodies. In certain embodiments, the antibody or its antigen-binding fragment comprises one or more CDRs, wherein the Jyotiai and Kabat CDRs have the same amino acid sequence. In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof comprising a combination of Kabat CDRs and Jyotiai CDRs.

[0092] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be identified according to the IMGT numbering system, as described in Lefranc MP, (1999) The Immunologist 7:132-136 and Lefranc 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. In a specific embodiment, provided herein are antibodies and antigen-binding fragments thereof comprising the IMGT VH and VL CDRs of the SAR114 and / or MEDI4893* antibodies, e.g., as described in LeFrançoise (1999) supra and LeFrançoise et al., (1999) supra).

[0093] In certain aspects, the CDR of an antibody or its antigen-binding fragment may be determined according to MacCallum RM et al., (1996) Journal of Molecular Biology 262: 732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains", Antibody Engineering, Kontermann and Dübel, ed., Chapter 31, pp. 422 to 439, Springer-Verlag, Berlin (2001). In a specific embodiment, an antibody or its antigen-binding fragment is provided herein, comprising VH and VL CDRs of SAR114 and / or MEDI4893* antibodies, determined by the method of MacCallum et al.

[0094] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions that represent a compromise between the Kabat CDRs and the Chothia structure loops, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In a specific embodiment, provided herein is an antibody or antigen-binding fragment comprising the VH and VL CDRs of the SAR114 and / or MEDI4893* antibodies, as determined by the AbM numbering scheme.

[0095] In another embodiment, the antibodies or antigen-binding fragments thereof (e.g., monoclonal antibodies or fragments) described herein may comprise a constant region (Fc) of any suitable class (IgG, IgA, IgD, IgM, and IgE) that has been modified to improve effector function (e.g., opsonophagocytic bacterial killing (OPK)) or the half-life of the first and / or second antibodies or antigen-binding fragments (e.g., monoclonal antibodies or fragments) present in the composition. For example, the antibodies or antigen-binding fragments thereof (e.g., monoclonal antibodies or fragments) described herein may comprise an Fc comprising a mutation that extends the half-life relative to the same antibody without the mutation, and wherein the mutation does not inhibit OPK activity relative to the same antibody or antigen-binding fragment (mutation) without the mutation. Fc region engineering is widely used in the art to extend the half-life of therapeutic antibodies and prevent degradation in vivo. In some embodiments, the Fc region of an IgG antibody or antigen-binding fragment may be modified to increase the affinity of the IgG molecule for the neonatal Fc receptor (FcRn), which mediates IgG catabolism and protects the IgG molecule from degradation. The Fc region of any antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) described herein may comprise one or more amino acid substitutions or modifications that improve or extend antibody half-life or effector function (e.g., by increasing the affinity of the IgG molecule for FcRn). Suitable Fc region amino acid substitutions or modifications are known in the art and include, for example, the triple substitution M252Y / S254T / T256E (referred to as "YTE") (see, e.g., U.S. Patent No. 7,658,921; U.S. Patent Application Publication No. 2014 / 0302058; and Yu et al., Antimicrobial Agents and Chemotherapy, 61(1):e01020-16 (2017)). In another embodiment, the Fc region may be derived from the high affinity FcRn binding Fc variant N3E-YTE (see, e.g., Borrok et al., Journal of Biochemistry, 290(7):4282-4290 (2015)), which comprises C H 2 and YTE mutations of cysteine ​​residues at positions 432 and 437. For example, the N3E-YTE variant may lack the YTE mutation (referred to as "N3E"), or may be substituted at Fc residue 432 (using Kabat numbering) with, for example, the sequence CSWHLC (referred to as "N3"; SEQ ID NO: 19), CSFHLC (referred to as "N3F"; SEQ ID NO: 20), or CSYHLC (referred to as "N3Y"; SEQ ID NO: 21). The N3, N3F, and N3Y Fc variants specifically display enhanced pharmacokinetic (PK) properties (e.g., serum persistence) and effector functions (e.g., opsonophagocytic bacterial killing (OPK)) compared to the YTE variant.

[0096] The sequences of exemplary Fc variants are provided below.

[0097]

[0098] The present invention provides antibodies and antigen-binding fragments thereof (e.g., antibodies and antigen-binding fragments comprising CDR, VH and / or VL (heavy chain and / or light chain) or Fc variant sequences listed in the table above) that bind to ClfA and have IC50s against ClfA001, ClfA002 and ClfA004 within 2 μg / ml of each other in a fibrinogen binding inhibition assay. For example, the IC50s of the antibodies or antigen-binding fragments thereof against ClfA001, ClfA002 and ClfA004 are within 2 μg / ml of each other. 50 The binding affinity (K) of the antibody or its antigen-binding fragment to ClfA001, ClfA002 and ClfA004 can be between 1 μg / ml and 5 μg / ml. D ) can be between 200pM and 350pM.

[0099] The present invention provides antibodies and antigen-binding fragments thereof (e.g., antibodies and antigen-binding fragments comprising CDR, VH and / or VL (heavy chain and / or light chain) or Fc variant sequences listed in the table above) that bind to ClfA and have a monomer purity that decreases by no more than 5% after exposure to conventional white light at 2 kLux / hr at 23°C for 14 days.

[0100] The present invention is not limited to compositions comprising ClfA binding antibodies or antigen binding fragments and the above-described AT binding antibodies or antigen binding fragments. In fact, the present invention also separately provides antibodies or antigen binding fragments thereof (e.g., monoclonal antibodies or fragments), which comprise: (a) a heavy chain polypeptide comprising a CDR1 amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, and (b) a light chain polypeptide comprising a CDR1 amino acid sequence of SEQ ID NO: 4, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6.

