Patent Grant
10781246
The present invention is directed to staphylococcal leukotoxin binding molecules and fusion constructs. The present invention is further directed to methods of treating, preventing, and diagnosing staphylococcal infection in a subject using the binding molecules and fusion constructs described herein.
Bacterial infections caused by Staphylococcus bacteria (i.e., a “staph infection”) are very common in the general population. About 25% of individuals commonly carry Staphylococcus bacteria on their skin or in their nose. Most of the time, these bacteria do not cause or problem or may cause a relatively minor skin infection. However, staph infections can turn deadly if the bacteria invade deeper into an individual's body, for example, entering the bloodstream, joints, bones, lungs or heart. In the past, a lethal staph infection might have occurred in a person who was hospitalized or had a chronic illness or weakened immune system. Now, it is increasingly common for an otherwise healthy individual to develop life-threatening staph infections. Importantly, many staph infections have become recalcitrant to antibiotic treatment due to infection with strains that exhibit true antibiotic resistance or reduced susceptibility to existing antibiotics. Such reductions in antibiotic effectiveness are typically more pronounced in patients with weakened immune systems due to immune senescence or immune compromization caused by co-morbidities or co-administered pharmaceutical agents or other medical procedures.
Staphylococcus aureus, often referred to as “staph,” Staph. aureus,” or “S. aureus,” is a major human pathogen, producing a multitude of virulence factors making it able to cause several types of infection, from superficial lesions to toxinoses and life-threatening systemic conditions such as endocarditis, osteomyelitis, pneumonia, meningitis and sepsis (reviewed in Miller and Cho, “Immunity Against Staphylococcus aureus Cutaneous Infections,” Nat. Rev. Immunol. 11:505-518 (2011)). Although most individuals encounter S. aureus shortly after birth (Holtfreter et al., “Towards the Immune Proteome of Staphylococcus aureus—The Anti-S. aureus Antibody Response,” Int. J. Med. Microbiol. 300:176-192 (2010)) and possess both antibodies against S. aureus and the ability to increase anti-S. aureus titers after infection, these antibodies are often not protective against recurrent S. aureus infections (Foster TJ, “Immune Evasion by Staphylococci,” Nat. Rev. Microbiol. 3:948-958 (2005)). In the United States alone, an annual mortality of more than 20,000 is attributed to methicillin-resistant S. aureus (MRSA), exceeding deaths caused by influenza, viral hepatitis, and HIV/AIDS (Foster, TJ., “Immune Evasion by Staphylococci,” Nat. Rev. Microbiol. 3:948-958 (2005); Klevens et al., “The Impact of Antimicrobial-Resistant, Health Care-Associated Infections on Mortality in the United States,” Clin. Infect. Dis. 47:927-930 (2008)).
The pathogen produces a variety of molecules that presumably facilitate survival in or on the human host. Bi-component, pore-forming leukotoxins are among the secreted virulence factors produced by S. aureus. These toxins are secreted as water soluble monomers which oligomerize, and insert pores into the plasma membrane of host cells, most notably polymorphonuclear leukocytes (PMNs) and mononuclear phagocytes (Alonzo F. and Torres V., “Staphylococcus aureus Bi-component leukotoxins,” Microbiol. Mol. Biol. Rev. 78(2): 199-230 (2014)). These pores disrupt cellular osmotic balance and membrane potential leading to death of the targeted cells. In the case of Leukotoxin ED (LukED), the targeting, binding, and killing of host phagocytic cells occurs via the cellular target CCR5, CXCR1 and CXCR2 located on the surface of the phagocytes (Alonzo III et al., “Staphylococcus aureus Leucocidin ED Contributes to Systemic Infection by Targeting Neutrophils and Promoting Bacterial Growth In Vivo,” Mol. Microbiol. 83:423-435 (2012); Alonzo III et al. “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430)51-55 (2012); and Reyes-Robles et al., “Staphylococcus aureus Leukotoxin ED Targets the Chemokine Receptors CXCR1 and CXCR2 to Kill Leukocytes and Promote Infection,” Cell Host & Microbe 14:453-459 (2013)). Indeed, when the cellular target of LukED, CCR5, is not present on host immune cells, the host animal is resistant to the otherwise lethal S. aureus infection (Alonzo III et al. “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430):51-55 (2012)). In recent studies, the Duffy antigen receptor for chemokines (DARC) was also identified as a receptor for LukED and is necessary for LukED-mediated hemolysis of erythrocytes (Spaan et al., “Staphylococcus aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes,” Cell Host & Microbe 18(3): p. 363-370 (2015)).
Leukotoxin AB (LukAB) can also kill host phagocytic cells, and its cytolytic activity can be exerted both from the outside and the inside of the cell, i.e., after the microorganism is phagocytosed into the host cell (Dumont et al., “Staphylococcus aureus LukAB Cytotoxin Kills Human Neutrophils by Targeting the CD11b Subunit of the Integrin Mac-1,” PNAS 110(26):10794-10799 (2013)). Due to the contribution both of these leukotoxins have to pathogenesis, they have been considered critical S. aureus virulence factors (Alonzo III and Torres, “Bacterial Survival Amidst an Immune Onslaught: The Contribution of the Staphylococcus aureus Leukotoxins,” PLOS Path 9(2):e1003143 (2013)).
Another critical factor for the pathogenic success of S. aureus depends on the properties of its surface proteins (Clarke et al., “Surface Adhesins of Staphylococcus aureus,” Adv. Microb. Physiol. 51:187-224 (2006); Patti et al., “MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues,” Annu. Rev. Microbiol. 48:585-617 (1994); and Patti et al., “Microbial Adhesins Recognizing Extracellular Matrix Macromolecules,” Curr. Opin. Cell Biol. 6:752-758 (1994)). S. aureus employs microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that adhere to and colonize host tissues via recognition of collagen, heparin-related polysaccharides, fibrinogen, and/or fibronectin of host cells. S. aureus expresses a subset of MSCRAMMs containing a serine-aspartate dipeptide repeat (SDR) domain, including clumping factor A (ClfA), clumping factor B (ClfB), SdrC, SdrD, and SdrE (Becherelli et al. “Protective Activity of the CnaBE3 Domain Conserved Among Staphylococcus aureus Sdr Proteins,” PLoS One 8(9): e74718 (2013)). S. epidermidis also expresses three members of this family, SdrF, SdrG, and SdrH (McCrea et al., “The Serine-Aspartate Repeat (Sdr) Protein Family in Staphylococcus Epidermidis,” Microbiology 146:1535-1546 (2000)). These proteins share a similar structure comprising an N-terminal ligand-binding A domain followed by the SDR domain, which contains between 25-275 serine-aspartate dipeptide repeats. The C-terminal portion of these proteins contains the LPXTG-motif, which facilitates cell wall anchoring by the transpeptidase sortase A. The serine-aspartate dipeptide regions in these proteins are modified by the sequential addition of glycans by two glycosyltransferases. First, SdgB appends N-acetylglucosamine (GlcNAc) on serine residues within the serine-aspartate dipeptide regions, followed by SdgA modification of the glycoprotein, resulting in disaccharide moieties. This glycosylation protects SDR-containing staphylococcal proteins from Cathepsin G-mediated degradation (Hazenbos et al., “Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins,” PLoS Pathog 9(10):e1003653 (2013)).
Protein A, located on the surface of S. aureus, also contributes to staphylococcal escape from protective host immune responses by capturing the Fc domain of host IgG, as well as the Fab domain of the VH3 clan of IgG and IgM (Sjodahl et al., “Repetitive Sequences in Protein A from Staphylococcus aureus. Arrangement of Five Regions Within the Protein, Four Being Highly Homologous and Fc-Binding,” Eur. J. Biochem. 73:343-351 (1997); and Cary et al., “The Murine Clan V(H) III Related 7183, J606 and S107 and DNA4 Families Commonly Encode for Binding to a Bacterial B cell Superantigen,” Mol. Immunol. 36:769-776 (1999)). In addition, S. aureus expresses a second immunoglobulin binding protein referred to as the second binding protein for immunoglobulins (Sbi) (Zhang et al., “A Second IgG-Binding Protein in Staphylococcus aureus,” Microbiology 144:985-991 (1998) and Atkins et al., “S. aureus IgG-binding Proteins SpA and Sbi: Host Specificity and mechanisms of Immune Complex Formation,” Mol. Immunol. 45:1600-1611 (2008)). Sbi is either secreted or associated with the cell envelope (Smith et al., “The Sbi Protein is a Multifunctional Immune Evasion Factor of Staphylococcus aureus” Infection & Immunity 79:3801-3809 (2011) and Smith et al., “The Immune Evasion Protein Sbi of Staphylococcus aureus Occurs both Extracellularly and Anchored to the Cell Envelope by Binding to Lipotechoic Acid” Mol. Microbiol. 83:789-804 (2012)) and shares a pair of conserved helices with Protein A involved in binding to the Fc region of IgG proteins (Atkins et al., “S. aureus IgG-binding Proteins SpA and Sbi: Host Specificity and mechanisms of Immune Complex Formation,” Mol. Immunol. 45:1600-1611 (2008)). Binding of IgGs to these proteins via the CH3 region of the Fc is thought to sequester antibodies on the cell surface of S. aureus in an orientation that prevents effective Fc-mediated opsonization of bacteria by neutrophils and therein serve as key immune evasion factors.
S. aureus also secretes a number of proteases that have been implicated in immune evasion. Rooijakkers et al. demonstrated that S. aureus secretion of staphylokinase, a plasminogen activator protein, led to the activation of plasmin that cleaved both surface-bound IgG and complement C3b, ultimately reducing immune-mediated S. aureus destruction (Rooijakkers et al., “Anti-Opsonic Properties of Staphylokinase,” Microbes and Infection 7:476-484 (2005)). S. aureus also secretes the serine protease glutamyl endopeptidase V8 (GluV8) that can directly cleave human IgG1 in the lower hinge region between E233 and L234 (EU numbering (Edelman et al., “The Covalent Structure of an Entire GammaG Immunoglobulin Molecule,” PNAS 63:78-85 (1969), Brezski et al., “Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs,” J. Immunol. 181:3183-3192 (2008)). It was also recently demonstrated that human anti-S. aureus IgGs are rapidly cleaved when bound to the surface of S. aureus (Fernandez Falcon et al., “Protease Inhibitors Decrease IgG Shedding From Staphylococcus aureus, Increasing Complement Activation and Phagocytosis Efficiency,” J. Med. Microbiol. 60:1415-1422 (2011)).
Taken together, these studies indicate that S. aureus utilizes a number of mechanisms that could adversely affect standard IgG1-based monoclonal antibody (mAb) therapeutics, either by directly cleaving the mAb, sequestering of the mAb by Protein A or Sbi binding on the Staph cell surface, or by killing off the very effector cells required for therapeutic efficacy. It is therefore not surprising that presently there are no mAb-based therapies targeting S. aureus that have achieved final approval for use in humans. Thus, there remains a need for methods and compositions that can treat staphylococcal infection, which (i) evade protein A and Sbi binding, (ii) escape staph-induced proteolysis, (iii) can neutralize leukotoxins and (iv) are capable of opsonizing and delivering S. aureus to phagocytes. The present application meets these and other needs.
A first aspect of the present disclosure is directed to a binding molecule comprising one or more modified fibronectin type III (FN3) domains, each modified FN3 domain having one or more loop regions that comprise one or more staphylococcal leukotoxin binding regions.
Another aspect of the present disclosure is directed to a binding molecule comprising one or more modified fibronectin type III (FN3) domains, each modified FN3 domain having one or more loop regions that comprise one or more staphylococcal hemolysin binding regions.
A second aspect of the present disclosure is directed to a fusion construct. The fusion construct comprises a first portion comprising one or more binding molecules described herein, and a second portion coupled to said first portion. The second portion of the fusion construct comprises a second binding molecule, a pharmaceutically active moiety, a prodrug, a pharmaceutically-acceptable carrier, a diagnostic moiety, a cell penetrating enhancer moiety, and/or a half-life extending modulating moiety.
Another aspect of the present disclosure is directed to a pharmaceutical composition comprising the binding molecules and/or fusion constructs as described herein.
Other aspects of the present disclosure are directed to methods of treating, preventing, and diagnosing a staphylococcal infection in a subject using the binding molecules, fusion constructs, and/or pharmaceutical compositions described herein.
Another aspect of the present disclosure is directed to a recombinant Leukocidin B (LukB) polypeptide comprising an amino acid sequence corresponding to amino acid residues 1-109 of SEQ ID NO: 1026 coupled to amino acid residues 152-305 of SEQ ID NO: 1026, where the LukB polypeptide does not comprise one or more amino acid residues corresponding to amino acid residues 110-151 of SEQ ID NO: 1026.
Another aspect of the present disclosure is directed to a recombinant Leukocidin A (LukA) polypeptide comprising an amino acid sequence corresponding to amino acid residues 1-134 of SEQ ID NO: 1018 coupled to amino acid residues 175-324 of SEQ ID NO: 1018, where the LukA polypeptide does not comprise one or more amino acid residues corresponding to amino acid residues 135-174 of SEQ ID NO: 1018.
Another aspect of the present disclosure is directed to a vaccine composition. The vaccine composition comprises a recombinant Leukocidin B (LukB) polypeptide comprising an amino acid sequence corresponding to amino acid residues 1-109 of SEQ ID NO: 1026 coupled to amino acid residues 152-305 of SEQ ID NO: 1026, wherein said LukB polypeptide does not comprise one or more amino acid residues corresponding to amino acid residues 110-151 of SEQ ID NO: 1026. The vaccine composition further comprises a recombinant Leukocidin A (LukA) polypeptide comprising an amino acid sequence corresponding to amino acid residues 1-134 of SEQ ID NO: 1018 coupled to amino acid residues 177-324 of SEQ ID NO: 1018, wherein said LukA polypeptide does not comprise one or more amino acid residues corresponding to amino acid residues 135-174 of SEQ ID NO: 1018.
The staphylococcal leukotoxin binding molecules described herein are small (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides or glycosylated residues. These molecules have excellent biophysical properties (e.g., greater than 100 mg/mL expression, greater than 170 mg/mL solubility, greater than 82° C. melting temperature, low predicted immunogenicity, and stable in serum for more than one month), and can be engineered for improved stability. Other advantages over conventional therapeutics include the ability to administer locally, orally, or a cross the blood-brain barrier, the ability to express in E. coli allowing for increased expression of protein as a function of resources versus mammalian cell expression, the ability to be engineered into bispecific molecules that bind to multiple targets or multiple epitopes of the same target, the ability to be conjugated to drugs, polymers, and probes, the ability to be formulated to high concentrations, and the ability of such molecules to effectively penetrate diseased tissues. Accordingly, the binding molecules described herein comprising one or more staphylococcal leukotoxin binding domains offer a unique therapeutic, prophylactic, and diagnostic approach to combatting staphylococcal infection.
A first aspect of the present disclosure is directed to a binding molecule comprising one or more modified fibronectin type III (FN3) domains, where each modified FN3 domain has one or more loop regions that comprise one or more staphylococcal leukotoxin binding regions.
The FN3 domain is an evolutionary conserved protein domain that is about 100 amino acids in length and possesses a beta sandwich structure. The beta sandwich structure of human FN3 comprises seven beta-strands, referred to as strands A, B, C, D, E, F, G, with six connecting loops, referred to as loops AB, BC, CD, DE, EF, and FG that exhibit structural homology to immunoglobulin binding domains. Three of the six loops, i.e., loops DE, BC, and FG, correspond topologically to the complementarity determining regions of an antibody, i.e., CDR1, CDR2, and CDR3. The remaining three loops are surface exposed in a manner similar to antibody CDR3. In accordance with the present disclosure, one or more of the loop regions of each FN3 domain of the binding molecule are modified to comprise one or more staphylococcal leukotoxin binding regions.
The modified FN3 domain of the binding molecule of the present disclosure can be a FN3 domain derived from any of the wide variety of animal, yeast, plant, and bacterial extracellular proteins containing these domains. In one embodiment, the FN3 domain is derived from a mammalian FN3 domain. Exemplary FN3 domains include, for example and without limitation, any one of the 15 different FN3 domains present in human tenascin C, or the 15 different FN3 domains present in human fibronectin (FN) (e.g., the 10th fibronectin type III domain). Exemplary FN3 domains also include non-natural synthetic FN3 domains, such as those described in U.S. Pat. Publ. No. 2010/0216708 to Jacobs et al., which is hereby incorporated by reference in its entirety. Individual FN3 domains are referred to by domain number and protein name, e.g., the 3rd FN3 domain of tenascin (TN3), or the 10th FN3 domain of fibronectin (FN10).