[0101] The present invention also provides bispecific antibodies or antigen-binding fragments that bind (e.g., simultaneously) both ClfA and AT. The term "bispecific monoclonal antibody" (also referred to as a "dual specific" monoclonal antibody) refers to a monoclonal antibody that comprises two different antigen recognition domains and can therefore simultaneously bind to two different antigenic determinants. Monoclonal antibodies that recognize and bind to more than two different antigenic determinants are referred to in the art as "multispecific monoclonal antibodies." The first bispecific antibody was produced by somatic hybridization of two antibody-secreting cells, but the yield produced by random assembly of parental heavy and light chains was very low (Milstein, C. and A. Cuello, Immunol. Today, 5: 299-304 (1984)). The discovery of single-chain variable fragments (scFv) and advances in antibody engineering have resulted in new methods for developing bispecific antibodies (Orcutt et al., Protein Eng. Des. Sel., 23:221-228 (2010); and Coloma, MJ and SL Morrison, Nat. Biotechnol., 15:159-163 (1997)). There are at least 50 different bispecific antibody formats, depending on the scFv numbering and fusion position on the IgG scaffold (Kontermann, RE, MAbs, 4:182-197 (2012)). Bispecific and multispecific antibodies can be made in several different structural formats, including, but not limited to, tandem scFvs, diabodies, tandem diabodies, dual variable domain antibodies, and heterodimerization using motifs such as CH1 / Ck domains or Dock and Lock motifs (see, e.g., Chames, P. and D. Baty, Curr. Opin. Drug. Discov. Devel., 12:276-283 (2009)).In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody or fragment) that specifically binds to a Staphylococcus aureus ClfA protein and a Staphylococcus aureus alpha toxin (AT) protein (i.e., a bispecific antibody), comprising: (a) a first heavy chain polypeptide comprising a CDR1 amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, (b) a first light chain polypeptide comprising a CDR1 amino acid sequence of SEQ ID NO: 4, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6, (c) a second heavy chain polypeptide comprising a CDR1 amino acid sequence of SEQ ID NO: 7, a CDR2 amino acid sequence of SEQ ID NO: 8, and a CDR3 amino acid sequence of SEQ ID NO: 9, and (d) a second light chain polypeptide comprising a CDR1 amino acid sequence of SEQ ID NO: 10, a CDR2 amino acid sequence of SEQ ID NO: 11, and a CDR3 amino acid sequence of SEQ ID NO: 12. In another embodiment, the first heavy chain polypeptide and the first light chain polypeptide of the bispecific antibody or antigen-binding fragment thereof (e.g., monoclonal antibody or fragment) comprise the variable region amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and the second heavy chain polypeptide and the second light chain polypeptide of the bispecific antibody or antigen-binding fragment thereof (e.g., monoclonal antibody or fragment) comprise the variable region amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16, respectively. Such bispecific (optionally, monoclonal) antibodies (antibodies comprising SAR114 and MEDI4893 or MEDI4893* sequences) may have reduced AT-neutralizing activity compared to the AT-neutralizing activity of MEDI4893 or MEDI4893*, for example, as a result of the strong binding of SAR114 to ClfA. This is in contrast to other bispecific (optionally monoclonal) antibodies that bind to S. aureus ClfA and AT proteins (e.g., antibodies comprising 11H10 and MEDI4893 or MEDI4893* sequences), which have AT-neutralizing activity that is not significantly reduced compared to the AT-neutralizing activity of MEDI4893 or MEDI4893*.Methods for producing bispecific or multispecific (optionally, monoclonal) antibodies are known in the art and described, for example, in Holliger et al., Proc. Acad. Sci. USA, 90(14):6444-6448 (1993); Brinkmann, U. and Contemann, MAbs, 9(2):182-212 (2017); and Segal, DM and Bast, BJ. 2001. Production of Bispecific Antibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1–2.13.16).

[0102] The antibodies or antigen-binding fragments (e.g., monoclonal antibodies or fragments) described herein may be or may be obtained from human antibodies, humanized antibodies, non-human antibodies, or chimeric antibodies. A "chimeric" antibody refers to an antibody or fragment thereof that comprises human and non-human regions. A "humanized" antibody is an antibody that comprises 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, rodents (e.g., mice or rats). Humanized antibodies may comprise one, two, or three CDRs obtained or derived from non-human antibodies. Fully human antibodies do not contain any amino acid residues obtained or derived from non-human animals. It should be understood that fully human and humanized antibodies have a lower risk of inducing an immune response in humans than mouse or chimeric antibodies (see, e.g., Harding et al., mAbs, 2(3):256-26 (2010)). In one embodiment, the antibodies or antigen-binding fragments described herein are fully human antibodies.

[0103] Human antibodies, non-human antibodies, chimeric antibodies, or humanized antibodies can be obtained by any means, including in vitro sources (e.g., hybridomas or recombinant antibody-producing cell lines) and in vivo sources (e.g., rodents, human tonsils). Methods for producing antibodies are known in the art and described, for example, in Kohler et al. and Milstein, Eur. J. Immunol., 5:511-519 (1976); Harlow and Lane (Eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Press (CSH Press) (1988); and Janeway et al. (Eds.), Immunobiology, 5th ed., Garland Press, New York, NY (2001). In certain embodiments, human antibodies or chimeric antibodies can be produced using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE TM 、Kirin TC MOUSE TM KyowaKirin KM-MOUSE TM (See, e.g., Lonberg, Nature Biotechnology, 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). Humanized antibodies can be produced 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, NJ (2009)), including, for example, grafting non-human CDRs onto human antibody scaffolds (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, humanized antibodies can be produced using the methods described, for example, in U.S. Patent Application Publication No. 2011 / 0287485A1.

[0104] The invention also provides one or more isolated nucleic acid sequences encoding a ClfA binding antibody, an AT binding antibody, or an antibody that binds both ClfA and AT as described herein, or an antigen-binding fragment thereof (optionally, wherein the antibody or fragment is monoclonal). The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, that is, polynucleotides, which may be single-stranded or double-stranded and may contain non-natural or altered nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. The terms include analogs of RNA or DNA as equivalents, which are made from nucleotide analogs and modified polynucleotides (such as (but not limited to) methylated and / or capped polynucleotides). Nucleic acids are typically linked by phosphate bonds to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).

[0105] The invention further provides one or more vectors comprising one or more nucleic acid sequences encoding a ClfA binding antibody, an AT binding antibody, or an antibody that binds both ClfA and AT as described herein, or an antigen-binding fragment thereof (optionally, wherein the antibody or fragment is monoclonal). The vector can be, for example, a plastid, an episome, an adhesin, a viral vector (e.g., a retrovirus or an adenovirus), or a bacteriophage. Suitable vectors and methods for preparing vectors are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor Laboratory, NY (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0106] In addition to the nucleic acid sequence encoding a CIfA binding antibody or antigen binding fragment, an AT binding antibody or antigen binding fragment, or an antibody or antigen binding fragment that binds both CIfA and AT as described herein (optionally, wherein the antibody or fragment is monoclonal), the vector desirably comprises expression control sequences, such as a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), and the like, which provide for expression of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0107] Vectors comprising nucleic acids encoding the amino acid sequences of antibodies or antigen-binding fragments described herein (e.g., amino acid sequences encoding the heavy and / or light chains of ClfA binding antibodies) (as the case may be, monoclonal antibodies or fragments) can be introduced into host cells (including any suitable prokaryotic or eukaryotic cells) capable of expressing the polypeptides encoded thereby. Thus, the present invention provides an isolated cell comprising a vector. Host cells that can be used include those that can be easily and reliably grown, have a reasonably fast growth rate, have a well-characterized expression system, and can be easily and efficiently transformed or transfected. Examples of suitable prokaryotic cells include, but are not limited to, cells from the genus Bacillus (e.g., Bacillus subtilis and Bacillus brevis), the genus Escherichia (e.g., E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include various strains of E. coli (e.g., K12, HB101 (ATCC No. 33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102). Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. In one embodiment, the vector is expressed in a mammalian cell. Several suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and CV-1 cell line (ATCC No. CCL70). Mammalian cells are ideally human cells. For example, the mammalian cell may be a human lymphoid or lymphoid-derived cell line, such as a cell line derived from pre-B lymphocytes, PER. cell lines (Crucell Holland BV, The Netherlands), or human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573).

[0108] The nucleic acid sequence encoding the amino acids of any antibody or antigen-binding fragment described herein (optionally, a monoclonal antibody or fragment) can be introduced into a cell by "transfection", "transformation" or "transduction". As used herein, "transfection", "transformation" or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA coprecipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten-promoted microprojectile bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after growing infectious particles in suitable packaging cells, many of which are commercially available.

[0109] The present invention provides a composition comprising an effective amount of any one or combination of the antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier. In one embodiment, for example, the composition may comprise a first antibody or antigen-binding fragment thereof (optionally, monoclonal) that specifically binds to the Staphylococcus aureus ClfA protein as described above, and a second antibody or antigen-binding fragment thereof (optionally, monoclonal) that specifically binds to the Staphylococcus aureus AT protein as described above, and a pharmaceutically acceptable carrier. Alternatively, the composition may comprise an antibody or antigen-binding fragment thereof that specifically binds to the Staphylococcus aureus ClfA protein or an antibody or antigen-binding fragment thereof that specifically binds to the Staphylococcus aureus AT protein and a pharmaceutically acceptable carrier. In yet another embodiment, the composition may comprise a nucleic acid sequence encoding a ClfA binding antibody or antigen-binding fragment, an AT binding antibody or antigen-binding fragment and / or an anti-ClfA / AT bispecific antibody or antigen-binding fragment, or one or more vectors comprising such nucleic acid sequences. In one embodiment, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition comprising a carrier (such as a pharmaceutically acceptable (e.g., physiologically acceptable) carrier), and a ClfA binding antibody or antigen binding fragment, an AT binding antibody or antigen binding fragment, an anti-ClfA / AT bispecific antibody or antigen binding fragment, a nucleic acid sequence or a vector. Any suitable carrier may be used in the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined in part by the specific site to which the composition may be administered and the specific method used to administer the composition. Optionally, the composition may be sterile. The composition may be stored frozen or freeze-dried and reconstituted in a suitable sterile carrier prior to use. The composition may be produced according to conventional techniques described, for example, in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0110] The composition desirably comprises a ClfA binding antibody and / or an AT binding antibody, and / or an anti-ClfA / AT bispecific antibody, or an antigen-binding fragment thereof (e.g., a monoclonal antibody or fragment) in an amount effective to treat or prevent a S. aureus infection. Thus, the present invention provides a method of treating or preventing a S. aureus infection in an individual (e.g., a human), the method comprising administering to an individual in need thereof a composition comprising any one or combination of the antibodies or antigen-binding fragments thereof (e.g., monoclonal antibodies or fragments) described herein, thereby treating or preventing a S. aureus infection in the individual. The present invention also provides a use of a ClfA binding antibody or antigen-binding fragment, an AT binding antibody or antigen-binding fragment, and / or an anti-ClfA / AT bispecific antibody or antigen-binding fragment described herein, or a composition comprising any one or combination of the antibodies or fragments thereof described herein in the manufacture of a medicament for treating or preventing a S. aureus infection. As described herein, S. aureus is a major human pathogen causing a wide range of clinical infections. Staphylococcus aureus is the main cause of bacteremia and infective endocarditis, as well as bone and joint infections, skin infections and soft tissue infections, pleural and lung infections, and device-related infections. About 30% of the population is infected with Staphylococcus aureus (Wertheim et al., Lancet Infect. Dis., 5:751-762 (2005)). Symptoms of Staphylococcus aureus skin infections include, for example, ecthyma, cellulits, and impetigo. Staphylococcus aureus can also cause food poisoning, blood poisoning (also known as bacteremia), toxic shock syndrome, and septic arthritis. The epidemiology, pathophysiology, and clinical manifestations of S. aureus infection are described in detail in, for example, Tong et al., Clin. Microbiol. Rev., 28(3):603-661 (2015), and the genomes of several different S. aureus strains have been sequenced (see, for example, GenBank / EMBL Accession Nos. BX571856, BX571857, BX571858, FN433596, FN433597, FN433598, HE681097, FR821777, FR821778, FR821779, and FR821780). As described herein, an individual (e.g., a human individual) may suffer from diabetes.

[0111] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. In one embodiment, the effect is therapeutic, that is, the effect partially or completely cures the disease and / or the adverse symptoms caused by the disease. To this end, the disclosed methods include administering a "therapeutically effective amount" of a ClfA binding antibody, an AT binding antibody, and / or an anti-ClfA / AT bispecific antibody, or an antigen-binding fragment thereof, or a composition comprising any one or a combination of the foregoing antibodies or fragments (including monoclonal antibodies or fragments). A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the necessary dosage and for the necessary period of time. A therapeutically effective amount can vary depending on factors such as the disease state, the 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. For example, a therapeutically effective amount of a CIfA binding antibody or antigen-binding fragment thereof, an AT binding antibody or antigen-binding fragment thereof, or a CIfA / AT bispecific antibody or antigen-binding fragment thereof is an amount that inhibits S. aureus-associated sepsis, inhibits S. aureus agglutination, inhibits thromboembolic lesion formation, neutralizes alpha toxin, induces opsonophagocytosis, inhibits S. aureus fibrinogen binding, inhibits S. aureus agglutination, or any combination of the foregoing in a human.

[0112] Alternatively, the pharmacological and / or physiological effects may be preventive, that is, the effects prevent, in whole or in part, the disease or its symptoms. In such aspects, the disclosed methods include administering a "prophylactically effective amount" of a ClfA binding antibody, an AT binding antibody and / or an anti-ClfA / AT bispecific antibody, or an antigen-binding fragment thereof (including a monoclonal antibody or fragment). A "prophylactically effective amount" refers to an amount effective to achieve a desired preventive result (e.g., preventing S. aureus infection or disease onset) at the necessary dosage and for the necessary period of time.

[0113] The therapeutic or preventive efficacy can be monitored by regularly evaluating the treated patient. For repeated administration over several days or longer, depending on the condition, the treatment can be repeated until the desired disease symptom suppression occurs. However, other dosage regimens may be useful and within the scope of the present invention. The desired dose can be delivered by administering the composition as a single bolus, by administering the composition as multiple boluses, or by administering the composition as a continuous infusion.

[0114] Methods of treating or preventing S. aureus infection may comprise administering a ClfA binding antibody as described herein, an AT binding antibody as described herein, both a ClfA binding antibody and an AT binding antibody as described herein, or a ClfA-AT bispecific antibody as described herein, or an antigen-binding fragment thereof. In embodiments where a ClfA binding antibody and an AT binding antibody or fragment (e.g., a monoclonal antibody or fragment) are administered to an individual, each antibody or fragment may be present in the same composition or in separate compositions. When separate compositions are administered to an individual, each composition may be administered simultaneously or sequentially in any order.

[0115] Compositions comprising an effective amount of an antibody described herein, or any one or combination of its antigen-binding fragments, encoding any of the foregoing nucleic acid sequences, or vectors comprising the nucleic acid sequences can be administered to an individual (such as a human) using standard administration techniques (including intravenous, intraperitoneal, subcutaneous, and intramuscular routes of administration). The composition may be suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In some embodiments, the composition is administered to an individual using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0116] CIfA binding antibodies or antigen binding fragments, AT binding antibodies or antigen binding fragments, and / or anti-CIfA / AT bispecific antibodies or antigen binding fragments, or compositions comprising the same can be administered alone or in combination with other drugs routinely used to treat S. aureus infections (e.g., as adjuvants). Compositions comprising CIfA binding antibodies or antigen binding fragments, AT binding antibodies or antigen binding fragments, or CIfA-AT bispecific antibodies or antigen binding fragments can be used in combination with, for example, one or more antibiotics such as penicillinase-resistant β-lactam antibiotics (e.g., oxacillin or flucloxacillin). Gentamicin can be used to treat serious infections such as endocarditis. However, most strains of S. aureus are now resistant to penicillin, and two in 100 people carry methicillin-resistant strains of S. aureus (MRSA). MRSA infections are typically treated with vancomycin, and mild skin infections can be treated with triple antibiotic ointment.