In one embodiment, the FN3 domain of the binding molecule is derived from the non-naturally occurring FN3 domain of Tencon (SEQ ID NO: 1017). Tencon was designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al., “Design of Novel FN3 Domains With High Stability by a Consensus Sequence Approach,” Protein Engineering, Design, and Selection 25:107-117 (2012), the disclosure of which is hereby incorporated by reference in its entirety). In another embodiment, the FN3 domain of the binding molecule is derived from the non-naturally occurring FN3 domain of Tencon-25 (SEQ ID NO: 1) as disclosed in Diem et al., “Selection of high-affinity Centyrin FN3 domains from a simple library diversified at a combination of strand and loop positions,” Protein Engineering, Design, and Selection 10:419-429 (2014), which is hereby incorporated by reference in its entirety).
Tencon domains, like other FN3 domains, have a beta-sandwich structure with the seven beta-strands, i.e., A, B, C, D, E, F, and G, linked by six loops, i.e., AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle, Proc. Natl. Acad. Sci. USA 89:8990-8992 (1992) and U.S. Pat. No. 6,673,901 to Koide et al., which are hereby incorporated by reference in their entirety). These loops span at or about amino acid residues 13-16 of SEQ ID NO: 1 (AB loop), amino acid residues 22-28 of SEQ ID NO: 1 (BC loop), amino acid residues 38-43 of SEQ ID NO:1 (CD loop), amino acid residues 51-54 of SEQ ID NO:1 (DE loop), amino acid residues 60-64 of SEQ ID NO: 1 (EF loop), and amino acid residues 75-81 of SEQ ID NO: 1 (FG loop). In accordance with the present disclosure, one or more of these loop regions or selected residues within one or more of these loop regions are modified for staphylococcal leukotoxin binding specificity and affinity. Suitable modifications include amino acid residue substitutions, insertions, and/or deletions. In one aspect, amino acid residues in at least one, at least two, at least three, at least four, at least five, or all six of the loop regions are altered for staphylococcal leukotoxin binding specificity and affinity. In one embodiment, one or more amino acid modifications within the loop regions at or about residues 22-28 (BC loop), 38-43 (CD loop), 51-54 (DE loop), and 75-81 (FG loop) of SEQ ID NO: 1 form the staphylococcal leukotoxin binding region. In another embodiment, one or more amino acid modification within the loop regions at or about residues 38-43 (CD loop) and 75-81 (FG loop) form the staphylococcal leukotoxin binding region. The modified one or more loop regions preferably interact with their target staphylococcal leukotoxin protein similar to an antibody CDR interaction with the protein.
As discussed above, FN3 domains contain two sets of CDR-like loops on the opposite faces of the molecule. The two sets of loops are separated by beta-strands that form the center of the FN3 structure. Like the loops, these beta-strands can be altered to enhance target molecule binding specificity and affinity. Preferably, some or all of the surface exposed residues in the beta strands are randomized without affecting (or minimally affecting) the inherent stability of the FN3 domain. One or more of the beta-strands can interact with a target protein. The beta-strands in a FN3 binding molecule provide a flat binding surface (compared to a curved binding surface found in protein scaffolds containing adjacent loops) that affects the target proteins, or specific epitopes on those target proteins, that can be bound effectively by the domain. In one aspect, at least a portion of one or more beta-strands of the FN3 domain is modified to interact with a staphylococcal leukotoxin protein. Suitable modifications include amino acid substitutions, insertions, and/or deletions. For example, one or more amino acid residues of the A beta strand (i.e., amino acid residues corresponding to residues 1-12 of SEQ ID NO: 1), the B beta strand (i.e., amino acid residues corresponding to residues 17-21 of SEQ ID NO: 1), the C beta strand (i.e., amino acid residues corresponding to residues 29-37 of SEQ ID NO: 1), the D beta strand (i.e., amino acid residues corresponding to residues 44-50 of SEQ ID NO: 1), the E beta strand (i.e., amino acid residues corresponding to residues 55-59 of SEQ ID NO: 1), the F beta strand (i.e., amino acid residues corresponding to residues 65-74 of SEQ ID NO: 1), or the G beta strand (i.e., amino acid residues corresponding to residues 82-89 of SEQ ID NO: 1) may be modified to generate staphylococcal leukotoxin binding domains or to enhance the specificity or affinity of leukotoxin binding. In one embodiment, one or more amino acid residues of the C beta strand and/or the F beta strand are modified for binding to a staphylococcal leukotoxin protein.
In one embodiment, the binding molecules of the present disclosure specifically bind to one or more staphylococcal leukotoxins, also referred to herein as staphylococcal leukocidins. Staphylococcal leukotoxins are a family bi-component toxins released by S. aureus, which damage membranes of host defense cells and erythrocytes by the synergistic action of two non-associated proteins or subunits, i.e., the S-subunit and F-subunit (see Menestrina et al., “Mode of Action of Beta-Barrel Pore-Forming Toxins of the Staphylococcal Alpha-Hemolysin Family,” Toxicol. 39(11):1661-1672 (2001). The binding molecules as described herein bind to one or more staphylococcal leukotoxins selected from leukotoxin A (LukA), leukotoxin B (LukB), leukotoxin AB (LukAB), leukotoxin D (LukD), leukotoxin E (LukE), leukotoxin ED (LukED), Panton-Valentine leukocidin S (LukS-PV), Panton-Valentine leukocidin F (LukF-PV), Panton-Valentine leukocidin (LukSF/PVL), gamma hemolysin A (HlgA), gamma hemolysin C (HlgC), gamma hemolysin B (HlgB), gamma hemolysin AB (HlgAB), and gamma-hemolysin BC (HlgBC). In one embodiment, the binding molecule binds to one or more of the staphylococcal leukotoxins selected from LukAB, LukD or LukE. In another embodiment, the binding molecules hereof are capable of specifically binding to a fragment of the above-mentioned proteins, where the fragment at least comprises a neutralizing epitope of the leukotoxin protein. Binding of the binding molecule of the present disclosure to a neutralizing epitope of the leukotoxin protein substantially or completely eliminates leukotoxin cytolytic and/or hemolytic activity. Neutralizing epitopes generally include regions of the leukotoxin protein involved in binding to a host cell membrane or receptor, regions of the leukotoxin protein involved in interacting with other leukotoxin proteins and oligomer formation, and regions of the leukotoxin protein involved in pore formation. The binding molecules of the present disclosure neutralize leukotoxin activity by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100% when compared to leukotoxin activity in the absence of the binding molecule.
As used herein “specifically binds” or “specific binding” refers to the ability of the FN3 containing binding molecule of the disclosure to bind to a predetermined antigen, i.e., a staphylococcal leukotoxin with a dissociation constant (KD) of about 1×10−6 M or less, for example about 1×10−7 M or less, about 1×10−8M or less, about 1×10−9 M or less, about 1×10−10M or less, about 1×10−11M or less, about 1×10−12M or less, or about 1×10−13 M or less. Typically the leukotoxin binding FN3 domain binds to its target leukotoxin(s) with a KD that is at least ten fold less than its KD for a nonspecific antigen (for example BSA or casein) as measured by surface plasmon resonance using for example a Proteon Instrument (BioRad). Thus, a monospecific, bispecific, or multispecific leukotoxin FN3 domain containing molecule as described herein specifically binds to each target leukotoxin with a binding affinity (KD) of at least 1×10−6 M or less. The isolated modified FN3 domain of the disclosure that specifically binds to a staphylococcal leukotoxin may, however, have cross-reactivity to other related bi-component toxins, for example to related S-subunit or F-subunit leukotoxins.
In one aspect, the binding molecule comprises a modified FN3 domain having one or more loop regions that comprise one or more LukE binding regions. This binding molecule is capable of binding a staphylococcal LukE protein having the amino acid sequence of SEQ ID NO: 1055 or fragments or homologs thereof. The LukE protein exemplified by SEQ ID NO: 1055 corresponds to the native mature LukE protein sequence modified to contain an N-terminal histidine tag. Accordingly, the amino acid residue at position 12 of SEQ ID NO: 1055 corresponds to the first amino acid residue of the mature LukE protein.
In one embodiment, binding of the LukE binding molecule to LukE effectively neutralizes LukE cytolytic and/or hemolytic activity, e.g., by inhibiting LukE and LukD interaction, inhibiting LukE binding to the plasma membrane of leukocytes, and/or inhibiting LukED pore formation. In accordance with this embodiment, the LukE binding molecule binds to one or more epitopes of LukE within a region of LukE comprising or consisting of amino acid residues corresponding to residues 22-33 of SEQ ID NO: 1055 (residues involved in LukE-LukD protein interaction), amino acid residues corresponding to residues 123-128 of SEQ ID NO: 1055 (residues involved in LukED membrane penetration), or amino acid residues corresponding to residues 137-142 of SEQ ID NO: 1055 (residues involved in LukED membrane penetration). In another embodiment, the LukE binding molecule binds to one or more epitopes of LukE within a region of LukE involved in LukE cell targeting. These regions comprise amino acid residues corresponding to residues 68-86 of SEQ ID NO: 1055, amino acid residues corresponding to residues 151-161 of SEQ ID NO: 1055, amino acid residues corresponding to residues 175-189 of SEQ ID NO: 1055, amino acid residues corresponding to residues 193-207 of SEQ ID NO: 1055, and amino acid residues corresponding to residues 248-278 of SEQ ID NO: 1055. As demonstrated in the Examples herein, amino acid residues 69-84 of SEQ ID NO: 1055 (corresponding to amino acid residues 86-101 of SEQ ID NO: 1054), and amino acid residues 252-275 of SEQ ID NO: 1055 (corresponding to amino acid residues 269-292 of SEQ ID NO: 1054) contain neutralizing epitopes of LukE.
In one embodiment, a LukE binding molecule as described herein comprises a C strand and a CD loop region having the amino acid sequence
DSFX32IX34YX36EX38X39X40X41X42E (SEQ ID NO: 993), where
X32 is any amino acid residue,
X34 is E or a functionally equivalent amino acid residue,
X36 is any amino acid residue,
X38 is any amino acid residue,
X39 is W or a functionally equivalent amino acid residue,
X40 is any amino acid residue,
X41 is W or a functionally equivalent amino acid residue,
X42 is any amino acid residue;
and an F strand and FG loop region having the amino acid sequence of TX66YX68VX70IX72GVKG X77 X78 X79 SX81 (SEQ ID NO: 994), where
X66 is any amino acid residue,
X68 is any amino acid residue,
X70 is F or a functionally equivalent amino acid residue,
X72 is G or a functionally equivalent amino acid residue,
X77 is any amino acid residue,
X78 is any amino acid residue,
X79 is any amino acid residue,
X81 is any amino acid residue.
In one embodiment, the LukE binding molecule as described herein comprises the a C strand and a CD loop region of SEQ ID NO: 993 and the F strand and FG loop region of SEQ ID NO: 994 as described above and binds to a neutralizing epitope of LukE in one of the regions of LukE noted above, i.e., amino acid residues 22-33, 123-128, 137-142, 68-86, 151-161, 175-189, 193-207, and 248-278 of SEQ ID NO:1055.
In another embodiment, the LukE binding molecule as described herein comprises the amino acid sequence of LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF X32I X34Y X36E X38 X39 X40 X41 X42EAI
X46LTVPGSERSYDLTGLKPGT X66Y X68V X701 X72GVKG X77 X78 X79 S X81 X82L X84A
X86FTT (SEQ ID NO: 989), where
X32 is any amino acid residue,
X34 is E or a functionally equivalent amino acid residue,
X36 is any amino acid residue,
X38 is any amino acid residue,
X39 is W or a functionally equivalent amino acid residue,
X40 is any amino acid residue,
X41 is W or a functionally equivalent amino acid residue,
X42 is any amino acid residue,
X46 is any amino acid residue,
X66 is any amino acid residue,
X68 is any amino acid residue,
X70 is F or a functionally equivalent amino acid residue,
X72 is G or a functionally equivalent amino acid residue,
X77 is any amino acid residue,
X78 is any amino acid residue,
X79 is any amino acid residue,
X81 is any amino acid residue,
X82 is any amino acid residue,
X84 is any amino acid residue, and
X86 is any amino acid residue.
In accordance with this aspect, exemplary LukE binding molecules comprise any one of the amino acid sequences of SEQ ID NOs: 25 (Luk26) and 739-814.
In some embodiments, these LukE binding molecules comprise an initiator methionine residue linked to the N-terminus or a cysteine residue linked to the C-terminus to facilitate expression and/or conjugation to another moiety (e.g., another leukotoxin binding FN3 domain, a half-life extending moiety, or other therapeutic moiety).
In another embodiment, the LukE binding molecule as described herein binds LukE with one or more amino acid residues corresponding to residues E34, W39, W41, F70, and G72 of SEQ ID NO: 25 (Luk26). Accordingly, the FN3 domain binding molecule containing a LukE binding region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 25, where residues E34, W39, W41, F70, and G72 of SEQ ID NO: 25 remain fixed or are substituted with functionally equivalent amino acid residues.
In another embodiment, a LukE binding molecule described herein comprises a C strand and a CD loop region having the amino acid sequence
DSFX32IX34YX36EX38X39X40X41GE (SEQ ID NO: 995) where
X32 is any amino acid residue,
X34 is any amino acid residue,
X36 is any amino acid residue,
X38 is W or a functionally equivalent amino acid residue,
X39 is any amino acid residue,
X40 is any amino acid residue, and
X41 is any amino acid residue;
and an F strand and FG loop region having the amino acid sequence of TEYX68VX70IX72GVKGG X78 X79 SX81 (SEQ ID NO: 996), where
X68 is L or a functionally equivalent amino acid residue,
X70 is D or a functionally equivalent amino acid residue,
X72 is Y or a functionally equivalent amino acid residue,
X78 is any amino acid residue,
X79 is W or a functionally equivalent amino acid residue, and
X81 is Y or a functionally equivalent amino acid residue.
In one embodiment, the LukE binding molecule as described herein comprises the a C strand and a CD loop region of SEQ ID NO: 995 and the F strand and FG loop region of SEQ ID NO: 996 as described above and binds to a neutralizing epitope of LukE in one of the regions of LukE noted above, i.e., amino acid residues 22-33, 123-128, 137-142, 68-86, 151-161, 175-189, 193-207, and 248-278 of SEQ ID NO:1055.
In one embodiment, the LukE binding molecule as described herein comprises the amino acid sequence of LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF X32I X34Y X36E X38 X39 X40 X41 GEAI VLTVPGSERSYDLTGLKPGT EY X68V X701 X72GVKG G X78 X79 S X81 PLSAIFTT (SEQ ID NO: 990) where,
X32 is any amino acid residue,
X34 is any amino acid residue,
X36 is any amino acid residue,
X38 is W or a functionally equivalent amino acid residue,
X39 is any amino acid residue,
X40 is any amino acid residue,
X41 is any amino acid residue,
X68 is L or a functionally equivalent amino acid residue,
X70 is D or a functionally equivalent amino acid residue,
X72 is Y or a functionally equivalent amino acid residue,
X78 is any amino acid residue,
X79 is W or a functionally equivalent amino acid residue, and
X81 is Y or a functionally equivalent amino acid residue.
In accordance with this embodiment, exemplary LukE binding molecules include any one of the amino acid sequences of SEQ ID Nos: 26 (Luk27) and 815-827. In some embodiments, these LukE binding molecules comprise an initiator methionine residue linked to the N-terminus or a cysteine residue linked to the C-terminus to facilitate expression and/or conjugation to another moiety (e.g., another leukotoxin binding FN3 domain, a half-life extending moiety, or other therapeutic moiety).
In one embodiment, the LukE binding molecule as described herein binds LukE with one or more amino acid residues corresponding to residues W38, L68, D70, Y72, W79, and Y81 of SEQ ID NO: 26 (Luk27). Accordingly, the FN3 domain containing a LukE binding region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 26, where residues W38, L68, D70, Y72, W79, and Y81 of SEQ ID NO: 26 remain fixed or are substituted with functionally equivalent amino acid residues.
In another embodiment, the LukE binding molecule described herein comprises a FG loop region having the amino acid sequence X75 X76 X77 X78 X79 X80X81 X82 X83 X84 X85 X86 (SEQ ID NO: 997) where
X75 is any amino acid residue,
X76 is any amino acid residue,
X77 is I or a functionally equivalent amino acid residue,
X78 is any amino acid residue,
X79 is any amino acid residue,
X80 is G or a functionally equivalent amino acid residue,
X81 is W or a functionally equivalent amino acid residue,
X82 is L or a functionally equivalent amino acid residue,
X83 is D or a functionally equivalent amino acid residue,
X84 is F or a functionally equivalent amino acid residue,
X85 is V or a functionally equivalent amino acid residue, and
X86 is F or a functionally equivalent amino acid residue.
In one embodiment, the FG loop region of SEQ ID NO: 997 can contain one or more amino acid insertions. For example, amino acid insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues can be made at or around positions X75 and X76 of SEQ ID NO: 997 or SEQ ID NO: 991. Likewise, amino acid insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues can be made at or around positions X78 and X79 of SEQ ID NO: 997 or SEQ ID NO: 991.
In one embodiment, the LukE binding molecule as described herein comprises the FG loop region of SEQ ID NO: 997 as described above and binds to a neutralizing epitope of LukE in one of the regions of LukE noted above, i.e., amino acid residues 22-33, 123-128, 137-142, 68-86, 151-161, 175-189, 193-207, and 248-278 of SEQ ID NO:1055.
In accordance with this embodiment, the LukE binding molecule as described herein comprises the amino acid sequence of LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTG LKPGTEYTVSIYGV X75 X76 X77 X78 X79 X80X81 X82 X83 X84 X85 X86 SNPLSAIFTT (SEQ ID NO: 991), where
X75 is any amino acid residue,
X76 is any amino acid residue,
X77 is I or a functionally equivalent amino acid residue
X78 is any amino acid residue,
X79 is any amino acid residue,
X80 is G or a functionally equivalent amino acid residue
X81 is W or a functionally equivalent amino acid residue
X82 is L or a functionally equivalent amino acid residue,
X83 is D or a functionally equivalent amino acid residue,
X84 is F or a functionally equivalent amino acid residue,
X85 is V or a functionally equivalent amino acid residue, and
X86 is F or a functionally equivalent amino acid residue.
In accordance with this embodiment, exemplary LukE binding molecules comprise any one of the amino acid sequences of SEQ ID Nos: 37 (Luk38) and 828-839. In some embodiments, these LukE binding molecules comprise an initiator methionine residue linked to the N-terminus or a cysteine residue linked to the C-terminus to facilitate expression and/or conjugation to another moiety (e.g., another leukotoxin binding FN3 domain, a half-life extending moiety, or other therapeutic moiety).
In another embodiment, the LukE binding molecule as described herein binds LukE with one or more amino acid residues corresponding to residues 177, G80, W81, L82, D83, F84, V85, and F86 of SEQ ID NO: 37 (Luk38). In one embodiment, the FN3 domain containing a LukE binding region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 37.
Additional exemplary LukE binding molecules are disclosed herein. Accordingly, an exemplary LukE binding molecule of the present disclosure comprises any one of the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 113, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 155, 363, 364, 366, 367, 368, 369, 375, 376, 388, and 586, or an amino acid sequence that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 113, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 155, 363, 364, 366, 367, 368, 369, 375, 376, 388, and 586. In some embodiments, these LukE binding molecules comprise an initiator methionine residue linked to the N-terminus or a cysteine residue linked to the C-terminus to facilitate expression and/or conjugation to another moiety (e.g., another leukotoxin binding FN3 domain, a half-life extending moiety, or other therapeutic moiety).
In another aspect, the binding molecule described herein comprises a FN3 domain having one or more modified loop regions that comprise one or more LukA and/or LukB binding regions. This binding molecule is capable of binding a LukA protein having the amino acid sequence of SEQ ID NO: 671 or a fragment or homolog thereof, and/or a LukB protein having the amino acid sequence of SEQ ID NO: 11 or a fragment or homolog thereof. In one embodiment, the binding molecule neutralizes LukAB activity, e.g., by inhibiting LukA and LukB interaction or by inhibiting LukAB binding to the host immune cells. In accordance with this embodiment, the binding molecule binds to one or more neutralizing epitopes of LukA and/or neutralizing epitopes of LukB. As described in more detail in the Examples herein, amino acid residues 201-224 and 260-269 of SEQ ID NO: 11 comprise regions of LukB that contain neutralizing epitopes.
In one embodiment, a LukAB binding molecule as described herein comprises a C strand and a CD loop region having the amino acid sequence DSFX32IX34YX36EX38X39X40X41X42E (SEQ ID NO: 998), where X32 is W or a functionally equivalent amino acid residue,
X34 is any amino acid residue,
X36 is E or a functionally equivalent amino acid residue,
X38 is K or a functionally equivalent amino acid residue,
X39 is F or a functionally equivalent amino acid residue,
X40 is Y or a functionally equivalent amino acid residue,
X41 is R or a functionally equivalent amino acid residue, and
X42 is any amino acid residue;
and an F strand and FG loop region having the amino acid sequence of TX66YX68VX70IX72GVKG X77 X78 X79 SX81 (SEQ ID NO: 999), where
X66 is any amino acid residue,
X68 is K or a functionally equivalent amino acid residue,
X70 is W or a functionally equivalent amino acid residue,
X72 is any amino acid residue,
X77 is any amino acid residue,
X78 is any amino acid residue,
X79 is any amino acid residue, and
X81 is W or a functionally equivalent amino acid residue.
In one embodiment, the LukAB binding molecule as described herein comprises the C strand and a CD loop region of SEQ ID NO: 998, and the F strand and FG loop region of SEQ ID NO: 999 as described above and binds to a neutralizing epitope of LukB in one of the regions of LukB identified above, i.e., amino acid residues 201-224 and/or 260-269 of SEQ ID NO:11.
In accordance with this embodiment, an exemplary LukAB binding molecule as described herein comprises the amino acid sequence of LPAPKNLVVSRVTEDSARLSWTAPDAAFDSF X32I X34Y X36E X38 X39 X40 X41 X42EAI X46LTVPGSERSYDLTGLKPGTX66Y X68V X701 X72GVKG X77 X78 X79 S X81 X82L X84A X86FTT (SEQ ID NO: 992), where
X32 is W or a functionally equivalent amino acid residue,
X34 is any amino acid residue,
X36 is E or a functionally equivalent amino acid residue,
X38 is K or a functionally equivalent amino acid residue,
X39 is F or a functionally equivalent amino acid residue,
X40 is Y or a functionally equivalent amino acid residue,
X41 is R or a functionally equivalent amino acid residue,
X42 is any amino acid residue,
X42 is any amino acid residue,
X46 is any amino acid residue,
X66 is any amino acid residue,
X68 is K or a functionally equivalent amino acid residue,
X70 is W or a functionally equivalent amino acid residue,
X72 is any amino acid residue,
X77 is any amino acid residue,
X78 is any amino acid residue,
X79 is any amino acid residue,
X81 is W or a functionally equivalent amino acid residue,
X82 is any amino acid residue
X84 is any amino acid residue, and
X86 is any amino acid residue.
In accordance with this embodiment, exemplary LukAB binding molecules comprise any one of the amino acid sequences of SEQ ID Nos: 14 (Luk17) and 672-738. In some embodiments, these LukAB binding molecules comprise an initiator methionine residue linked to the N-terminus or a cysteine residue linked to the C-terminus to facilitate expression and/or conjugation to another moiety (e.g., another leukotoxin binding FN3 domain, a half-life extending moiety, or other therapeutic moiety).
In another embodiment, the LukAB binding molecule as described herein binds LukAB with one or more amino acid residues corresponding to residues W32, T34, E36, K38, F39, Y40, R41, A44, V46, E66, K68, W70, V72, W81, and P82 of SEQ ID NO: 14 (Luk17). In one embodiment, the FN3 domain containing a LukAB binding region comprises an amino acid sequence that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14, where amino acid residues W32, T34, E36, K38, F39, Y40, R41, A44, V46, E66, K68, W70, V72, W81, and P82 of SEQ ID NO: 14 remain fixed or are substituted with functionally equivalent amino acid residues.
Additional exemplary LukAB binding molecules are disclosed herein. Accordingly, an exemplary LukAB binding molecule of the present disclosure comprises any one of the amino acid sequences of SEQ ID NOs: 15, 16, 17, 145, 156, 158, 167, 214, 226, 247, 282, 286, 316, 370, 386, 388, 392, 446, 454, 462, 530, 540, 568, 574, 584, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666 as well as an amino acid sequence that is at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 15, 16, 17, 145, 156, 158, 167, 214, 226, 247, 282, 286, 316, 370, 386, 388, 392, 446, 454, 462, 530, 540, 568, 574, 584, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666 In some embodiments, these LukAB binding molecules comprise an initiator methionine residue linked to the N-terminus or a cysteine residue linked to the C-terminus to facilitate expression and/or conjugation to another moiety (e.g., another leukotoxin binding FN3 domain, a half-life extending moiety, or other therapeutic moiety).
Another aspect of the present disclosure is directed to a binding molecule comprising one or more modified fibronectin type III (FN3) domains, where the modified FN3 domain contains one or more loop regions that comprise one or more staphylococcal hemolysin binding regions. In one embodiment, the binding molecule described herein comprises a FN3 domain having one or more modified loop regions that comprise one or more staphylococcal alpha-hemolysin (Hla) binding regions. This binding molecule is capable of binding an Hla protein having the amino acid sequence of SEQ ID NO: 1086 or fragments or homologs thereof. In one embodiment, the binding molecule neutralizes Hla activity, e.g., Hla mediated pore formation and cell death. In accordance with this embodiment, the binding molecule binds to one or more neutralizing epitopes of Hla.
In accordance with this embodiment, exemplary Hla binding molecules comprise any one of the amino acid sequences of SEQ ID Nos: 1097, 1099, 1112, 1142, and 1100, as well as amino acid sequences that are at least 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1097, 1099, 1112, 1142, and 1100. In some embodiments, these Hla binding molecules comprise an initiator methionine residue linked to the N-terminus or a cysteine residue linked to the C-terminus to facilitate expression and/or conjugation to another moiety (e.g., another leukotoxin binding FN3 domain, a half-life extending moiety, or other therapeutic moiety).
In one embodiment, the binding molecule as disclosed herein is a monospecific binding molecule. In another embodiment, the binding molecule as disclosed herein is a bispecific binding molecule. A bispecific binding molecule of the present disclosure is a molecule comprising a first FN3 domain having a first staphylococcal leukotoxin binding region and a second FN3 domain having a second staphylococcal leukotoxin binding region that is distinct from the first staphylococcal leukotoxin binding region. In another embodiment, the binding molecule of the present disclosure is a multispecific binding molecule. A multispecific binding molecule as described herein is a molecule having at least a first, a second, and a third FN3 domain, each FN3 domain having a distinct staphylococcal leukotoxin binding region, i.e., the binding molecule comprises at least first, second, and third staphylococcal leukotoxin binding regions that are each distinct from each other. Bispecific and multispecific binding molecules as disclosed herein can be generated by covalently linking any first staphylococcal leukotoxin binding FN3 domain and any second or third or more staphylococcal leukotoxin binding FN3 domains directly or via a linker. Suitable linkers include peptides composed of repetitive modules of one or more of the amino acids, such as glycine and serine or alanine and proline. Exemplary linker peptides include, e.g., (Gly-Gly)n, (Gly-Ser)n, (Gly3-Ser)n, (Ala-Pro)n whereinn is an integer from 1-25. The length of the linker may be appropriately adjusted as long as it does not affect the function of the binding molecule. The standard 15 amino acid (Gly4-Ser)3 linker peptide has been well-characterized (e.g., within the context of an antibody single-chain Fv (scFv) domain) and has been shown to adopt an unstructured, flexible conformation. In addition, this linker peptide does not interfere with assembly and binding activity of the domains it connects (Freund et al., “Characterization of the Linker Peptide of the Single-Chain Fv Fragment of an Antibody by NMR Spectroscopy,” FEBS 320:97 (1993), the disclosure of which is hereby incorporated by reference in its entirety).
In one embodiment, a bispecific binding molecule of the present disclosure comprises a first FN3 domain having a staphylococcal LukE binding region, e.g., any of the FN3 domain LukE binding regions described supra, coupled to a second FN3 domain having a different leukotoxin binding region. In one embodiment, the second FN3 domain comprises a LukAB binding region, e.g., any of the FN3 domain LukAB binding regions described supra. In one embodiment, the bi-specific binding molecule has a FN3 domain binding LukE that comprises an amino acid sequence of any one of SEQ ID NOs: 989, 990, or 991, and the FN3 domain binding LukAB that comprises an amino acid sequence of SEQ ID NO: 992. In another embodiment, the FN3 domain binding LukE comprises any one of the amino acid sequences selected from SEQ ID NOs: 25-59, 113,116-136, 155, 363, 364, 366-369, 375, 376, 388, 586, and 739-839, and the FN3 domain binding LukAB comprises any one of the amino acid sequences selected from SEQ ID NOs: 14, 15, 16, 17, 145, 156, 158, 167, 214, 226, 247, 282, 286, 316, 370, 386, 388, 392, 446, 454, 462, 530, 540, 568, 574, 584, 587-666 and 672-738.
The FN3 domains specifically binding a staphylococcal leukotoxin as described herein can be modified to improve their properties such as thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison to highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase alpha-helix propensity, engineering of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol, 12: 371-375 (2001), which is hereby incorporated by reference in its entirety). High thermal stability may increase the yield of the expressed protein, improve solubility or activity, decrease immunogenicity, and minimize the need of a cold chain in manufacturing. Residues that can be substituted to improve thermal stability of Tencon (SEQ ID NO: 1017) or Tencon 25 (SEQ ID NO: 1) include, without limitation, residues at positions 11, 14, 17, 37, 46, 73, or 86, and are described in U.S. Patent Publication No. 2011/0274623 to Jacobs et al., which is hereby incorporated by reference in its entirety. Substitutions corresponding to these residues can be incorporated to the FN3 domains or the monospecific, bispecific, or multispecific FN3 domain containing binding molecules of the disclosure.
The binding molecule of the present disclosure is preferably an “isolated” binding molecule. “Isolated” when used to describe the binding molecule disclosed herein, means a binding molecule that has been identified, separated and/or recovered from a component of its production environment. Preferably, the isolated binding molecule is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the binding molecule will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, an isolated binding molecule will be prepared by at least one purification step.
As described supra, amino acid sequence modifications of the binding molecules described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the binding molecule. Amino acid sequence variants of the binding molecules are prepared by introducing appropriate nucleotide changes into the binding molecules nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequences of the binding molecules. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as abolishment of protein A binding and FcγRI binding, or protease-resistance.
Exemplary modifications are for example conservative substitutions or functionally equivalent amino acid residue substitution and include those that will result in variants with similar characteristics to those of the parent binding molecules and fusion constructs described infra. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. Alternatively, the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981, which is hereby incorporated by reference in its entirety). Non-conservative substitutions can be made to the binding molecules that involve substitutions of amino acid residues between different classes of amino acids to improve properties of the binding molecules and fusion constructs. Whether a change in the amino acid sequence of a polypeptide or fragment thereof results in a functional homolog can be readily determined by assessing the ability of the modified binding molecule to produce a response in a fashion similar to the unmodified binding molecule using assays described herein.
Another aspect of the present disclosure is directed to a fusion construct comprising a first portion comprising one or more binding molecules as described herein, and a second portion coupled to said first portion. The second portion of the fusion construct may comprise a second binding molecule, a pharmaceutically active moiety, a prodrug, a pharmaceutically-acceptable carrier, a diagnostic moiety, a cell penetrating enhancer moiety, a half-life extending modulating moiety, and any combination thereof.
In accordance with this aspect of the present disclosure, the first and second portions of the fusion construct are covalently coupled either directly or via a linker as described supra. The first and second portions may be directly fused and generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the portions directly or via a peptide or other linker to recombinantly produce fusion constructs as described herein.
In one embodiment, the second portion of the fusion construct of the present disclosure comprises a half-life extending moiety. Exemplary half-life extending moieties include, without limitation, albumin, albumin variants (see e.g., U.S. Pat. No. 8,822,417 to Andersen et al., U.S. Pat. No. 8,314,156 to Desai et al., and U.S. Pat. No. 8,748,380 to Plumridge et al., which are hereby incorporated by reference in their entirety), albumin-binding proteins and/or domains, transferrin and fragments and analogues thereof (see e.g., U.S. Pat. No. 7,176,278 to Prior et al., which are hereby incorporated by reference in their entirety), Fc regions and variant Fc regions (see e.g., U.S. Pat. No. 8,546,543 to Lazar et al., U.S. Patent Publication No. 20150125444 to Tsui, and U.S. Pat. No. 8,722,615 to Seehra et al., which are hereby incorporated by reference in their entirety).