[0117] In addition to therapeutic uses, any one or combination of the antibodies described herein may be used for diagnostic or research applications. In this regard, the CIfA binding antibody or antigen binding fragment, AT binding antibody or antigen binding fragment, or CIfA-AT bispecific antibody or antigen binding fragment may be used in an assay to monitor an individual for S. aureus infection. Research applications include, for example, methods using the CIfA binding antibody or antigen binding fragment, AT binding antibody or antigen binding fragment, or CIfA-AT bispecific antibody or antigen binding fragment and a label to detect S. aureus in a sample (e.g., in a human fluid or in a cell or tissue extract). The CIfA binding antibody or antigen binding fragment, AT binding antibody or antigen binding fragment, or CIfA-AT bispecific antibody or antigen binding fragment may be used with or without modification (e.g., covalently or non-covalently labeled with a detectable moiety). For example, the detectable moiety may be a radioactive isotope (e.g., 3 H. 14 C. 32 P. 35 S or 125 I), fluorescent or chemiluminescent compounds (e.g., fluorescein isothiocyanate, rhodamine or luciferin), enzymes (e.g., alkaline phosphatase, β-galactosidase or horseradish peroxidase) or promotor. Any method known in the art for coupling antibodies or antigen-binding fragments thereof individually to detectable moieties can be used in the context of the present invention (see, e.g., Hunter et al., Nature, 194:495-496 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Meth., 40:219-230 (1981); and Nygren, J., Histochem. And Cytochem., 30:407-412 (1982)).

[0118] Any one or combination of the antibodies, or antigen-binding fragments thereof (e.g., monoclonal antibodies or fragments), nucleic acid sequences encoding any of the foregoing, vectors comprising the nucleic acid sequences, or compositions comprising any of the foregoing described herein may be provided in a kit (that is, a packaged combination of predetermined amounts of reagents and instructions for performing a diagnostic assay). If the CIfA binding antibody or antigen-binding fragment, AT binding antibody or antigen-binding fragment, or CIfA-AT bispecific antibody or antigen-binding fragment is labeled with an enzyme, the kit desirably includes the substrate and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives may be included in the kit, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), and the like. The relative amounts of the various reagents may be varied to provide concentrations of the reagents in solution that generally optimize the sensitivity of the assay. The reagents may be provided as dry powders (typically freeze-dried), including excipients that, when dissolved, will provide a reagent solution having a suitable concentration.

[0119] The following examples further illustrate the present invention but, of course, should not be construed as limiting the scope of the present invention in any way.

[0120] Example 1

[0121] The example demonstrates the selection and characterization of a monoclonal antibody that specifically binds to the S. aureus CIfA protein.

[0122] It has been previously reported that in a Staphylococcus aureus lethal bacteremia model, prophylaxis with a combination of an anti-S. aureus alpha toxin (AT) monoclonal antibody (mAb) (referred to as "MEDI4893*," described in International Patent Application Publication Nos. WO 2012 / 109285, and "LC10," described in WO 2014 / 074540) and an anti-ClfA mAb (referred to as "11H10") provided enhanced protection and isolate coverage relative to prophylaxis with a single mAb (Tkachik et al., Int. J. Acad. Sci., 7(3).pii:e00528-16 (2016)). (It should be noted that MEDI4893, which contains a YTE mutation not present in MEDI4893*, was not used in mice because, although the YTE mutation increases IgG half-life in humans, it decreases serum exposure in mice.) Although 11H10 showed potent anti-ClfA activity, in a fibrinogen binding inhibition assay, 11H10 exhibited a greater than 1000-fold reduced affinity (K) for the ClfA founder sequence ClfA002 relative to the other ClfA founder sequences ClfA001 and ClfA004. on Below the detection limit, ND in Table 1) and about 40-fold increased IC 50 ,like Figure 1A and Table 1. ClfA002 is expressed by prominent S. aureus hospital-acquired MRSA (HA-MRSA; USA100 or sequence type 5 (ST5)) (Sharma-Kuinkel et al., J. Clin. Microbiol., 53:227-236 (2015); and Mendes et al., J. Clin. Microbiol., 50:3694-3702 (2012)).

[0123] Table 1. Anti-ClfA mAbs: Correlation between affinity and in vitro activity

[0124]

[0125]

[0126] To increase the potential coverage of clinical isolates, human tonsil B cell libraries were screened for more broadly reactive anti-ClfA mAbs. Specifically, memory B cells were isolated from cryopreserved lymphocytes isolated from tonsils using phycoerythrin (PE)-Cy7 labeled CD19 microbeads (BD Biosciences, San Jose, CA), followed by staining with anti-PE beads (Miltenyi Biotec, Inc., San Jose, CA), and depletion of IgM, IgD, and IgA-bearing cells by cell sorting on a FACSAria (BD Biosciences, San Jose, CA). Cells were immortalized under asexual propagation conditions using Epstein Barr virus (EBV) as described in Traggiai et al., Nat. Med., 10:871-875 (2004). Two weeks later, culture supernatants were screened for the presence of ClfA001-specific monoclonal antibodies using a 384-well format ELISA assay. Briefly, serial dilutions of anti-ClfA mAb (1:2 or 1:600) were added to ClfA-coated plates, followed by the addition of biotinylated 11H10 (1:600). Percent competition was calculated as 100*(OD mAb+11H10biot ) / (OD 11H10biot )). Positive cultures were expanded in complete RPMI medium and selected for their ability to bind with high affinity to CIfA genotypes 001, 002 and 004. VH and VL sequences were retrieved by RT-PCR.

[0127] Through these efforts, a monoclonal antibody (termed "SAR114") was identified that displayed high affinity for CIfA001, 002, and 004 (KD = 1.15-44.7 pM, Table 1) and potent inhibition of fibrinogen binding by the three significant founder CIfA genotypes (IC 50 ~20μM), such as Figure 1 As shown in B. SAR114 also exhibited opsonophagocytic killing (OPK) activity against several clinical isolates of S. aureus, such as Figure 3 As shown in A-3F (see, e.g., Tkachik et al., supra, for methods of determining OPK killing) and the improvement in inhibition of bacterial agglutination in human plasma compared to 11H10, as follows Figure 2 , Figure 4 and shown in Table 2). Agglutination inhibition in human plasma was determined by culturing 112 clinical isolates of S. aureus in tryptic soy mixture (TSB) overnight, washing in PBS, and suspending in ice-cold PBS to one-tenth of the original volume. Anti-ClfA monoclonal antibodies were serially diluted (2-fold) starting at 200 μg / ml in 30 μl PBS and mixed with 30 μl of citrated human plasma in a 96-well U-bottom plate (ThermoFisher Scientific, Waltham, MA). Bacteria (30 μl) were added and incubated at 37°C for 5 minutes. Each well was visually evaluated and the lowest monoclonal antibody concentration in the case of bacterial agglutination was recorded. R347 (human anti-gp120 monoclonal antibody) was used as an isotype control human IgG1 (c-IgG). The human negative control monoclonal antibody (c-IgG) did not show any inhibition up to 200 μg / ml.

[0128] Table 2: Minimum SAR114 concentration required to inhibit bacterial agglutination.

[0129]

[0130]

[0131] Data are representative of three independent experiments using the same donor as plasma source.

[0132] The heavy chain polypeptide of the SAR114 antibody was determined to comprise the variable region amino acid sequence of SEQ ID NO: 13, the CDR1 amino acid sequence of SEQ ID NO: 1, the CDR2 amino acid sequence of SEQ ID NO: 2, and the CDR3 amino acid sequence of SEQ ID NO: 3. The light chain polypeptide of the SAR114 antibody was determined to comprise the variable region amino acid sequence of SEQ ID NO: 14, the CDR1 amino acid sequence of SEQ ID NO: 4, the CDR2 amino acid sequence of SEQ ID NO: 5, and the CDR3 amino acid sequence of SEQ ID NO: 6. It was found that 11H10 and SAR114 competed for binding to ClfA001 by ELISA and in competition assays based on Octet, as shown in FIG. Figure 5 As shown in A and 5B. Briefly, mAb diluted in PBS at 5 μg / ml was captured on aminopropylsilane (APS) biosensors for 7 min. The coated biosensors were moved to wells containing blocking buffer (PBS, 1 mg / ml BSA (Sigma-Aldrich, St. Louis)) for 6 min to block free sensor binding sites, incubated with 2.5 μg / ml ClfA001 diluted in blocking buffer for 7 min, and finally moved to wells containing competing mAb diluted in blocking buffer at 5 μg / ml. Data were analyzed using the Data Acquisition and Analysis software (Pall ForteBio LLC, Fremont, CA). Association of no competing mAb results in competition and thus recognition of the same antigenic site, whereas no competition is observed when association of a second mAb is detected.

[0133] The results of the examples indicate that anti-CIfA mAbs 11H10 and SAR114 bind overlapping epitopes on CIfA001, suggesting that the differences in their activities against CIfA002 may be due to different binding affinities.

[0134] Example 2

[0135] The examples describe the generation of SAR114-N3, which has an increased half-life compared to SAR114.

[0136] It is well known that the Fc region of an antibody plays a role in its half-life, and Fc engineering has been used to manipulate the half-life of therapeutic biologics. For example, the triple amino acid substitution M252Y / S254T / T256E (referred to as the "YTE" substitution) has been engineered into the Fc region of antibodies (including the anti-Staphylococcus aureus MEDI4893 antibody), and it can result in a 3- to 4-fold increase in half-life. However, the YTE mutation has also been shown to result in reduced binding to C1q and FcγR and reduced effector function (such as ADCC and CDC activity). (Monnet C. et al., Front Immunol. 6:39 (2015)). The YTE mutation also reduces opsonophagocytic killing (OPK) of anti-bacterial antibodies. (See Figure 6 , showing that YTE substitution reduces OPK of the anti-Pseudomonas antibody Cam004). Thus, although an anti-ClfA antibody is desired to have an extended half-life, YTE mutation is not suitable for SAR114.

[0137] Borrok et al. (Journal of Biological Chemistry, 290(7):4282-4290 (2015)) studied the effects of other Fc alterations, including the "N3" variant. The "N3" variant modified to include the sequence CSWHLC (SEQ ID NO: 19) in the CH3 domain instead of the wild-type sequence (LHNHYT; SEQ ID NO: 22) at the same position. The Fc variant also increased half-life (see Fig. 9 , above), but if Figure 6 As shown in , OPK killing by the anti-bacterial antibodies Cam004 (upper panel) or 2F4 (lower panel) was not reduced.

[0138] Therefore, the effect of the N3 mutation on the binding of SAR114 was evaluated. In these experiments, the kinetic rate / affinity (K) of binding of the parental and Fc variant antibodies SAR-114 and SAR114-N3 to CLFA001, CLFA002 and CLFA004 proteins, respectively, was determined using the Biacore platform. D The results are shown in Table 3 below.

[0139] Table 3. SAR-114 and SAR114-N3 binding affinities for the three major ClfA genotypes

[0140] Capture sample <![CDATA[Ka(M -1 s -1 )]]> <![CDATA[Kd(s -1 )]]> <![CDATA[K D (pM)]]> SAR114-N3 CLFA001 3.383E+6 7.725E-4 228 SAR114-N3 CLFA002 3.921E+6 10.41E-4 265 SAR114-N3 CLFA004 2.387E+6 7.558E-4 316 SAR114 CLFA001 3.911E+6 6.013E-5 15.4 SAR114 CLFA002 3.816E+6 1.821E-4 47.7 SAR114 CLFA004 2.968E+6 6.210E-5 20.9

[0141] Kinetic fits to a 1:1 binding model were adequate. Both SAR114 and SAR114-N3 bound to CLFA001, CLFA002, and CLFA004 with similar affinities. However, SAR114 binding to all CLFA proteins (K D) binds about 10-fold stronger than SAR114-N3. The weaker binding of SAR114-N3 can be attributed to a faster dissociation rate.

[0142] The ability of SAR114-N3 to inhibit fibrinogen binding was also evaluated. In these assays, ClfA binding to fibrinogen was determined in the presence of serial dilutions (200 to 0.5 μg / ml) of SAR114, SAR114-N3, or control IgG antibodies. The results are shown below Figure 7 and Table 4.

[0143] Table 4. Inhibition of fibrinogen binding by SAR-114 and SAR114-N3

[0144] IC50(μg / ml) SAR114 SAR114-N3 ClfA001 2.576 2.134 ClfA002 2.910 3.108 ClfA004 1.720 2.516

[0145] These data demonstrate that SAR114-N3 inhibits the binding of the three major ClfA genotypes to fibrinogen.

[0146] Example 3

[0147] The Examples describe the generation of SAR114-N3Y, which has improved stability over SAR114-N3.

[0148] However, the "N3" variant contains tryptophan (W434), which contributes to enhanced FcRn affinity, but also results in photosensitivity, such that normal light conditions result in approximately 20% monomer loss within a week, and high light conditions result in more than 60% monomer loss within the same period. Tryptophan (W434) was substituted with non-oxidizable hydrophobic residues (F, Y, L, I, V, A, and S), and their effects on SAR114 half-life, OPK, and photosensitivity were evaluated. The F and Y substitutions were most similar to SAR114 N3 in terms of binding, and both had similar half-life extensions as the N3 changes (see Fig. 9 , upper figure). SAR114 antibodies with changes in F ("N3F"; SEQ ID NO: 20) and Y ("N3Y"; SEQ ID NO: 21) were subjected to photostability assessment. In the assessment, mAbs were exposed to CWL (conventional white light) at 2 kLux / hr (intensity measurement) for 14 days at 23°C, and the observed monomer purity is summarized in Table 5 below.

[0149] Table 5. Stability of SAR114-N3F and SAR114-N3Y

[0150]

[0151] The results confirmed that N3Y has excellent photostability. Agglutination and fibrinogen potency assays also indicated that N3Y has better activity than N3F, as Figure 8 As shown in . Determination of antibody concentrations in mice confirmed that both N3Y and N3F had similar pK values ​​to the N3 mutant ( Fig. 9 , upper panel), and OPK killing of the N3Y variant was unchanged ( Fig. 9 , figure below).

[0152] Example 4

[0153] The Examples describe the effects of SAR114 or SAR114 N3Y anti-CIfA monoclonal antibodies alone or in combination with anti-alpha toxin (AT) monoclonal antibodies in a murine bacteremia model.

[0154] Groups of 10 6- to 8-week-old female BALB / c mice (Envigo, Huntingdon, Cambridgeshire, United Kingdom) were passively immunized by intraperitoneal (IP) injection of isotype control IgG (c-IgG), SAR114 anti-ClfA mAb, and / or MEDI4893* anti-AT mAb. LD 90 Mice were challenged with clinical isolates of Staphylococcus aureus. Survival was monitored over two weeks. Statistical analysis of specific anti-staphylococcal antigens and c-IgG was performed using the log-rank (Mantel-Cox) test. Data were considered statistically different if p < 0.05.

[0155] Prophylaxis with SAR114 (15 mg / kg (mpk)) resulted in increased survival after challenge with S. aureus isolates representing sequence types (ST) ST8, ST5, or ST30, which were confirmed to encode ClfA genotypes ClfA001, 002, and 004, respectively, compared to isotype control IgG (c-IgG). Fig.10 Similar to the combination of 11H10 and MEDI4893* antibodies, prophylaxis with a combination of SAR114 and MEDI4893* (7.5 mg / kg (mpk) each) significantly increased survival relative to c-IgG after challenge with all strains tested and provided benefits over the corresponding single monoclonal antibodies against certain strains (Tkachik et al., supra).