Other second portion half-life extending moieties of the fusion construct include, without limitation, polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. A pegyl moiety may for example be added to the bispecific or monospecific molecules of the disclosure by adding a cysteine residue to the C-terminus of the molecule and attaching a pegyl group to the cysteine using methods well known in the art.
In another embodiment, the second portion of the fusion construct comprises a pharmaceutically active moiety, such as, e.g., a cytokine, a toxin, a chemokine, an antibacterial peptide, an antibiotic, an enzyme, a peptide or protein with specific target binding characteristics, a fluorescent dye, a photosensitizer, a radionuclide, a contrast agent for medical imaging, a toxic compound, a pro-coagulant factor, an enzyme for pro-drug activation, an albumin binder, an albumin, an IgG binder or polyethylene glycol.
In another embodiment, the second portion of the fusion construct comprises a cell penetrating peptide (CPPs). CPPs translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway and have been used successfully for intracellular delivery of macromolecules, including antibodies, peptides, proteins, and nucleic acids, with molecular weights several times greater than their own. Several commonly used CPPs, including polyarginines, transportant, protamine, maurocalcine, and M918 are known in the art (see Stewart et al., “Cell-Penetrating Peptides as Delivery Vehicles for Biology and Medicine,” Organic Biomolecular Chem 6:2242-2255 (2008), which is hereby incorporated by reference in its entirety). In another embodiment, the second portion of the fusion construct comprises a cell penetrating enhancer moiety. Suitable cell penetrating enhancer moieties include, without limitation, oligo-arginyl derivatives (Bersani et al., Bioconjug. Chem. 23(7):1415-25 (2012), which is hereby incorporated by reference in its entirety), and corona-like (guanidyl)-oligosaccharidic derivatives (see WO2012/097876 To Caliceti et al., which is hereby incorporated by reference in its entirety).
In another embodiment, the second portion of the fusion construct comprises a diagnostic moiety. Suitable diagnostic moieties are those that facilitate the detection, quantitation, separation, and/or purification of the fusion construct. Suitable diagnostic moieties include, without limitation, purification tags (e.g., poly-histidine (His6), glutathione-S-transferase (GST-), or maltose-binding protein (MBP-)), fluorescent tags (e.g., chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red), an enzymatic tag, a radioisotope or radioactive label, a contrast agent suitable for imaging, or a photosensitize.
In another embodiment, the second portion of the fusion construct comprises a second binding molecule. In one aspect, the second binding molecule is an antibody or antibody binding domain thereof. As used herein, an “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to, at least one, at least two, or at least three complementarity determining region (CDR) of a heavy or light chain, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof. Antibodies encompass full antibodies, digestion fragments, specified portions and variants thereof, including, without limitation, portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including, without limitation, single chain antibodies, single domain antibodies (i.e., antibody fragments comprising merely one variable domain, which might be VHH, VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains). Functional fragments include antigen-binding fragments that bind to a particular target. For example, antibody fragments capable of binding to a particular target or portions thereof, include, but are not limited to, Fab (e.g., by papain digestion), Fab′ (e.g., by pepsin digestion and partial reduction) and F(ab′)2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc′ (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments.
Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F(ab′)2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and/or hinge region of the heavy chain. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
Furthermore, the term “antibody” encompasses monoclonal and polyclonal antibodies, as well as human, humanized, or chimeric antibodies, and derivatives or variants of the antibodies described herein which display the same specificity as the described antibodies. Examples of “antibody variants” include humanized variants of non-human antibodies, “affinity matured” antibodies (see e.g., Hawkins et al., “Selection of Phage Antibodies by Binding Affinity. Mimicking Affinity Maturation,” J. Mol. Biol. 254:889-896 (1992) and Lowman et al., “Selecting High-Affinity Binding Proteins by Monovalent Phage Display,” Biochemistry 30:10832-10837 (1991), each of which is hereby incorporated by reference in its entirety) and antibody mutants with altered effector function(s) (see, e.g., U.S. Pat. No. 5,648,260 to Winter et al., Kontermann and DUbel, A
In another embodiment, the second binding molecule comprises an antibody mimetic, i.e., an organic compound, often a peptide, polypeptide, or protein that binds specifically to an antigen, but is structurally unrelated to an antibody. Exemplary antibody mimetics include, without limitation, affibodies (scaffold based on the Z domain of Protein A), affilins (scaffold based on the structure of gamma crystallin or ubiquitin), affimers (scaffold based on the structure of cystatins), affitins (scaffold derived from the DNA binding protein Sac7d), alphabodies (scaffold based on a coiled coil structure), anticalins (scaffold derived from lipocalins), avimers (scaffold derived from A domains of various membrane receptors), DARPins (scaffold derived from ankyrin repeat motif), fynomers (scaffold derived from SH3 domain of Fyn), and Kunitz domain (scaffold derived from Kunitz domains of various protease inhibitors).
The second binding molecule may be a monospecific, bispecific, or multi-specific binding molecule, i.e., the second binding molecule has one, two, or multiple distinct binding sites for binding one or more distinct protein targets. The second binding molecule may be a monovalent, bivalent, or multi-valent, i.e., the second binding molecule has one, two, or multiple binding sites for a particular target molecule. For example, the second binding molecule may comprise an antibody that is monospecific, bispecific, or tri-specific, and the antibody may be monovalent, bivalent, or multi-valent. In one embodiment, the antibody is a mono-specific, bivalent antibody.
In one embodiment, the carboxy terminus of the second binding molecule is coupled to the amino terminus of the binding molecule of the present disclosure. In another embodiment the carboxy terminus of the second binding molecule is coupled to the carboxy terminus of the binding molecule of the present disclosure. When the second binding molecule comprises an antibody, one or more of the same or distinct binding molecules may be coupled to the light chain and/or the heavy chain of the antibody, or binding fragments thereof. In one embodiment, one or more of the same or distinct binding molecules are coupled to one or more of the heavy chains of an antibody. In another embodiment, one or more of the same or distinct binding molecules are coupled to one or more of the light chains of an antibody. In another embodiment, one or more distinct binding molecules are coupled to one or more of the light chains and the heavy chains of an antibody.
In one aspect, the second binding molecule is resistant to proteolytic degradation by a staphylococcal protease that cleaves wild-type IgG1 (such as the staphylococcal protease, Staphylococcus aureus V8 protease, that cleaves wild-type IgG1 between or at residues 222-237 (EU numbering) within SEQ ID NO: 60) (see U.S. Pat. No. 8,871,204 to Strohl et al., which is hereby incorporated by reference in its entirety).
In one aspect, the second binding molecule is not capable of specific binding to human FcγRI, is not capable of specific binding to Protein A, and is not capable of specific binding to Sbi. In one aspect, the second binding molecule is capable of specific binding to FcRn.
In one embodiment, the second binding molecule of the fusion construct has binding specificity for a staphylococcal protein. In one embodiment, the second binding molecule has binding specificity for a staphylococcal virulence factor. As used herein a “virulence factor” refers to a molecule expressed by staphylococcal protein that enables the bacteria to achieve colonization of a niche in the host (including adhesion to cells), immunoevasion (i.e., evasion of the host's immune response), immunosuppression (i.e., inhibition of the host's immune response), entry into and exit out of cells (if the pathogen is an intracellular one), and/or obtain nutrition from the host. The virulence factor may be encoded on mobile genetic elements, such as bacteriophages, and can easily be spread through horizontal gene transfer. Non-limiting examples of Staphylococcus aureus virulence factors include hyaluronidase, protease, coagulase, lipases, deoxyribonucleases, enterotoxins and other toxins. For purposes of this disclosure, staphylococcal surface proteins, such as SDR-containing proteins, e.g., ClfA, ClfB, SdrC, SdrD, SdrE, SdrF, SdrG and SdrH, are also considered virulence factors.
In one embodiment, the second binding molecule of the fusion construct of the present disclosure is capable of binding to a glycosylated staphylococcal surface protein. Exemplary second binding molecules that bind a glycosylated staphylococcal surface protein include the antibodies and antibody binding domain fragments disclosed in U.S. Pat. Nos. 8,460,666 and 8,211,431 to Throsby et al., which is hereby incorporated by reference in its entirety. In one embodiment, the antibody or antibody binding domain that binds a glycosylated staphylococcal surface protein has an immunoglobulin heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 60, 62, 64 or 66. In another embodiment, the antibody or antibody binding domain that binds a glycosylated staphylococcal surface protein has an immunoglobulin light chain comprising an amino acid sequence of SEQ ID NOs: 61, 63, 65 or 67. Alternatively, the second binding molecule comprises an antibody having (a) a heavy chain having the amino acid sequence of SEQ ID NOs:60, 62, 64 or 66; and (b) a light chain having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67. In another embodiment, the second binding molecule comprises an antibody having (1) a heavy chain having the amino acid sequence of SEQ ID NO:60, and a light chain having the amino acid sequence of SEQ ID NO:61; (2) a heavy chain having the amino acid sequence of SEQ ID NO:62, and a light chain having the amino acid sequence of SEQ ID NO:63; (3) a heavy chain having the amino acid sequence of SEQ ID NO:64, and a light chain having the amino acid sequence of SEQ ID NO:65; (4) a heavy chain having the amino acid sequence of SEQ ID NO:66, and a light chain having the amino acid sequence of SEQ ID NO:67; (5) a heavy chain having the amino acid sequence of SEQ ID NO:68, and a light chain having the amino acid sequence of SEQ ID NO:69; (6) a heavy chain having the amino acid sequence of SEQ ID NO:70, and a light chain having the amino acid sequence of SEQ ID NO:71; (7) a heavy chain having the amino acid sequence of SEQ ID NO:72, and a light chain having the amino acid sequence of SEQ ID NO:73; (8) a heavy chain having the amino acid sequence of SEQ ID NO:74, and a light chain having the amino acid sequence of SEQ ID NO:75; (9) a heavy chain having the amino acid sequence of SEQ ID NO:76, and a light chain having the amino acid sequence of SEQ ID NO:77; or (10) a heavy chain having the amino acid sequence of SEQ ID NO:78, and a light chain having the amino acid sequence of SEQ ID NO:79.
In one embodiment, the fusion construct of the present disclosure comprises a binding molecule having a LukE binding region coupled to an immunoglobulin heavy chain, where the heavy chain comprises a variable region that binds to a glycosylated staphylococcal surface protein. Exemplary fusion constructs according to this aspect of the present disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 848-851, 900, or 903. In one embodiment, these exemplary fusion constructs further comprise an immunoglobulin light chain variable region. Suitable light chains include, without limitation, light chains having an amino acid sequence of any one of SEQ ID NO: 61, 63, 65 or 67.
In another embodiment, the fusion construct of the present disclosure comprises a binding molecule having a LukE binding region coupled to an immunoglobulin light chain, where the light chain comprises a variable region that binds to a glycosylated staphylococcal surface protein. An exemplary fusion construct according to this aspect comprises an amino acid sequence of SEQ ID NO: 980. In one embodiment, these exemplary fusion constructs further comprise an immunoglobulin heavy chain variable region or heavy chain. Suitable heavy chains include, without limitation, heavy chains having an amino acid sequence of any one of SEQ ID NOs: 60, 62, 64 or 66.
In one embodiment, the fusion construct of the present disclosure comprises a binding molecule having a LukAB binding region coupled to an immunoglobulin heavy chain, where the heavy chain comprises a variable region that binds to a glycosylated staphylococcal surface protein. Exemplary fusion constructs according to this aspect of the present disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 70 and 72. In one embodiment, these exemplary fusion constructs further comprise an immunoglobulin light chain. Suitable light chains include, without limitation, light chains having an amino acid sequence of any one of SEQ ID NO: 61, 63, 65 or 67.
In another embodiment, the fusion construct of the present disclosure comprises a binding molecule having a LukAB binding region coupled to an immunoglobulin light chain, where the light chain comprises a variable region that binds to a glycosylated staphylococcal surface protein. Exemplary fusion constructs according to this aspect of the present disclosure comprise an amino acid sequence of SEQ ID NO: 979. In one embodiment, these exemplary fusion constructs further comprise an immunoglobulin heavy chain variable region or heavy chain. Suitable heavy chains include, without limitation, heavy chains having an amino acid sequence of any one of SEQ ID NO: 60, 62, 64 or 66.
In another embodiment, the fusion construct of the present disclosure comprises a binding molecule having a LukE binding region and a LukAB binding region coupled to an immunoglobulin heavy chain, where the heavy chain comprises a variable region that binds to a glycosylated staphylococcal surface protein. Exemplary fusion constructs according to this aspect of the disclosure comprise an amino acid sequence of any one of SEQ ID NOs: 852-859, 887, 888, 893, 894, 906, 920, 931-956, 961, 976, and 984-988. In one embodiment, these exemplary fusion constructs further comprise an immunoglobulin light chain variable region or light chain comprising a glycosylated staphylococcal surface protein binding domain. Suitable immunoglobulin light chains include, without limitation, light chains having an amino acid sequence of any one of SEQ ID NO: 61, 63, 65 or 67.
Another aspect of the present disclosure is directed to nucleic acid molecules encoding the binding molecules and fusion constructs described herein. The nucleic acid molecules of the present disclosure include isolated polynucleotides, portions of expression vectors or portions of linear DNA sequences, including linear DNA sequences used for in vitro transcription/translation, vectors compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion and/or display of the compositions or directed mutagens thereof.
In one embodiment isolated polynucleotides of the present disclosure include those encoding the binding molecules described supra. Exemplary isolated polynucleotide molecules include those encoding a FN3 domain that comprises a LukE binding region having any one of the amino acid sequences of SEQ ID NOs: 25-59, 113,116-136, 155, 363, 364, 366-369, 375, 376, 388, 586, and 739-839. In another embodiment, exemplary polynucleotides include those encoding a FN3 domain that comprises a LukAB binding region having any one of the amino acid sequences of SEQ ID NOs: 14, 15, 16, 17, 145, 156, 158, 167, 214, 226, 247, 282, 286, 316, 370, 386, 388, 392, 446, 454, 462, 530, 540, 568, 574, 584, 587-666 and 672-738. In another embodiment, exemplary polynucleotides include those encoding a FN3 domain that comprises an Hla binding region having any one of the amino acid sequences of SEQ ID Nos: 1097, 1099, 1112, 1142, and 1100.
In other embodiment, exemplary polynucleotides include isolated polynucleotides encoding the antibody-binding molecule fusion constructs described herein. For example, exemplary isolated polynucleotides include those encoding a fusion construct comprising a binding molecule having a LukE binding domain coupled to an immunoglobulin heavy chain comprising a glycosylated staphylococcal surface protein binding domain (e.g., polynucleotides encoding amino acid sequences of SEQ ID NOs: 848-851, 900 or 903), and a LukE binding domain coupled to an immunoglobulin light chain comprising a glycosylated staphylococcal surface protein binding domain (e.g., a polynucleotide encoding an amino acid sequence of SEQ ID NO: 980). Exemplary isolated polynucleotides also include those encoding a fusion construct comprising a binding molecule having a LukAB binding domain coupled to an immunoglobulin heavy chain comprising a glycosylated staphylococcal surface protein binding domain (e.g., polynucleotides encoding amino acid sequences of SEQ ID NOs: 70 and 72), and a LukAB binding domain coupled to an immunoglobulin light chain comprising a glycosylated staphylococcal surface protein binding domain (e.g., a polynucleotide encoding an amino acid sequence of SEQ ID NO: 979). Exemplary isolated polynucleotides also include those encoding a fusion construct comprising a binding molecule having LukE and LukAB binding domains coupled to a immunoglobulin heavy chain comprising a glycosylated staphylococcal surface protein binding region (e.g., polynucleotides encoding amino acid sequences of SEQ ID NOs: 852-859, 887, 888, 893, 894, 906, 920, 931-956, 961, 976, and 984-988).
The polynucleotides of the disclosure may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer and assembled into complete single or double stranded molecules. Alternatively, the polynucleotides of the disclosure may be produced by other techniques such a PCR followed by routine cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.
The polynucleotides described herein may comprise at least one non-coding sequence, such as a promoter or enhancer sequence, intron, polyadenylation signal, a cis sequence facilitating RepA binding, and the like. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids that encode for example a marker or a tag sequence such as a histidine tag or an HA tag to facilitate purification or detection of the protein, a signal sequence, a fusion protein partner such as RepA, Fc or bacteriophage coat protein such as pIX or pIII.
Another embodiment of the disclosure is a vector comprising at least one polynucleotides as described herein. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotides of the invention into a given organism or genetic background by any means. Such vectors may be expression vectors comprising nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell-based, or cell-free systems well known in the art.
Another embodiment of the present disclosure is a host cell comprising the above described vectors. The binding molecules and/or fusion constructs disclosed herein can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art (see e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), which are hereby incorporated by reference in their entirety).
The host cell chosen for expression may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of glycosylating polypeptides, e.g. a prokaryotic cell or organism, such as BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any of the natural or engineered E. coli spp, Klebsiellaspp., or Pseudomonas spp strains.
Another aspect of the disclosure is directed to a method of producing and isolating the binding molecules and fusion constructs as described herein. This method involves culturing the isolated host cell of the disclosure under conditions such that the binding molecules or fusion constructs are expressed, and purifying the expressed binding molecules or fusion constructs from the host cell culture.
The binding molecules and fusion constructs described herein can be purified from recombinant cell cultures by well-known methods, for example by protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography, or high performance liquid chromatography (HPLC).
Purified or isolated binding molecules and fusion constructs as described herein may be linked to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The binding molecules and/or fusion constructs may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatine-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in R
For therapeutic use, the binding molecules and fusion constructs as described herein may be prepared as pharmaceutical compositions containing an effective amount of the binding molecules as an active ingredient in a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of binding molecule or fusion construct as described herein in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. R
The binding molecules and fusion constructs described herein can be used in non-isolated or isolated form. Furthermore, the binding molecules and fusion constructs hereof can be used alone or in a mixture comprising at least one other binding molecule or fusion construct hereof. In other words, the binding molecules and fusion constructs can be used in combination, e.g., as a pharmaceutical composition comprising two or more binding molecules hereof, two or more fusion constructs, a binding molecule and fusion construct, and variants or fragments thereof. For example, binding molecules and/or fusion constructs having different, but complementary activities can be combined in a single therapy to achieve a desired therapeutic effect, but alternatively, binding molecules and fusion constructs having identical activities can also be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect. Optionally, the mixture further comprises at least one other therapeutic agent. In one aspect, the other therapeutic agent may be an anti-infective agent, an antibiotic agent, and/or an antimicrobial agent that is useful in the prophylaxis and/or treatment of a staphylococcal infection. In another aspect, the other therapeutic agent may be any agent that is useful in the prophylaxis and/or treatment of a condition associated with a staphylococcal infection.
The binding molecules, fusion constructs, or pharmaceutical compositions containing the same can be used for the treatment, prevention or amelioration of a staphylococcal infection. The staphylococcal infection may be caused by any Staphylococcus spp. In one aspect, the staphylococcal infection is caused by Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). Accordingly, the present disclosure provides a method for the treatment, prevention or amelioration of a staphylococcal infection that involves administering to a subject in need thereof a binding molecule, fusion construct, or compositions containing the same as described herein.
In accordance with this aspect, the target “subject” encompasses any animal, for example, a mammal, such as a human. In the context of administering a composition of the disclosure for purposes of preventing a staphylococcal infection in a subject, the target subject encompasses any subject that is at risk of becoming infected with Staphylococcus or developing a staphylococcal infection. Susceptible subjects include infants and juveniles, as well as immunocompromised juvenile, adults, and elderly adults. However, any infant, juvenile, adult, or elderly adult or immunocompromised individual at risk for developing a staphylococcal infection can be treated in accordance with the methods described herein. In the context of administering a composition of the disclosure for purposes of treating a staphylococcal infection in a subject, the target subject encompasses any subject infected with Staphylococcus. Particularly suitable subjects include those at risk of infection, susceptible to infection, or those infected with methicillin-resistant S. aureus (MRSA) or methicillin sensitive S. aureus (MSSA). Other suitable subjects include those subjects which may have or are at risk for developing a condition resulting from a Staphylococcus infection, i.e., a staphylococcal associated condition, such as, for example, skin wounds and infections, tissue abscesses, folliculitis, osteomyelitis, pneumonia, scalded skin syndrome, septicemia, septic arthritis, myocarditis, endocarditis, and toxic shock syndrome.
In one embodiment, the binding molecules, fusion constructs, or pharmaceutical compositions containing the same are administered prophylactically to prevent, delay, or inhibit the development of staphylococcal infection in a subject at risk of developing a staphylococcal infection or associated condition. In one aspect, prophylactic administration of one or more binding molecules described herein is effective to fully prevent S. aureus infection in an individual. In other embodiments, prophylactic administration is effective to prevent the full extent of infection that would otherwise develop in the absence of such administration, i.e., substantially prevent, inhibit, or minimize staphylococcal infection in an individual.
In another embodiment, the binding molecules, fusion constructs, or pharmaceutical compositions as described herein are administered therapeutically to an individual having a staphylococcal infection to inhibit the progression and further development of the infection, i.e., to inhibit and/or prevent the spread of the infection to other cells in an individual, decrease infection, and to treat or alleviate one or more symptoms of infection.
Therapeutically effective amounts of the binding molecules and fusion constructs described herein are determined in accordance with standard procedures, which take numerous factors into account, including, for example, the concentrations of the binding molecules or fusion constructs in a pharmaceutical composition, the mode and frequency of administration, the severity of the Staphylococcus infection to be treated (or prevented), and subject details, such as age, weight and overall health and immune condition. General guidance can be found, for example, in the publications of the International Conference on Harmonization and in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company 1990), which is hereby incorporated by reference in its entirety. A clinician may administer a composition comprising the binding molecules or fusion constructs described herein in a single dose or in accordance with a multi-dosing protocol until a dosage is reached that provides the desired or required prophylactic or therapeutic effect. The progress of this therapy can be easily monitored by conventional assays. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dosage at relatively short intervals (e.g., as little as 15 minutes, 30 minutes, 60 minutes, 90 minutes or even 2 or 3 hours) is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease.
The therapeutically effective amount, i.e., the dosage sufficient for a subject having a staphylococcal infection that is sufficient to slow or prevent the spread or severity of staphylococcal infection, and/or the dosage sufficient to prevent, alleviate (either partially or completely) a staphylococcal infection associated condition. Such therapeutically effective amounts vary by individual, but may range from 0.1 to 10 mg/kg body weight, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg. A fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 400, 300, 250, 200, or 100 mg/m2. Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat infection, but 10, 12, 20 or more doses may be given depending on the severity of infection. Administration of binding molecules or fusion constructs of the present disclosure may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose.
The therapeutic compositions of the present disclosure can be administered alone or as part of a combination therapy in conjunction with one or more other active agents, depending upon the nature of the Staphylococcus infection that is being treated. Such additional active agents include anti-infective agents, antibiotic agents, and antimicrobial agents that are readily known in the art.
The mode of administration of the binding molecules, fusion constructs, and pharmaceutical compositions described herein may be any suitable route that delivers the binding molecule(s) or fusion construct(s) to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal); using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by, for example, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.
The binding molecules and fusion constructs provided herein can also be used in methods for diagnosing a staphylococcal infection in a subject. In one aspect, the method for diagnosing a staphylococcal infection involves contacting a binding molecule or fusion construct as described herein with a sample from the subject to be diagnosed, and detecting at least the presence or the absence of one or more staphylococcal leukotoxins in the sample. In another aspect, the method for diagnosing a staphylococcal infection involves contacting a binding molecule or fusion construct as described herein with a sample from the subject to be diagnosed, and detecting the presence or the absence of one or more staphylococcal leukotoxins in the sample and the presence or absence of one or more other staphylococcal proteins, such as, e.g., a glycosylated staphylococcal surface protein. A staphylococcal infection is diagnosed in the subject based on this detection. In other words, the detection of the one or more staphylococcal leukotoxins alone or in combination with another staphylococcal protein indicates a positive diagnosis of a staphylococcal infection.
In accordance with this aspect, the sample from the subject may comprise a blood, tissue, cell, serum, or any other biological sample.
Another aspect relates to a method for the detection of a staphylococcal infection in a sample. This method involves contacting the binding molecule or fusion construct as described herein with a sample, and detecting the presence or the absence of a at least one or more staphylococcal leukotoxins. Optionally, the presence or absence of one or more staphylococcal leukotoxins and one or more additional staphylococcal proteins, e.g., glycosylated staphylococcal surface proteins, can be detected using one or more of the fusion constructs described herein. Detection of the one or more staphylococcal leukotoxins alone or in combination with another staphylococcal protein indicates the presence of Staphylococcus in the sample. In accordance with this aspect, the sample may be any biological sample obtained from the environment, an animal, or a human.
Methods described herein involving the detection of a staphylococcal leukotoxin alone or in combination with another staphylococcal protein in a sample from a subject or elsewhere involve the use of a detectably labeled binding molecule or fusion construct. Accordingly, in one aspect the binding molecule or fusion construct as described herein may be coupled to a detectable label. Suitable detectable labels are well known in the art and include detectable tags (e.g., a poly-histidine (His6) tag, a glutathione-S-transferase (GST-) tag, or a maltose-binding protein (MBP-) tag); radioactive labels (e.g., carbon (14C) or phosphorous (32P)); fluorescent labels (e.g., fluorescein and derivatives thereof, fluorescein isothiocyanate, rhodamine and derivatives thereof, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); luminescent labels (e.g., luminol); bioluminescent labels (e.g., luciferase, luciferin, and aequorin); or enzymatic labels (e.g., luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase). Alternatively, the binding molecule can be bound by a detectable label, for example, bound by a secondary antibody that contains a detectable label.
Detection assays for detecting the labeled binding molecule or fusion construct bound to a staphylococcal leukotoxin and/or another staphylococcal protein in a sample are well known in the art and include, for example, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescent activated cell sorting (FACS).
Furthermore, the binding molecules and fusion constructs of the present disclosure can be used for the prevention of a staphylococcal infection. This method involves contacting the binding molecule or fusion construct as described herein with a sample from a subject, and detecting a staphylococcal leukotoxin and/or another staphylococcal protein, e.g., a glycosylated staphylococcal surface protein as a result of the contacting. If a staphylococcal leukotoxin and/or another staphylococcal protein is detected in the subject sample, then an agent suitable for preventing staphylococcal infection is administered to the subject. Exemplary prophylactic agents include, but are not limited to, the binding molecules described herein, one or more antibiotics (e.g., mupirocin, nafcillin, cefazolin, dicloxacillin, clindamycin, vancomycin, linezolid, rifampin, sulfamethoxazole-trimethoprim), and/or other anti-infective agents that are effective against staphylococcal infection.
Another aspect of the present disclosure is directed to Leukocidin B (LukB) and Leukocidin A (LukA) polypeptides comprising stem domain mutations and/or deletions. The stem domains of the bi-component leukocidins are structural domains involved in the formation of the active oligomeric, pore forms of the toxins that pierce the host cell membrane and cause cell death via osmotic lysis. As demonstrated in the Examples herein, the stem domain variants of LukA and LukB retain the neutralizing epitope recognized by the LukAB binding molecule (Luk17) described herein. Accordingly, since the stem domain variants do not exhibit cytolytic activity, yet present a neutralizing epitope, they make ideal components of a S. aureus vaccine composition.
In one embodiment, the recombinant LukB polypeptide stem domain variant comprises an amino acid sequence corresponding to amino acid residues 1-109 of SEQ ID NO: 1026 coupled to amino acid residues 152-305 of SEQ ID NO: 1026. The LukB polypeptide does not comprise one or more amino acid residues corresponding to amino acid residues 110-151 of SEQ ID NO: 1026. In other words, one or more amino acid residues corresponding to amino acid residues 110-151 of SEQ ID NO: 1026 is deleted or mutated, thereby rendering the stem domain of the LukB polypeptide inactive or non-functional. When two or more amino acid residues are deleted or mutated, the two or more amino acid residues may be contiguous or non-contiguous. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 contiguous or non-contiguous amino acid residues within the stretch of amino acid residues corresponding to 110-151 of SEQ ID NO: 1026 are deleted or mutated in the LukB polypeptide as described herein. In one embodiment, the LukB polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 122-126 of SEQ ID NO: 1026. In another embodiment, the LukB polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 130-134 of SEQ ID NO: 1026. In another embodiment, the LukB polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 108-150 of SEQ ID NO: 1026. In another embodiment, the LukB polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 110-150 of SEQ ID NO: 1026. In another embodiment, the LukB polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 110-151 of SEQ ID NO: 1026. The remaining portions of the LukB polypeptide, e.g., amino acid residues corresponding to and comprising residues 1-109 of SEQ ID NO: 1026 and 152-305 of SEQ ID NO: 1026 can be coupled directly or via a short linker. Suitable linkers include, without limitation, glycine-rich (e.g. G3-5) or glycine/serine-rich (e.g. GSG, GSGS, (SGG)2, GSNG) linker sequences.
Exemplary LukB polypeptides in accordance with this aspect of the disclosure include, without limitation, LukB polypeptides comprising an amino acid sequence of SEQ ID NO: 1029, SEQ ID NO: 1030, SEQ ID NO: 1031, SEQ ID NO: 1032, SEQ ID NO: 1150, or SEQ ID NO: 1151.
The recombinant Leukocidin A (LukA) polypeptide stem domain variant comprises an amino acid sequence corresponding to amino acid residues 1-134 of SEQ ID NO: 1018 coupled to amino acid residues 175-324 of SEQ ID NO: 1018. The LukA polypeptide does not comprise one or more amino acid residues corresponding to amino acid residues 135-174 of SEQ ID NO: 1018. In other words, one or more amino acid residues corresponding to amino acid residues 135-174 of SEQ ID NO: 1018 is deleted or mutated, thereby rendering the stem domain of the LukA polypeptide inactive or non-functional. When two or more amino acid residues are mutated or deleted, the two or more amino acid residues may be contiguous or non-contiguous. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous or non-contiguous amino acid residues within the stretch of amino acid residues corresponding to 135-174 of SEQ ID NO: 1018 are deleted or mutated in the LukA polypeptide as described herein. In one embodiment, the LukA polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 144-149 of SEQ ID NO: 1018. In another embodiment, the LukA polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 135-174 of SEQ ID NO: 1018. The remaining portions of the LukA polypeptide, i.e., amino acid residues corresponding to residues 1-134 or SEQ ID NO: 1018 and 175-324 of SEQ ID NO: 1018 can be coupled directly or via a suitable linker. Suitable linkers include, without limitation, glycine-rich (e.g. G3-5) or glycine/serine-rich (e.g. GSG, GSGS, (SGG)2, GSNG) linker sequences.
The recombinant LukA polypeptide as described herein may further comprise a glutamic acid to alanine substitution at the amino acid residue corresponding to amino acid residue 323 of SEQ ID NO: 1018 (DuMont et al., “Identification of a Crucial Residue Required for Staphylococcus aureus LukAB Cytotoxicity and Receptor Recognition,” Infect Immun. 82(3):1268-76 (2014), which is hereby incorporated by reference in its entirety).
Exemplary LukA polypeptides in accordance with this aspect of the disclosure include, without limitation, LukA polypeptides comprising an amino acid sequence of SEQ ID NO: 1022, SEQ ID NO: 1023, SEQ ID NO: 1024, SEQ ID NO: 1025, or SEQ ID NO: 1149.
Another aspect of the present disclosure is directed to a vaccine composition comprising the LukB and LukA stem domain variants as described supra. In other words, the vaccine composition comprises a recombinant Leukocidin B (LukB) polypeptide comprising an amino acid sequence corresponding to amino acid residues 1-109 of SEQ ID NO: 1026 coupled to amino acid residues 152-305 of SEQ ID NO: 1026, wherein said LukB polypeptide does not comprise one or more amino acid residues corresponding to amino acid residues 110-151 of SEQ ID NO: 1026. The vaccine composition further comprises a recombinant Leukocidin A (LukA) polypeptide comprising an amino acid sequence corresponding to amino acid residues 1-134 of SEQ ID NO: 1018 coupled to amino acid residues 177-324 of SEQ ID NO: 1018, wherein said LukA polypeptide does not comprise one or more amino acid residues corresponding to amino acid residues 135-174 of SEQ ID NO: 1018. Exemplary LukB and LukA stem domain variants are described supra.
The vaccine composition may further comprise one or more adjuvants. Suitable adjuvants are known in the art and include, without limitation, flagellin, Freund's complete or incomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsion, dinitrophenol, iscomatrix, and liposome polycation DNA particles.