[0156] The results of these experiments demonstrate that the anti-CIfA SAR114 monoclonal antibody is functional in vivo and suggest that the combination of SAR114 with an anti-AT monoclonal antibody provides broader strain coverage for S. aureus prevention.

[0157] Example 5

[0158] The example describes the effect of SAR114 anti-ClfA monoclonal antibody alone or in combination with anti-alpha toxin (AT) monoclonal antibody in a diabetic murine lethal bacteremia model.

[0159] Six-week-old diabetic BKS.Cg-Dock7 mice were treated with isotype control IgG (c-IgG), SAR114 anti-ClfA mAb, and / or MEDI4893* anti-AT mAb via intraperitoneal (IP) injection. m + / +Lepr db / J male mice were immunized. 24 hours later, LD 90 Mice were challenged with 5e7 CFU of S. aureus clinical isolate SF8300. Survival was monitored over two weeks. Fig.11 , above. Ten animals were euthanized 48 hours later to obtain Fig.11 , middle image) and liver ( Fig.11 , lower figure) were used for bacterial counts. Statistical analysis of specific anti-staphylococcal antigens and c-IgG was performed using the log-rank (Mantel-Cox) test. Data were considered statistically different if p < 0.05.

[0160] Prophylaxis with SAR114 (15 mg / kg (mpk)) resulted in increased survival after challenge compared to isotype control IgG (c-IgG), and prophylaxis with a combination of SAR114 and MEDI4893* (7.5 mg / kg (mpk) each) significantly increased survival relative to c-IgG after challenge. Fig.11 , upper figure. The combination of SAR114 and MEDI4893* (7.5 mg / kg (mpk) each) also significantly reduced kidney ( Fig.11 , middle image) and liver ( Fig.11 , below).

[0161] The results of these experiments demonstrate that the anti-ClfA SAR114 monoclonal antibody is functional in vivo and indicate that the combination of SAR114 and anti-AT monoclonal antibodies protects against CA-MRSA SF8300-induced lethal bacteremia in diabetic db / db mice.

[0162] Example 6

[0163] The example describes the effect of SAR114 anti-ClfA monoclonal antibody alone or in combination with anti-alpha toxin (AT) monoclonal antibody on pro-inflammatory cytokine concentrations in a murine bacteremia model.

[0164] Cohorts of 6-week-old diabetic (db) BKS.Cg-Dock7 mice were injected intraperitoneally (IP) with isotype control IgG (c-IgG), SAR114 anti-ClfA mAb, and / or MEDI4893* anti-AT mAb. m + / +Lepr db / J male mice (n=20) and non-diabetic C57 / B6 (B6) male mice (n=20) were immunized. LD 90 Mice were challenged with the clinical isolate SF8300 of Staphylococcus aureus. Ten animals in each group were euthanized after 8 or 24 hours, and blood was collected from cardiac puncture. Proinflammatory cytokines were measured from plasma using the Mesoscale Multiplex Proinflammatory Cytokines Kit. Statistical differences between groups were analyzed using the Mann-Whitney U test, and if p < 0.05, they were considered statistically different.

[0165] As shown in Table 6, the combination of SAR114 and MEDI4893* significantly reduced IL-6, TNF-α, and KC concentrations at 24 hours in diabetic db / db mice.

[0166] Table 6: SAR114 and MEDI4893* reduce pro-inflammatory cytokines

[0167]

[0168] Data are representative of three independent experiments.

[0169] The combination also significantly reduced IL-6, TNF-α, and KC concentrations at 24 hours in non-diabetic C57 / B6 mice.

[0170] Example 7

[0171] The Example describes the effect of SAR114 anti-ClfA monoclonal antibody alone or in combination with anti-alpha toxin (AT) monoclonal antibody on liver injury in a diabetic murine bacteremia model.

[0172] Six-week-old diabetic BKS.Cg-Dock7 mice were injected intraperitoneally (IP) with isotype control IgG (c-IgG), SAR114 anti-ClfA monoclonal antibody (15 mg / kg (mpk)), MEDI4893* anti-AT monoclonal antibody (15 mpk), or a combination of SAR114+MEDI4893* (15 mpk each). m + / +Lepr db / J male mice (n=10) were immunized. 24 hours later, LD90 Mice were challenged with the clinical isolate of Staphylococcus aureus SF8300. Mice were euthanized 48 h after infection and the livers were harvested. Gross pathology was recorded by photographs ( Fig.12 , left), and liver sections were stained with hematoxylin / eosin after fixation with 10% formalin ( Fig.12 , right). SAR114 antibody, MEDI4893* antibody, and the combination of SAR114+MEDI4893* all prevent liver damage in diabetic mice exposed to S. aureus.

[0173] Example 8

[0174] The Examples describe the effects of the SAR114 anti-ClfA monoclonal antibody alone or in combination with an anti-alpha toxin (AT) monoclonal antibody in a murine diabetic bacteremia model.

[0175] Six-week-old diabetic BKS.Cg-Dock7 mice were treated with isotype control IgG (c-IgG), SAR114 anti-ClfA mAb, and / or MEDI4893* anti-AT mAb via intraperitoneal (IP) injection. m + / +Lepr db / J male mice (n=10) were immunized. 24 hours later, LD 90 Mice were challenged with clinical isolates of Staphylococcus aureus. Survival was monitored over a two-week period. Fig.13 .

[0176] The combination of SAR114 and MEDI4893* (7.5 mg / kg (mpk) each) increased survival relative to c-IgG following challenge with most strains tested and also provided benefit over the corresponding single monoclonal antibodies in most strains.

[0177] The results of these experiments demonstrate that the anti-CIfA SAR114 mAb is functional in vivo and suggest that the combination of SAR114 and anti-AT mAb provides broader strain coverage for the prevention of S. aureus in diabetic mice.

[0178] Example 9

[0179] The Examples describe the generation of a bispecific monoclonal antibody that specifically binds to both CIfA and AT and its in vitro efficacy.

[0180] Because passive immunization using a combination of anti-ClfA monoclonal antibodies and anti-AT monoclonal antibodies provides benefits for strain coverage of lethal bacteremia and retains anti-AT protective capacity in mouse skin necrosis and pneumonia models (Tkachik et al., supra), bispecific antibodies (BiSAbs) against ClfA and AT were generated to determine whether such bispecific antibodies provide benefits over the combination of the corresponding individual antibodies. To this end, BiSAbs were engineered as previously described (see, e.g., Dimasi et al., J. Mol. Biol., 393:672-692 (2009); and Coloma, MJ and SL Morrison, Nature Biotechnology, 15:159-163 (1997)). Briefly, anti-ClfA mAbs 11H10 or SAR114 were used as IgG scaffolds, and MEDI4893* was grafted in scFv format. MEDI4893*scFv was synthesized in VL-VH format with a 20 amino acid (GGGGSx4) linker between the light and heavy variable domains (GeneArt, Thermo Fisher Scientific, Waltham, MA). The "BiS2" antibody was constructed by fusing the MEDI893*scFv sequence to the N-terminus of the heavy chain of 11H10 or SAR114 anti-ClfA IgG1. The "BiS3" construct was generated by appending the linker-scFv of MEDI4893* to the C-terminus of the heavy chain of 11H10 or SAR114. The BiS2 and BiS3 constructs were constructed in Fig.14 Schematically illustrated in Figure 2. BiS2 and BiS3 molecules were expressed by transient transfection in 293 cells, purified by protein A affinity chromatography, and refined by size exclusion chromatography. The integrity of each molecule was assessed by mass spectrometry and by intact mass and peptide mapping to verify the correct formation of the engineered and endogenous disulfide bonds. The BiS2 and BiS3 formats were selected because the scFvs are located at different positions on the IgG and the only way to determine whether one format has an advantage over the other is to empirically test their antibody specificity of interest.