The vaccine composition as described herein may be prepared by formulating the recombinantly produced LukA and LukB stem domain variants with a pharmaceutically acceptable carrier and optionally a pharmaceutically acceptable excipient. As used herein, the terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives and solubilizing agents) are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Examples of pharmaceutically acceptable carriers include water, e.g., buffered with phosphate, citrate and another organic acid. Representative examples of pharmaceutically acceptable excipients that may be useful include antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; adjuvants; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
Another aspect of the present disclosure relates to a method of immunizing a subject against a Staphylococcus aureus infection. This method involves administering the vaccine composition comprising the LukA and LukB stem domain variants, in an amount effective to immunize against S. aureus infection in the subject. A suitable subject for treatment in accordance with this aspect of the present invention is a subject at risk of developing a S. aureus infection.
In accordance with this aspect, a therapeutically effective amount of the vaccine composition for administration to a subject to immunize against S. aureus infection is the amount necessary to generate a humoral (i.e., antibody mediated) immune response. The generated humoral response is sufficient to prevent or at least reduce the extent of S. aureus infection that would otherwise develop in the absence of such response. Preferably, administration of a therapeutically effective amount of the vaccine composition described herein induces a neutralizing immune response against S. aureus in the subject. To effectuate an effective immune response in a subject, the composition may further contain one or more additional S. aureus antigens or an adjuvant as described supra. In an alternative embodiment, the adjuvant is administered separately from the composition to the subject, either before, after, or concurrent with administration of the composition of the present invention.
For purposes of this aspect the disclosure, the target “subject” encompasses any animal, preferably a mammal, more preferably a human. In the context of administering a vaccine composition for purposes of preventing a S. aureus infection in a subject, the target subject encompasses any subject that is at risk of being infected by S. aureus. Particularly susceptible subjects include infants and juveniles, as well as immunocompromised juvenile, adults, and elderly adults. However, any infant, juvenile, adult, or elderly adult or immunocompromised individual at risk for S. aureus infection can be treated in accordance with the methods and vaccine composition described herein. Particularly suitable subjects include those at risk of infection with methicillin-resistant S. aureus (MRSA) or methicillin sensitive S. aureus (MSSA).
Therapeutically effective amounts of the vaccine composition comprising LukA and LukB for immunization will depend on whether an adjuvant is co-administered, with higher dosages being required in the absence of adjuvant. The amount of LukAB for administration sometimes varies from 1 μg-500 μg per patient and more usually from 5-500 μg per injection for human administration. Occasionally, a higher dose of 1-2 mg per injection is used. Typically about 10, 20, 50 or 100 μg is used for each human injection. The timing of injections can vary significantly from once a day, to once a year, to once a decade. Generally an effective dosage can be monitored by obtaining a fluid sample from the subject, generally a blood serum sample, and determining the titer of antibody developed against LukAB, using methods well known in the art and readily adaptable to the specific antigen to be measured. Ideally, a sample is taken prior to initial dosing and subsequent samples are taken and titered after each immunization. Generally, a dose or dosing schedule which provides a detectable titer at least four times greater than control or “background” levels at a serum dilution of 1:100 is desirable, where background is defined relative to a control serum or relative to a plate background in ELISA assays.
Examples are provided below to illustrate the present disclosure. These examples are not meant to constrain the present disclosure to any particular application or theory of operation.
The specificity of mAb 5133 for glycosylated forms of recombinant variants of the S. aureus SdrC protein, a member of the Serine-Aspartate Repeat (SDR) family, was previously demonstrated in a series of western blot analyses (PCT Application Publication No. WO2015089073 to Torres et al., which is hereby incorporated by reference in its entirety). Specifically, following incubation of purified, recombinant SdrC proteins with whole cell lysates prepared from S. aureus strain JE2 (Fey et al., “A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes,” mBio 4(1):e00537-12 (2013), which is hereby incorporated by reference in its entirety), specific protein bands were detected via western blot in contrast to those detected following incubation with lysates prepared from S. aureus NE105, an otherwise-isogenic derivative of JE2 that lacks expression of the SdgB glycosyltransferase (PCT Application Publication No. WO2015089073 to Torres et al., which is hereby incorporated by reference). Further, incubation of purified, recombinant SdrC proteins with a recombinant form of the SdgB glycosyltransferase similarly yielded the mAb 5133 specific epitope in a manner that was dependent on the presence of uridine diphosphate N-acetylglucosamine (UDP-GlcNac) (PCT Application Publication No. WO2015089073 to Torres et al., which is hereby incorporated by reference). In order to further define a minimal epitope for mAb 5133, a synthetic peptide was employed as an in vitro substrate for the SdgB glycosyltransferase that contains ten (10) copies of the Serine-Aspartate dipeptide sequence but lacks any additional protein sequence derived from any of the five members of the SDR family of proteins of S. aureus (Cheng et al., “Variation of Serine-Aspartate Repeats in Membrane Proteins Possibly Contributes to Staphylococcal Microevolution,” PLoS ONE 7(4): e34756 (2012); Becherelli et al., “Protective Activity of the CnaBE3 Domain Conserved Among Staphylococcus aureus Sdr Proteins,” PLoS One 8(9): e74718 (2013), which are hereby incorporated by reference in their entirety).
Procedure. Recombinant variants of the S. aureus SDR family members Clumping Factor A (ClfA) and Clumping Factor B (ClfB) were engineered in vector pET29a(+) such that each bears an amino-terminal S-tag (Merck KGaA; Raines et al., “The S-Tag Fusion System for Protein Purification,” Methods Enzymol. 326:362-367 (2000), which is hereby incorporated by reference in its entirety), thrombin cleavage site, a carboxyl-terminal poly-histidine affinity tag, and correspond to SEQ ID NOs: 668 and 669, respectively. These recombinant proteins were purified to apparent homogeneity following inducible over-expression in Escherichia coli and purification via Ni-NTA agarose resin (Qiagen 1018244) plus S•Tag™ Thrombin Purification Kit (EMD Millipore 69232). A recombinant form of the S. aureus SdgB glycosyltransferase bearing a C-terminal poly-histidine (His)6 affinity tag (SEQ ID NO: 99) was similarly expressed in E. coli and purified via Ni-NTA affinity chromatography. For in vitro glycosylation reactions, 100 g of recombinant SDR proteins [SdrC4/SEQ ID NO: 100, ClfA/SEQ ID NO: 668 and ClfB/SEQ ID NO: 669) were incubated +/−30 μg of Uridine diphosphate N-acetylglucosamine (UDP-GlcNac), +/−4 μg of recombinant SdgB/SEQ ID NO:99 in a final volume of 100 μl 100 mM Tris pH 7.5 or 100 μl 100 mM Tris pH 7.5 plus 10% glycerol at 37 C.° for 1 hour. High binding 96-well ELISA plates (Nunc) were coated with recombinant proteins SDR proteins +/− SdgB-mediated glycosylation at 5 μg/mL in PBS and incubated overnight at 4° C. Plates were washed three times with ELISA wash buffer (0.15M NaCl, 0.02% Tween-20) and blocked with blocking buffer (Superblock Thermo 37515) for one hour at ambient temperature. In separate dilution plates, test articles were serially diluted three-fold in blocking buffer starting at 1 μM or 10 μM. ELISA plates were washed three times with ELISA wash buffer and antibody dilutions were transferred from the dilution plates to the ELISA plates and incubated for one hour at ambient temperature. ELISA plates were washed three times with ELISA wash buffer and a secondary goat anti-human Fc gamma-specific-HRP (Jackson Immunoresearch 109-035-098) was diluted 1:10,000 in blocking buffer and added to the plates. Plates were incubated with secondary antibody for one hour at ambient temperature then washed four times with ELISA wash buffer. POD Chemiluminescence substrate (Roche-cat#11582950001) was then added to the plates and absorbance was read immediately on the Perkin Elmer EnVision Multilabel Reader at 405 nm. The data were analyzed using GraphPad Prism. Values were transformed to a log scale and fit using a non-linear regression sigmoidal dose-response equation resulting in an eleven point binding curve for each antibody against each antigen.
For peptide studies, a twenty eight (28) residue peptide of the amino acid sequence: (N-terminus)-SDSDSDSDSDSDSDSDSDSDHHHHHHHH-(C-terminus) (SEQ ID NO: 670) was synthesized (New England Peptide, Inc., Gardner, Mass.). The peptide contains ten copies of the SD dipeptide repeat element followed by an eight residue poly-Histidine sequence and was additionally modified such as to bear an amino-terminal biotin moiety. This peptide is herein referred to as the “SD peptide” and has a measured molecular weight of 3475 Daltons. For in vitro glycosylation reactions, 100 g of the SD peptide was incubated with 4 μg of recombinant SdgB protein in 100 μl of 100 mM Tris pH 7.5 containing 10% glycerol and 30 μg of uridine diphosphate N-acetylglucosamine (UDP-GlcNac) at 37 C.° for 1 hour. Analysis of the extent of in vitro glycosylation was determined by matrix-assisted laser desorption/ionization (MALDI) analysis. The binding of mAb 5133 and mAb 5133-based fusion proteins to the SD peptide (+/− glycosylation) was determined using a plate-based ELISA format wherein the SD peptide (+/− glycosylation) was captured on high binding 96-well ELISA plates (Nunc) coated with streptavidin at 5 μg/mL in PBS and incubated overnight at 4° C. Detection of bound test articles was performed using an HRP-conjugated F(ab′)2 fragment donkey anti-human IgG (H+L) (Jackson Immunoresearch 709-006-149 lot 112932) and detection of streptavidin plate-bound SD peptide (+/− glycosylation) by use of an HRP-conjugated anti-polyhistidine antibody (R&D Systems MAB050H polyhistidine HRP MAb Clone AD1.1.10). POD Chemiluminescence substrate (Roche-cat#11582950001) was then added to the plates and absorbance was read immediately on the Perkin Elmer EnVision Multilabel Reader at 405 nm. The data were analyzed using GraphPad Prism. Values were transformed to a log scale and fit using a non-linear regression sigmoidal dose-response equation resulting in an eleven point binding curve for each antibody against the SD peptide (+/− glycosylation) antigen.
Results.
MALDI analysis of in vitro SdgB-mediated glycosylation of the SD peptide revealed a series of discrete species that differ by ˜203 Daltons indicative of the addition of individual GlcNac units to Serine residues with up to ten apparent sites of glycosylation per monomer peptide. As shown in
Summary.
These data indicate (i) that the epitope target of mAb 5133 and mAb 5133-FN3 fusion proteins can be defined minimally as a peptide sequence containing as few as ten copies of the SD repeat sequence that has been modified by the S. aureus SdgB glycosyltransferase in the presence of UDP-GlcNac, and (ii) that no other sequences from the SdrC protein, or other S. aureus SDR family members [Clumping Factor A (ClfA), Clumping Factor B (ClfB), SdrD or SdrE], are necessary components of the minimal antigen epitope recognized by mAb 5133.
The roles that individual SDR proteins and the leukotoxins LukAB and LukED play in establishing and/or maintaining S. aureus infections in different tissue sites is predicted to vary. In some settings, glycosylated forms of the SDR proteins may be important in serving as adhesins to host tissues or soluble factors in the systemic blood circulation or other fluids in either the host extracellular environment or sub-cellular bodies in the host intracellular environment via specific host surface receptors (Hazenbos et al., “Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins,” PLoS Pathog. 9(10):e1003653 (2013); Thomer et al., “N-Acetylglucosaminylation of Serine-Aspartate Repeat Proteins Promotes Staphylococcus aureus Bloodstream Infection,” J. Biol. Chem. 289(6):3478-86 (2014), which are hereby incorporated by reference in their entirety). In contrast, the leukotoxins LukAB and LukED likely play distinct roles in mediating invasive disease through their targeted cytolytic activity against specific classes of immune cells (Yoong & Torres, “The Effects of Staphylococcus aureus Leukotoxins on the Host: Cell Lysis and Beyond,” Curr. Opin. Microbiol. 16(1):63-9 (2013), which is hereby incorporated by reference in its entirety). Similarly, the roles of these different virulence factors in facilitating S. aureus survival in, or escape from, the intracellular environment of host cells (e.g., the phagolysosome) is not fully characterized although it has been established that LukAB facilitates the escape of bacteria engulfed within human polymorphonuclear leukocytes (PMNs) (Dumont et al., “Staphylococcus aureus Elaborates Leukocidin AB to Mediate Escape From Within Human Neutrophils,” Infect. Immun. 81(5):1830-41 (2013), which is hereby incorporated by reference in its entirety). Considering the possibility that both SDR family proteins and the leukotoxins LukAB and LukED are important common virulence factors in some infection settings, it was important to demonstrate that mAb 5133-FN3 fusion proteins can engage multiple protein targets simultaneously. Herein, the interaction of mAb 5133-FN3 fusion proteins with purified target antigen proteins is described as determined using the Biacore surface plasmon resonance (SPR) methodology.
Procedure.
The leukotoxin binding FN3 domains of the mAb5133-FN3 fusion proteins used in this and in other Examples described herein were developed as described in PCT Application Publication No. WO2015089073 to Torres et al., which is hereby incorporated by reference in its entirety. Characteristics of the mAB5133-FN3 fusion constructs utilized herein are provided in Table 1. In these studies mAb 5133-based mAbs and mAb-5133-FN3 fusion proteins were captured as ligands on goat anti-human Fc antibody (Jackson ImmunoResearch product #109-005-098) modified CM4 sensor chips (Biacore Life Sciences product BR-1005-34) using the Biacore T200 instrument. Binding studies employed as target analytes (i) a single SdgB glycosylated preparation of the SdrC4 protein (SEQ ID NO: 100) as the V-region target antigen referred to herein as SdrC4GlcNac, (ii) a recombinant polyhistidine-tagged variant of LukE (SEQ ID NO: 13) prepared from S. aureus, and (iii) a recombinant polyhistidine-tagged, toxoid variant of LukAB (LukA variant of SEQ ID NO: 10 and LukB of SEQ ID NO: 11) prepared from S. aureus that bears an E323A mutation (DuMont et al., “Identification of a Crucial Residue Required for Staphylococcus aureus LukAB Cytotoxicity and Receptor Recognition,” Infect Immun. 82(3): 1268-76 (2014), which is hereby incorporated by reference in its entirety). Binding studies employed 0. μM filtered, de-gassed PBS/Tween/EDTA, pH7.4 buffer (Bio-Rad Phosphate buffered saline, pH 7.4, 0.005% Tween 20 (GE Healthcare product BR100054), 3 mM EDTA) as both the ligand immobilization running buffer (IRB) and the Biacore running buffer (BRB). The kinetic binding data was obtained using the “single cycle kinetics” mode on the Biacore T200 instrument at an analyte flow rate of 60 μL/minute and dissociation monitored for 900 seconds. In summary, data analysis was performed by (i) subtracting the curves generated by buffer injection (average) from the reference-subtracted curves for analyte injections to correct for buffer contribution to the signal and instrument noise (Myszka, “Improving Biosensor Analysis,” J. Mol. Recognition. 12:279-284 (1999), which is hereby incorporated by reference in its entirety) and (ii) the resulting processed data generated for kinetic and affinity determinations analyzed using the T200 BIAevaluation software with kinetic data analyzed using a simple 1:1 binding model for LukAB and LukE and a two-state model for SdrC4GlcNac (Jonsson & Malmqvist, “Real Time Biospecific Interaction Analysis: The Integration of Surface Plasmon Resonance Detection, General Biospecific Interface Chemistry and Microfluidics Into One Analytical System,” Advances in Biosensor, 2:291-336 (1992); Morton & Myszka, “Kinetic Analysis of Macromolecular Interactions Using Surface Plasmon Resonance Biosensors,” Methods in Enzymol. 295:268-294 (1998); Svitel et al., “Probing the Functional Heterogeneity of Surface Binding Sites by Analysis of Experimental Binding Traces and the Effect of Mass Transport Limitation,” Biophysical Journal, 92:1742-1758 (2007); Drake et al., “Characterizing High-Affinity Antigen/Antibody Complexes by Kinetic- and Equilibrium-Based Methods,” Anal Biochem 328(1):35-43 (2004), which are hereby incorporated by reference in their entirety).
Results.