[0181] To understand whether the BiSAbs retain the functional activity of the corresponding single monoclonal antibodies, the ability of each BiSAb to inhibit AT-dependent rabbit red blood cell (RBC) lysis and inhibit fibrinogen binding to ClfA001, ClfA002 and ClfA004 was evaluated. Rabbit RBC hemolysis assays were performed by mixing serial dilutions (500 to 1.7 nM) of the BiSAbs and MEDI4893* with AT (0.1 μg / ml = 3 nM) and incubating with 50 μl washed rabbit RBCs (Peel Freeze) at 37°C for 1 hour. In some assays, the anti-AT scFv of the BisAbs was saturated with a 10M excess of ClfA (5 μM). The plate was then centrifuged at 1200 rpm for 3 minutes, and 50 μl of the supernatant was transferred to a new plate. Nonspecific human IgG1 R347 was used as a negative control (c-IgG) (see, e.g., Tkachik et al., Clinical and Vaccine Immunology, 19:377-385 (2012)). OD450 nm was measured using a spectrophotometer (Molecular Devices, Sunnyvale, CA). Inhibition of hemolysis was calculated using the following equation:

[0182] 100-(100×[ODAT+mAb] / [ODAT]).

[0183] For fibrinogen binding assays, NUNC MAXISORP TMPlates (Thermo Fisher Scientific, Waltham, MA) were coated overnight with 2 μg / ml human fibrinogen (Sigma-Aldrich, St. Louis, MO), washed three times with PBS containing 0.1% Tween 20 (wash buffer) and blocked with 200 μl / well casein (Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature (RT). After washing three times, the plates were incubated with a mixture of 50 μl Avi-tagged ClfA221-559 (2 μg / ml) and serial dilutions of anti-ClfA monoclonal antibody or BiS antibody in a final volume of 100 μl PBS for 1 hour at RT. In some analyses, the anti-ClfA IgG1 of BiSAb was saturated with 10M excess AT (6.6 mM). After washing, bound ClfA was detected using horseradish peroxidase (HRP)-conjugated streptavidin (1:20000, GE Healthcare, Chicago, IL) and 100 μl 3,3',5,5'-tetramethylbenzidine (TMB) substrate (KPL). The reaction was stopped after 10 min with 100 μl 0.2 M H2SO4. The plate was read at OD450 nm on a spectrophotometer. The percent inhibition of ClfA binding to fibrinogen was calculated using the following formula: 100-(100×[OD ClfA+mAb ] / [OD ClfA,nomAb ]).

[0184] 11H10-BiS2 and BiS3 antibodies and SAR114-BiS2 antibody showed IC values ​​similar to MEDI4893* in AT hemolysis assay 50 values, while SAR114-BiS3 showed reduced AT neutralization activity, as Fig.15 As shown in A. Both 11H10 BiSAb and SAR114 BiS3Ab showed IC values ​​similar to those of the respective parental anti-ClfA IgG in the fibrinogen binding inhibition assay. 50 , while SAR114 BiS2Ab lost some anti-ClfA002 activity but was still superior to 11H10, as Fig.15 B. Fig.16 and as shown in Table 7. The BiSAb also mediated opsonophagocytic bacterial killing (OPK) similar to that of the parental anti-ClfA IgG, as Fig.17 Importantly, saturation of anti-AT scFv in the presence of 10 M excess AT did not interfere with anti-ClfA activity in the fibrinogen binding assay, as shown in Fig.15 C. Similarly, saturation of ClfA binding with a 10 M excess of ClfA did not reduce the AT neutralization activity of the BiSAb in the hemolytic assay, as shown in Fig.15 As shown in D.

[0185] Table 7. IC values ​​of SAR114 and 11H10-BiS2 and BiS3 molecules in fibrinogen binding assay 50

[0186]

[0187]

[0188] The results of the described examples demonstrate that the anti-CIfA / AT BiS molecules retain in vitro functional activity similar to that of the parental IgG in most cases and that the activity is not diminished in the presence of a 10-fold molar excess of another antigen recognized by the BiSAb.

[0189] Example 10

[0190] The example describes the protective effect of an anti-ClfA / AT bispecific antibody in a lethal S. aureus bacteremia model.

[0191] Mice were passively immunized with anti-ClfA SAR114 antibody and MEDI4893* monoclonal antibody combination (7.5 mpk or 1 mpk each) or equimolar doses of BiSAb (9 or 1.2 mpk, respectively), followed by IV infection with S. aureus strain SF8300 24 hours later, as described in Example 4, and survival was monitored for 14 days. Both SAR114-BiSAbs at 9 mpk showed reduced but not significantly different protection compared to the monoclonal antibody combination at 7.5 mpk each (p=0.234 for BiS2 and p=0.412 for BiS3), as shown in Table 4. Fig.18 As shown in A. The monoclonal antibody combination at 1 mpk (p=0.0051, relative to c-IgG) and SAR114-BiS2 at 1.2 mpk (p=0.0336, relative to c-IgG) significantly increased survival relative to c-IgG. Consistent with the observed loss of in vitro AT neutralization activity (see Fig.15 A), SAR114-BiS3 did not significantly increase survival when given at 1.2 mpk (p=0.657, Fig.18 A). When targeting S. aureus strain 3049057 (MRSA, ST8) (a strain for which no single monoclonal antibody was sufficient to provide significant protection (see Fig.10 )), 1.2 mpk of the SAR114-BiS molecule did not significantly increase survival relative to c-IgG (p=0.4310), whereas an equimolar concentration of the monoclonal antibody combination (1 mpk) did increase survival, as shown in Fig.18The results suggest that the SAR114-BiS2 antibody is defective in vivo. Interestingly, passive immunization with 11H10-BiSAb resulted in protection similar to that of the monoclonal combination at both doses tested (9 mpk and 1.2 mpk) and was comparable to that of the monoclonal combination against two ClfA001 expressing lines, SF8300 and 3049057 (Fig. Fig.18 C and 18D) provided a significant increase in survival.

[0192] The results of the examples demonstrate that the anti-CIfA / AT BiSAbs do not provide a combination benefit over the corresponding single antibodies. In contrast, the SAR114 / MEDI4893* bispecific antibody exhibited a loss of protection at lower doses against strains where the corresponding single monoclonal antibodies were insufficient to provide protection.

[0193] Example 11

[0194] The experiments describe experiments examining the efficacy of the SAR114 / MEDI4893* bispecific antibody in a lethal pneumonia model.