Test article ligands captured on CM4 sensor chips via the goat anti-human Fc antibody were mAb 5133 PRASA A6 (Table 1: construct 4/SEQ ID NO: 66 HC plus SEQ ID NO:67 LC), mAb 5133 PRASA A6 HC-L4-AB (Table 1: construct 6/SEQ ID NO: 70 HC plus SEQ ID NO:71 LC), mAb 5133 PRASA A6 HC-L4-E (Table 1: construct 15/SEQ ID NO: 848 HC plus SEQ ID NO:71 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Table 1: construct 11/SEQ ID NO:856 HC plus SEQ ID NO:67 LC). In an initial phase of target binding (Phase A), SdrC4GlcNac (glycosylated SdrC4 protein at 450 nM) was bound to the immobilized ligands until apparent saturation. As shown in the Phase A of binding in
Analysis of these binding data allows for the quantitative assessment of the binding stoichiometry of each target antigen and these data are shown in
Finally, analysis of these binding data also allows for the determination of the binding affinity of each target antigen and these data are shown in
Summary.
These data provide evidence that mAb 5133-FN3 fusion proteins targeting LukAB and LukE can engage both toxin targets simultaneously while engaged via the V-region of the antibody portion with the SD-GlcNac antigen. This feature of mAb 5133-FN3 fusion proteins may be of significance in host tissues and/or organelles in some disease states mediated by S. aureus wherein roles for the glycosylated forms of the SDR family of adhesin proteins plus leukotoxins AB and/or ED are important in establishing and/or maintaining infection. Similarly, the apparent ability of mAb 5133-FN3 fusion proteins to bind both LukAB and LukE simultaneously may also be important in S. aureus disease states where both leukotoxins are expressed. The apparent binding stoichiometry of >1 for each toxin target antigen would presumably enable higher neutralization than afforded if the binding of a single LukAB molecule precluded binding of a second LukAB molecule or the binding of a single LukE molecule precluded binding of a second LukE molecule. Finally, the observation that the context of a FN3 domain in mAb-FN3 fusion proteins can have a significant impact on target affinity suggests that target engagement of mAb-FN3 fusion proteins may be optimized through modulating sequence context through exploring appendage at different positions to the light and/or heavy chains of the mAb and through exploring FN3 domain order in mAb-FN3 fusion proteins bearing tandem FN3 domains. Each of these findings was unexpected and could not be predicted a priori.
The length and nature of the linker sequences used in multi-specific fusion proteins can affect the activity and/or in vivo efficacy of the individual components, presumably by altering target (antigen) interactions and/or factors that influence in vivo pharmacokinetics-pharmacodynamics (PK-PD). Described herein is the synthesis and characterization of a series of 24 derivatives of a single mAb 5133-FN3 fusion protein (Construct 11 in Table 1; SEQ ID NO: 856 HC plus SEQ ID NO:67 LC), where the linker length between the carboxyl-terminus of the mAb heavy chain and the first FN3 domain (targeting LukE) is varied from zero to four copies of the G4S linker (hereinafter referred to as ‘Linker 1’), and similarly, the linker length between the carboxyl-terminus of the first FN3 domain (targeting LukE) and the amino-terminus of the second FN3 domain (targeting LukAB) is varied from zero to four copies of the G4S linker (hereinafter referred to as ‘Linker 2’).
Procedure.
Derivatives of the mAb 5133-FN3 fusion protein Construct 11 (CR5133 PRASA A6 HC-L4-E-L4-AB; SEQ ID NO:856 HC plus SEQ ID NO:67 LC) with variable linker lengths were synthesized by standard molecular methods and purified proteins prepared from transiently transfected Human Embryonic Kidney (HEK) 293 cells. The binding affinity of each leukotoxin target, i.e., LukE (SEQ ID NO: 13), and a LukAB toxoid variant (SEQ ID NOs: 10 and 11) that bears a mutation corresponding to E323A in the wild-type toxin (DuMont et al., “Identification of a Crucial Residue Required for Staphylococcus aureus LukAB Cytotoxicity and Receptor Recognition,” Infect Immun. 82(3): 1268-76 (2014), which is hereby incorporated by reference in its entirety), was determined by ELISA. Briefly, 100 μl of a 5 μg/mL solution of streptavidin (in PBS) was added per well of a 96 well White Maxisorp plate (Nunc-cat#436110) and incubated overnight at 4° C. Wells were washed 3× with TBST (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and blocked with 300 μL/well with StartingBlock T20 (Pierce cat#37543) and incubated 45-60 minutes at room temperature (RT). The plate was washed 3 times with TBST and 0.2 μg of biotinylated versions of the leukotoxin target antigens (in 100 μL) added to each test well and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was washed 3 times with TBST. In separate dilution plates, test articles were serially diluted three-fold in blocking buffer starting at 10 μM. ELISA plates were washed three times with ELISA wash buffer and antibody dilutions were transferred from the dilution plates to the ELISA plates and incubated for one hour at ambient temperature with gentle shaking. ELISA plates were washed three times with ELISA wash buffer and a secondary goat anti-human Fc gamma-specific-HRP (Jackson Immunoresearch 109-035-098) was diluted 1:10,000 in blocking buffer and added to the plates. Plates were incubated with secondary antibody for one hour at ambient temperature then washed four times with ELISA wash buffer. POD Chemiluminescence substrate (Roche-cat#11582950001) was then added to the plates and absorbance was read immediately on the Perkin Elmer EnVision Multilabel Reader at 405 nm. The data were analyzed using GraphPad Prism. Values were transformed to a log scale and fit using a non-linear regression sigmoidal dose-response equation resulting in an eleven point binding curve for each antibody against each antigen.
A mouse model of S. aureus kidney infections was used to assess the relative in vivo efficacy of the mAb-FN3 fusion linker variants. Briefly, female 5-6 week old Swiss Webster ND4 mice (Harlan Laboratories, Indianapolis, Ind.) were administered test articles (8 per group) via intra-peritoneal (IP injection) 24 hours prior to infection. Mice were subsequently infected with a pre-determined fixed concentration (˜10{circumflex over ( )}6 Log 10 CFU per mouse) of S. aureus Newman injected in a 100 μL volume retro-orbitally (under isoflurane anesthesia) using a 28 G, ½ inch needle. This concentration corresponded to the lowest bacterial inoculum that typically yields a robust kidney infection as defined by the resulting bacterial burden based on determinations of colony forming units (CFUs) measured from homogenates of explanted kidneys and the visual observation and enumeration of kidney surface abscess lesions. In all cases, test articles were administered 24 hours prior to infection. Mice were euthanized, and kidneys harvested 96 hour post infection. Kidneys were scored for severity of infection via visual lesion count. The kidneys were explanted, weighed, homogenized, and the resulting homogenates serially diluted and plated on bacterial growth media (Tryptic Soy Agar (TSA)) to determine terminal viable bacterial burdens per individual kidneys via the CFU endpoint. Mice were monitored daily for health observations. The kidney severity abscess score was recorded as: 1-2 visible lesions=severity score 1, 3-9 visible lesions=severity score 2, and >=10 visible lesions=severity score 3.
Results.
The influence of varying linker lengths on leukotoxin binding affinity could not be predicted a priori and the data in
Similarly, the influence of varying linker lengths on in vivo efficacy could not be predicted a priori and the data in
Summary.
The influence of varying linker lengths of mAb 5133-FN3 fusion proteins on in vitro and in vivo activities could not be predicted a priori. As exemplified herein through studies of leukotoxin target binding, toxin neutralization and in vivo efficacy of a broad panel of linker variants of a single mAb 5133-FN3 fusion protein, the nonobvious impact that such variations of Linker 1 and/or Linker 2 have on in vitro, ex vivo and in vivo activity was borne out. These studies highlight the potential for optimizing the activity of mAb 5133-FN3 fusion proteins through varying the G4S linker length employed between the carboxyl-terminus of the mAb heavy chain and the first FN3 domain and, where relevant, the linker length between the carboxyl-terminus of the first FN3 domain and the amino-terminus of the second FN3 domain.
The specificity in leukotoxin binding and neutralization observed for some FN3 variants implies that they interact in a highly specific manner. In an effort to define key molecular interactions between the FN3 protein and the LukE subunit, mutant derivatives of a series of three LukE-specific FN3 variants, specifically Luk26 (SEQ ID NO: 25), Luk27 (SEQ ID NO: 26) and Luk38 (SEQ ID NO: 37) were prepared in which residues that differ from the parental, wild-type TENCON FN3 protein (SEQ ID NO: 1) were each individually changed to Alanine to create a so-called ‘Alanine Scan’ set of variants across the putative LukE binding surface. Each variant was then assessed for (i) retention of binding to purified, recombinant LukE protein as determined in an ELISA format assay, and (ii) for LukED toxin neutralization activity in assays employing isolated primary human neutrophils.
Procedure.
Binding of the FN3 variants to purified, recombinant LukE protein (SEQ ID NO: 13) was determined by ELISA. Briefly, 100 μl of a 5 μg/mL solution of streptavidin (in PBS) was added per well of a 96 well White Maxisorp plate (Nunc-cat#436110) and incubated overnight at 4° C. Wells were then washed 3× with TBST (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and blocked with 300 μL/well with StartingBlock T20 (Pierce cat#37543) and incubated 45-60 minutes at room temperature (RT). The plate was then washed 3 times with TBST and 0.2 μg of a biotinylated preparation of LukE protein (in 100 μL) was added to each test well and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was then washed 3 times with TBST. In separate dilution plates, test articles were serially diluted three-fold in blocking buffer starting at 1 μM. 100 μL of titrated test articles were added to test wells and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was then washed 3 times with TBST. For detection of bound test articles, 100 μL/well of a polyclonal anti-FN3-HRP antibody diluted 1:5000 in Starting block T20 was added and the plate incubated for 45-60 min at RT with gentle shaking. The plate was then washed 3 times with TBST. To detect bound anti-FN3-HRP antibody, 100 μL/well of the POD Chemiluminescence substrate (Roche-cat#11582950001) was added immediately prior to reading plates and the plates read using a Paradigm or Envision reader within 15 minutes of the substrate addition. The data were analyzed using GraphPad Prism. Values were transformed to a log scale and fit using a non-linear regression sigmoidal dose-response equation resulting in an eleven point binding curve for each antibody against each antigen.
For LukED neutralization studies, the FN3 domain test articles (10 μg/mL in 100 μL reactions were incubated with purified, recombinant LukED (SEQ ID NOs: 12 & 13) for 30 mins at 4° C. Freshly isolated human primary polymorphonuclear leukocytes (hPMNs, 200,000 cells in RPMI+10 mM HEPES+0.1% HSA) were added to the mixture of toxin and FN3 domain protein to a final volume of 100 μl. Ethidium bromide was then added to the cells at 1:2000 final dilution and plates were read 30 and 60 mins post toxin addition. Following 1 hour intoxication in a 37° C. CO2 incubator, 25 μl of supernatant was carefully transferred to a new plate after spinning the plate down at 1500 RPM for 10 mins. Cell Titer reagent (Promega) was added to the remaining cells and incubated for 1.5 hours. The 25 μl of supernatant were mixed with equal amounts of CytoTox-ONE™ Assay reagent (Promega) that rapidly measures the release of lactate dehydrogenase (LDH) from cells with a damaged membrane. LDH released into the culture medium was measured with a 10-minute coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. For the neutralization experiments, LukED was used at a concentration of 72.5 nM (2.5 μg/mL per subunit).
Results.
As shown in
Similarly, as shown in
Finally, as shown in
Summary.
These data show that the Luk26, Luk27 and Luk38 FN3 domain proteins bind and neutralize LukED through a series of specific molecular interactions at their respective paratope-epitope surfaces. Further, the identification of residues in these LukE-specific FN3 proteins that when mutated to Alanine have no apparent impact on either LukE binding or LukED neutralization provides opportunities to further enhance the potency of LukE binding and LukED neutralization by changing these residues to alternate amino acids.
The specificity in leukotoxin binding and neutralization observed for some FN3 variants implies that they interact in a highly specific manner. In an effort to define key molecular interactions between the FN3 protein and the LukAB toxin, mutant derivatives of Luk17 (SEQ ID NO: 14) were prepared in which residues that differ from the parental, wild-type TENCON FN3 protein (SEQ ID NO: 1) were each individually changed to Alanine to create a so-called ‘Alanine Scan’ set of variants across the putative LukAB binding surface. Each variant was then assessed for (i) retention of binding to purified, recombinant LukAB protein as determined in an ELISA format assay, and (ii) for LukAB toxin neutralization activity in assays employing isolated primary human neutrophils.
Procedure.
Binding of the FN3 variants to a purified, recombinant polyhistidine-tagged, toxoid variant of LukAB (SEQ ID NOs: 10 and 11) prepared from S. aureus that bears a mutation corresponding to E323A in the wild-type toxin sequence (DuMont et al., “Identification of a Crucial Residue Required for Staphylococcus aureus LukAB Cytotoxicity and Receptor Recognition,” Infect. Immun. 82(3): 1268-76 (2014), which is hereby incorporated by reference in its entirety) was determined by ELISA. Briefly, 100 μl of a 5 μg/mL solution of streptavidin (in PBS) was added per well of a 96 well White Maxisorp plate (Nunc-cat#436110) and incubated overnight at 4° C. Wells were then washed 3× with TBST (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and blocked with 300 μL/well with StartingBlock T20 (Pierce cat#37543) and incubated 45-60 minutes at room temperature (RT). The plate was then washed 3 times with TBST and 0.2 μg of a biotinylated preparation of LukAB (E323A) protein (in 100 μL) was added to each test well and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was then washed 3 times with TBST. In separate dilution plates, test articles were serially diluted three-fold in blocking buffer starting at 10 μM. 100 μL of titrated test articles were added to test wells and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was then washed 3 times with TBST. For detection of bound test articles, 100 μL/well of a polyclonal anti-FN3-HRP antibody diluted 1:5000 in Starting block T20 was added and the plate incubated for 45-60 min at RT with gentle shaking. The plate was then washed 3 times with TBST. To detect bound anti-FN3-HRP antibody, 100 μL/well of the POD Chemiluminescence substrate (Roche-cat#11582950001) was added immediately prior to reading plates and the plates read using a Paradigm or Envision reader within 15 minutes of the substrate addition. The data were analyzed using GraphPad Prism. Values were transformed to a log scale and fit using a non-linear regression sigmoidal dose-response equation resulting in an eleven point binding curve for each antibody against each antigen.
For LukAB toxin neutralization studies, the FN3 domain test articles (40 μg per mL in a 100 μL reaction volume were incubated with purified, recombinant LukAB (SEQ ID NOs: 671 & 11) for 30 mins at 4° C. Freshly isolated human polymorphonuclear neutrophils (hPMNs, 200,000 cells in RPMI+10 mM HEPES+0.1% HSA) were added to the mixture of toxin and FN3 domain protein to a final volume of 100 μl. Ethidium bromide was then added to the cells at 1:2000 final dilution and plates were read 30 and 60 mins post toxin addition. Following 1 hour intoxication in a 37° C. CO2 incubator, 25 μl of supernatant was carefully transferred to a new plate after spinning the plate down 1500 RPM for 10 mins. Cell Titer reagent (Promega) was added to the remaining cells and incubated for 1.5 hours. The 25 μl of supernatant were mixed with equal amounts of CytoTox-ONE™ Assay reagent (Promega) that rapidly measures the release of lactate dehydrogenase (LDH) from cells with a damaged membrane. LDH released into the culture medium was measured with a 10-minute coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. For the neutralization experiments, LukAB was used at a final concentration of 33.75 nM (1.25 μg/mL of each subunit).
Results.
As shown in
Summary.
These data show that the Luk17 FN3 domain protein binds and neutralizes LukAB through a series of specific molecular interactions at the paratope-epitope surface. Further, the identification of residues in Luk17 that when mutated to Alanine have no apparent impact on either LukAB binding or neutralization provides opportunities to further enhance the potency of LukAB binding and neutralization by changing these residues to alternate amino acids.
In understanding the relative contributions of the variable (V) region and/or the anti-toxin FN3 components of mAb 5133-based FN3 fusion proteins with regard to efficacy in animal models of human S. aureus infections, a series of test articles were compared with regard to their relative efficacy in a mouse renal (kidney) infection model of disease. Specifically, a series of test articles were evaluated “head-to-head” that bear the same mAb 5133-derived V-region (targeting glycosylated forms of the SDR family of adhesins) but differ in their FN3 domain composition with regard to targeting of LukE alone or LukE and LukAB in combination. As an isotype IgG1 control, non-antistaphylococcal antibody, CNTO3930 (Construct 21 in Table 1; SEQ ID NO:104 HC plus SEQ ID NO:105 LC) was employed that targets the respiratory syncytial virus F (RSV-F) protein.
Procedure.