[0195] Since SAR114 binds CIfA001 with approximately 1000-fold higher affinity than 11H10 (Table 1), it is hypothesized that SAR114 binding to CIfA sequesters SAR114 / MEDI4893*BiSAb on the bacterial surface, resulting in poor capture and neutralization of AT when it is secreted. AT is a key virulence factor in S. aureus pneumonia (Bubeck, Waldenburg and O. Schneewind, J. Exp. Med., 205:287-294 (2008)), and passive immunization with anti-AT monoclonal antibodies alone protects mice from lethal S. aureus pneumonia (Foletti et al., J. Mol. Biol., 425(10):1641-1654 (2013); Hua et al., Antimicrobial Agents and Chemother., 58:1108-1117 (2014); and Rager and Bubeck, Waldenburg, Infection and Immunity, 77:2712-2718 (2009)). In addition, anti-ClfA monoclonal antibodies did not affect survival in a pneumonia model and the combination of anti-ClfA and anti-AT monoclonal antibodies provided protection similar to that of anti-AT mAb alone (Tkachik et al., supra). Therefore, to determine whether the reduced protection observed for SAR114-BiS2Ab in the lethal bacteremia model was caused by insufficient AT neutralization, female C57 / B6 mice (Jackson Laboratory, Bar Harbor, ME, USA) were injected IP with MEDI4893* alone or in combination with SAR114 or with SAR114 BiS2 or BiS3 molecules. SF8300 (1e AT) was used as described in Hua et al. (supra). 8 Pneumonia was induced by intranasal infection with 100 μg / mL CFU (100 μg / mL). Animal survival was monitored for 6 days. Statistical analysis relative to c-IgG was performed using the log-rank (Mantel-Cox) test. Data were considered statistically different if p < 0.05.

[0196] Passive immunization with MEDI4893* (15 mpk) alone or in combination with SAR114 resulted in 100% protection after challenge with SF8300. However, passive immunization with SAR114-BiS2 or BiS3 resulted in 30% and 0% survival, respectively. Fig.19As shown in A. Interestingly, passive immunization with 11H10BiS2, which has an affinity for ClfA reduced by about 1000-fold (Table 1), provided 100% survival. These results support the conclusion that ClfA bound to the bacterial surface sequesters SAR114-BiSAb, thereby impairing AT neutralization. To further test the hypothesis, mice were passively immunized with BiS2 molecules and then infected intranasally (IN) with the ClfA isogenic mutant SF8300Δclfa. Prophylaxis with SAR114-BiSAb provided protection against SF8300Δclfa, similar to MEDI4893*, as Fig.19 As shown in B.

[0197] The results of the described examples further demonstrate that binding of SAR114-BiSAb to surface-localized CIfA prevents efficient neutralization of soluble AT.

[0198] Example 12

[0199] The experiment describes an experiment to examine the pharmacokinetics (pK) of the SAR114 antibody in cynomolgus macaques. Monkeys were treated with 5 mg / kg SAR114, SAR114 N3F, or SAR114 N3Y by intravenous (IV) administration, and the antibody concentration in the blood was measured over 60 days. The results are shown in Fig. 20 and reported in Table 8 below.

[0200] Table 8: Cynomolgus monkey PK parameters.

[0201] Sar114 structure Clearance (mL / day / kg) <![CDATA[β-phase t 1 / 2 (days)]]> <![CDATA[AUC 最后 (μg×day / ml)]]> wild type 5.69±0.27 10.1±1.5 754±21 N3Y 2.14±0.17 23.7±2.4 1900±170 N3F 2.54±0.46 20.3±4.1 1690±254

[0202] The above data confirm that the modified form of SAR114 (and in particular SAR114 N3Y) exhibits increased half-life in primates. The above data are consistent with the predictions from the half-life extension studies of human FcRN transgenic mice. The effective extension of the half-life in primates indicates that the half-life of SAR114 N3Y will be appropriately extended and is very important for the correct administration, treatment and prevention of S. aureus-related diseases.

[0203] Example 13

[0204] The Example describes experiments examining the immunogenicity of N3Y Fc.

[0205] The immunogenicity of therapeutic proteins can cause problems, including neutralization, accelerated clearance of therapeutic agents, and / or adverse events. Although most human proteins (such as antibody framework regions) are non-immunogenic, mutations in the Fc region of antibodies present a potential risk of immune response. Due to the major role of helper T cells in immunogenic responses, functional activation analysis using human CD4 T cells is now considered a marker for immunogenicity prediction. Therefore, the effect of N3Y in the Fc region of IgG1 on T cell activation was analyzed.

[0206] In these experiments, PMBCs were isolated from 39 human whole blood collections using a Ficole gradient. CD8 cells were then extracted using positive selection and enriched by stimulation with 5 different peptide pools and amplification with IL-2 for 10 days in vitro. Cells were then re-stimulated with a single peptide pool in an ELIspot plate for CD4. Shown in Fig.21 The results in demonstrated that the NY3 mutant dose did not significantly increase immunogenicity compared to the wild-type Fc region.

[0207] 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.

[0208] Unless otherwise indicated herein or clearly contradicted by the context, the use of the terms "a" and "an" and "the" and "at least one" and similar indications in describing the present invention (especially in the context of the appended claims) are to be interpreted as including both the singular and the plural. Unless otherwise indicated herein or clearly contradicted by the context, the use of the term "at least one" in conjunction with a list of one or more items (e.g., "at least one of A and B") is to be interpreted as indicating a selection from one of the listed items (A or B) or any combination of two or more of the listed items (A and B). Unless otherwise noted, the terms "including", "having", "including", and "containing" are to be interpreted as open-ended terms (that is, meaning "including (but not limited to)") unless otherwise indicated herein. Unless otherwise indicated herein, the numerical ranges recited herein are merely intended as a shorthand method of individually referring to each individual value within the range, and each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. No language in this specification should be construed as indicating any non-claimed element as a necessary condition for practicing the present invention.

[0209] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for carrying out the present invention. A person of ordinary skill in the art will appreciate variations of the preferred embodiments after reading the above description. The inventor expects that those skilled in the art will be able to use the variations appropriately, and the inventor intends to implement the present invention in a manner other than as explicitly described herein. Therefore, the present invention includes all modifications and equivalents of the objects listed in the claims attached hereto as permitted by applicable law. In addition, unless otherwise specified herein or the context clearly conflicts, the present invention includes any combination of the above elements in all possible variations thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to Staphylococcus aureus (S. aureus) clot factor A (ClfA) protein, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementary determining region CDR 1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a variable light chain (VL) CDR 1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6, and wherein the antibody or antigen-binding fragment comprises a heavy chain constant domain comprising the amino acid sequence of CSYHLC (SEQ ID NO: 21).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:

13.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO:

14.

4. An antibody or antigen-binding fragment thereof that specifically binds to the ClfA protein of Staphylococcus aureus, wherein the antibody or antigen-binding fragment comprises (1) VH, (2) VL and (3) a heavy chain constant domain comprising the amino acid sequence of CSYHLC (SEQ ID NO:21); wherein the VH comprises the amino acid sequence of SEQ ID NO:

13.

5. An antibody or antigen-binding fragment thereof that specifically binds to the ClfA protein of Staphylococcus aureus, wherein the antibody or antigen-binding fragment comprises (1) VH, (2) VL and (3) a heavy chain constant domain comprising the amino acid sequence of CSYHLC (SEQ ID NO:21); wherein the VL comprises the amino acid sequence of SEQ ID NO:

14.

6. An antibody or antigen-binding fragment thereof that specifically binds to the ClfA protein of Staphylococcus aureus, wherein the antibody or antigen-binding fragment comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of SAR114.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the CDR is a Kabat-defined CDR, a Chaudier- defined CDR or an AbM-defined CDR.

8. The antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein the antibody or antigen-binding fragment comprises a heavy chain constant domain comprising the amino acid sequence of CSYHLC (SEQ ID NO: 21).

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the heavy chain constant domain comprises the amino acid sequence of MHEACSYHLCQKSLSLS (SEQ ID NO: 23).

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO: 24.

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