Female 5-6 week old Swiss Webster ND4 mice (Harlan Laboratories, Indianapolis, Ind.) were administered test articles (8 per group) via intra-peritoneal (IP) injection in a fixed dose volume of 200 μL/mouse 24 hours prior to infection. Mice were subsequently infected with a pre-determined fixed concentration (˜6.7×10{circumflex over ( )}6 Log10 CFU per mouse) of S. aureus Newman (Baba et al., “Genome Sequence of Staphylococcus aureus Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands,” J Bacteriol. 190(1):300-310 (2008), which is hereby incorporated by reference in its entirety) injected in a 100 μL volume retro-orbitally (under isoflurane anesthesia) using a 28 G, ½ inch needle. This infectious dose corresponds to the lowest bacterial inoculum that typically yields a robust kidney infection as defined by the resulting bacterial burden. Bacterial burden is determined by colony forming units (CFUs) measured from homogenates of explanted kidneys and the visual observation and enumeration of kidney surface lesions. In all cases, test articles were administered 24 hours prior to infection and mice euthanized and kidneys harvested 96 hour post infection. Mice were monitored daily for health observations. Kidneys were scored for severity of infection via visual lesion (abscess) count with the aid of a dissecting microscope, and then explanted, weighed, homogenized, and the resulting homogenates serially diluted and plated on bacterial growth media (Tryptic Soy Agar (TSA)) to determine terminal viable bacterial burdens per individual kidneys via the CFU endpoint. Mice were monitored daily for health observations. Statistical analysis of both the lesion data and bacterial burden (CFU) data was performed by calculating p-values using the Dunn's Method. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO:104 HC plus SEQ ID NO:105 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO:66 HC plus SEQ ID NO:67 LC), mAb 5133 PRASA A6 HC-L4-E (Construct 15 in Table 1; SEQ ID NO:848 HC plus SEQ ID NO:71 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO:856 HC plus SEQ ID NO:67 LC). All test articles were administered IP at a dose of 500 μg per animal 24 hours prior to infection.
Results.
Summary.
These data show that both the V-region and anti-toxin FN3 components of mAb5133-based FN3 fusion proteins are important contributors towards the overall efficacy observed in this mouse model of kidney infection. Treatment with mAb 5133 PRASA A6 resulted in efficacy improved over the non-antistaphylococcal mAb (CNTO3930) with a reduction in kidney lesion incidence of 50% and a reduced bacterial burden in the kidney of minus 0.8 log10 CFU/g tissue. However, addition of a single FN3 fusion protein targeting LukE as exemplified in mAb 5133 PRASA A6 HC-L4-E resulted in a further reduction in kidney lesion incidence (25%) and a further reduction in bacterial burden in the kidney of minus 1.8 log10 CFU/g tissue. However, addition of dual, tandem FN3 fusion proteins targeting LukE and LukAB as exemplified in mAb 5133 PRASA A6 HC-L4-E-L4-AB resulted in the highest overall reduction in bacterial burden in the kidney (minus 2.6 log10 CFU/g tissue compared to CNTO3930) and a reduction in kidney lesion incidence beyond the limit of detection (0/16). In toto, these data suggest that both the V-region and anti-toxin FN3 components of mAb5133-based FN3 fusion proteins contribute towards efficacy in this mouse kidney infection model and further that mAb5133-based FN3 fusion proteins targeting both LukE and LukAB exhibit the best overall efficacy. Finally, these data suggest that the antigenic target of mAb 5133 (glycosylated forms of the SDR family of adhesins) and leukotoxins ED and AB are all contributory virulence factors in this mouse kidney infection model.
In understanding the relative contributions of the V-region and/or the anti-leukotoxin FN3 components of mAb 5133-based FN3 fusion proteins with regard to efficacy in animal models of human S. aureus infections, a series of test articles were compared for their relative efficacy in a mouse renal (kidney) infection model of disease. Specifically, a series of test articles were evaluated “head-to-head” which differ only in their V-region component but are otherwise identical in their anti-toxin FN3 components as fused to the heavy chain of the mAb entity of the mAb-FN3 fusion protein. These included anti-staphylococcal antibodies targeting the iron regulated surface determinant B (IsdB) protein, (Ebert et al., “A Fully Human Monoclonal Antibody to Staphylococcus aureus Iron Regulated Surface Determinant B (IsdB) With Functional Activity In Vitro and In Vivo,” Human Antibodies 19(4): 113-28 (2010); Pancari et al., “Characterization of the Mechanism of Protection Mediated by CS-D7, a Monoclonal Antibody to Staphylococcus aureus Iron Regulated Surface Determinant B (IsdB),” Frontiers in Cellular and Infection Microbiology 2(36): 1-13 (2012), which are hereby incorporated by reference in their entirety), the immunoglobulin-G binding protein Protein-A, lipoteichoic acid (Weisman et al., “Phase 1/2 Double-Blind, Placebo-Controlled, Dose Escalation, Safety, and Pharmacokinetic Study of Pagibaximab (BSYX-A110), an Antistaphylococcal Monoclonal Antibody for the Prevention of Staphylococcal Bloodstream Infections, in Very-Low-Birth-Weight Neonates,” Antimicrob Agents & Chemotherapy 53(7):2879-86 (2009), which is hereby incorporated by reference in its entirety) or an uncharacterized S. aureus cell surface antigen (mAb CR6526-based FN3 fusion protein). As controls, the anti-RSV V-region derived from CNTO3930 that targets the respiratory syncytial virus F (RSV-F) protein was included in the context of both a mAb-FN3 fusion protein and as an isotype IgG1 control antibody, CNTO3930.
Procedure.
Female 5-6 week old Swiss Webster ND4 mice (Harlan Laboratories, Indianapolis, Ind.) were administered test articles (8 per group) via intra-peritoneal (IP injection) 24 hours prior to infection. Mice were subsequently infected with a pre-determined fixed concentration (˜10{circumflex over ( )}6 Log 10 CFU per mouse) of S. aureus Newman (Baba et al., “Genome Sequence of Staphylococcus aureus Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands,” J Bacteriol. 190(1):300-310 (2008), which is hereby incorporated by reference in its entirety) injected in a 100 μL volume retro-orbitally (under isoflurane anesthesia) using a 28 G, ½ inch needle. The administered concentration corresponds to the lowest bacterial inoculum that typically yields a robust kidney infection as defined by the resulting bacterial burden. Bacterial burden is determined by colony forming units (CFUs) measured from homogenates of explanted kidneys and the visual observation and enumeration of kidney surface lesions. In all cases, test articles were administered 24 hours prior to infection and mice euthanized and kidneys harvested 96 hour post infection. Kidneys were scored for severity of infection via visual lesion count. The kidneys were then explanted, weighed, homogenized, and the resulting homogenates serially diluted and plated on bacterial growth media (Tryptic Soy Agar (TSA)) to determine terminal viable bacterial burdens per individual kidneys via the CFU endpoint. Mice were monitored daily for health observations. The kidney severity abscess score was recorded as: 1-2 visible lesions=severity score 1, 3-9 visible lesions=severity score 2, and >=10 visible lesions=severity score 3. Statistical analysis of both the lesion data and bacterial burden (CFU) data was performed by calculating p-values using the Dunn's Method. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO:104 HC plus SEQ ID NO:105 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO:66 HC plus SEQ ID NO:67 LC), ProA3 PRASA A6 HC-L4-E-L4-AB (Construct 16 in Table 1; SEQ ID NO:HC 868 plus SEQ ID NO:841 LC), ProA9 PRASA A6 HC-L4-E-L4-AB (Construct 17 in Table 1; SEQ ID NO:HC 880 plus SEQ ID NO:842 LC), IsdB PRASA A6 HC-L4-E-L4-AB (Construct 18 in Table 1; SEQ ID NO:HC 921 plus SEQ ID NO:844 LC), mAb 6526 PRASA A6 HC-L4E-L4-AB (Construct 23 in Table 1; SEQ ID NO:HC 923 plus SEQ ID NO: 845 LC), RSV PRASA A6 HC-L4-E (Construct 20 in Table 1; SEQ ID NO:927 HC plus SEQ ID NO:843 LC), LTA PRASA A6 HC-L4E-L4-AB (Construct 19 in Table 1; SEQ ID NO:HC 925 plus SEQ ID NO:846 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO:856 HC plus SEQ ID NO:67 LC). All test articles were administered IP at a dose of 500 μg per animal 24-hours prior to infection.
Results.
Summary.
These data support show that the V-region target of mAb 5133 and mAb 5133-based FN3 fusion proteins affords efficacy improved over other anti-staphylococcal mAbs and mAb-FN3 fusion proteins targeting different S. aureus surface-expressed antigens including Protein-A, lipoteichoic acid, Iron Sulfur Determinant B (IsdB) and that targeted by mAb CR-6526.
Patients with serious, diagnosed S. aureus infections that may benefit clinically from administration of an anti-staphylococcal biologic agent will likely be receiving antibiotic therapy. Hence, it is of interest to understand whether the co-administration of an anti-staphylococcal biologic agent either interferes with antibiotic activity, has no impact, or possibly enhances antibiotic effectiveness. To address this, a series of biologic test articles were administered to mice in the context of sub-therapeutic doses of the commonly used, first-line anti-MRSA antibiotic, vancomycin, and efficacy assessed in a mouse renal (kidney) infection model of disease. As an isotype IgG1 control, non-antistaphylococcal antibody, CNTO3930 (SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC) was employed that targets the respiratory syncytial virus F (RSV-F) protein.
Procedure.
Female 5-6 week old Swiss Webster ND4 mice (Harlan Laboratories, Indianapolis, Ind.) were administered test articles (8 per group) via intra-peritoneal (IP injection) 24 hours prior to infection. Mice were subsequently infected with a pre-determined fixed concentration (˜10{circumflex over ( )}6 Log 10 CFU per mouse) of S. aureus Newman (Baba et al., “Genome Sequence of Staphylococcus aureus Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands,” J. Bacteriol. 190(1):300-310 (2008), which is hereby incorporated by reference in its entirety), injected in a 100 μL volume retro-orbitally (under isoflurane anesthesia) using a 28 G, ½ inch needle. The administered concentration corresponds to the lowest bacterial inoculum that typically yields a robust kidney infection as defined by the resulting bacterial burden. Bacterial burden is reflected by determinations of colony forming units (CFUs) measured from homogenates of explanted kidneys and the visual observation and enumeration of kidney surface (abscess) lesions. In all cases, test articles were administered 24 hours prior to infection and mice euthanized and kidneys harvested 96 hour post infection. Kidneys were scored for severity of infection via visual lesion count. The kidneys were then explanted, weighed, homogenized, and the resulting homogenates serially diluted and plated on bacterial growth media (Tryptic Soy Agar (TSA)) to determine terminal viable bacterial burdens per individual kidneys via the CFU endpoint. Mice were monitored daily for health observations. Statistical analysis of both the lesion data and bacterial burden (CFU) data was performed by calculating p-values using the Dunn's Method. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO:104 HC plus SEQ ID NO:105 LC), mAb 5133 PRASA (Construct 2 in Table 1; SEQ ID NO: 62 HC plus SEQ ID NO:63 LC), mAb 5133 PRASA A6 HC-L4-E (Construct 15 in Table 1; SEQ ID NO:848 plus SEQ ID NO:71 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO:856 HC plus SEQ ID NO:67 LC). All test articles were administered IP at a dose of 500 μg per animal 24-hours prior to infection. Where administered, vancomycin was dosed at 3.125 mgs/kg IP one and three hours post-infection.
Results.
Summary.
These data show that biologic agents that target glycosylated forms of the SDR family of adhesin proteins and neutralize leukotoxins LukAB and LukED may be used in combination with standard-of-care antibiotic agents like vancomycin to afford enhanced therapeutic benefit for patients diagnosed with serious S. aureus infections.
In understanding the relative contributions of the V-region and/or the anti-leukotoxin FN3 components of mAb 5133-based FN3 fusion proteins with regard to efficacy in animal models of human S. aureus infections, a series of test articles were compared for their relative efficacy in a mouse model of bacteremia disease. Specifically, a series of test articles were evaluated “head-to-head” that bear the same mAb 5133-derived V-region (targeting glycosylated forms of the SDR family of adhesins) but differ in their FN3 domain composition. As an isotype IgG1 control, non-antistaphylococcal antibody, CNTO3930 (SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC) was employed that targets the respiratory syncytial virus F (RSV-F) protein.
Procedure.
Female 5-6 week old Swiss Webster ND4 mice (Harlan Laboratories, Indianapolis, Ind.) were administered test articles (4-8 per group) via the retro-orbital (RO) route in a fixed dose volume of 100 μL/mouse 4 hours prior to infection. Mice were subsequently infected with a fixed concentration (1×105.4 Log10 CFU per mouse) of S. aureus Newman injected in a 200 μL volume via the intra-peritoneal (IP) route using a 27 G, ½ inch needle. Two hours later, mice were euthanized by CO2 asphyxiation and immediately bled by cardiac puncture into lithium heparin tubes and neat blood plus serial dilutions in phosphate buffered saline (PBS) immediately plated on Tryptic Soy Agar (TSA) plates. Then 2-5 mLs of sterile saline was injected via the intra-peritoneal (IP) route, gently mixed by inverting the mouse several times, and the peritoneal fluid collected by opening the peritoneum and withdrawing the fluid using a 1 cc syringe. After collecting the peritoneal fluid, the spleen was collected, weighed, and homogenized. Spleen homogenates and the peritoneal fluid were serially diluted with sterile saline and plated on bacterial growth media (TSA) to determine the terminal viable bacterial burdens in both peritoneal fluid and spleens. Statistical analysis was performed by unpaired t-test using GraphPad Prism, version 5.0. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO:104 HC plus SEQ ID NO:105 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO:66 HC plus SEQ ID NO:67 LC), mAb 5133 PRASA A6 HC-L4-E-L1-AB (Construct 12 in Table 1; SEQ ID NO: 952 HC plus SEQ ID NO:67 LC), mAb 5133 PRASA A6 HC-L4-E-L1-AB-FLAG (Construct 13 in Table 1; SEQ ID NO:965 HC plus SEQ ID NO:63 LC), mAb 5133 PRASA A6 HC-L4-E-L4-AB-FLAG (Construct 14 in Table 1; SEQ ID NO:970 HC plus SEQ ID NO:63 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO:856 HC plus SEQ ID NO:67 LC). All test articles were administered RO at a dose of 500 μg per animal.
Results.
Summary.
These data show that mAb 5133-FN3 fusion proteins that target glycosylated forms of the SDR family of adhesin proteins and neutralize leukotoxins LukAB and LukED may have therapeutic utility in the treatment of S. aureus mediated bacteremia.
In understanding the relative contributions of the V-region and/or the anti-toxin FN3 components of mAb 5133-based FN3 fusion proteins with regard to efficacy in animal models of human S. aureus infections, a series of test articles were compared for their relative efficacy in a mouse renal (kidney) infection model of disease. Specifically, a series of test articles were evaluated “head-to-head” that bear the same mAb 5133-derived V-region (targeting glycosylated forms of the SDR family of adhesins) but differ in their FN3 domain composition. As an isotype IgG1 control, non-antistaphylococcal antibody, CNTO3930 (SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC) was employed that targets the respiratory syncytial virus F (RSV-F) protein.
Procedure.
Female 5-6 week old Swiss Webster ND4 mice (Harlan Laboratories, Indianapolis, Ind.) were administered test articles (8 per group) via intra-peritoneal (IP) injection in a fixed dose volume of 200 μL/mouse 24 hours prior to infection. Mice were subsequently infected with a pre-determined fixed concentration (˜6.8×10{circumflex over ( )}6 Log10 CFU per mouse) of S. aureus Newman (Baba et al., “Genome Sequence of Staphylococcus aureus Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands,” J. Bacteriol. 190(1):300-310 (2008), which is hereby incorporated by reference in its entirety) injected in a 100 μL volume retro-orbitally (under isoflurane anesthesia) using a 28 G, ½ inch needle. This infectious dose corresponds to the lowest bacterial inoculum that typically yields a robust kidney infection as defined by the resulting bacterial burden. In all cases, test articles were administered 24 hours prior to infection and mice euthanized and kidneys harvested 96 hour post infection. Mice were monitored daily for health observations. Kidneys were scored for severity of infection via visual lesion (abscess) count with the aid of a dissecting microscope, and then explanted, weighed, homogenized, and the resulting homogenates serially diluted and plated on bacterial growth media (Tryptic Soy Agar (TSA)) to determine terminal viable bacterial burdens per individual kidney via the CFU endpoint. The kidney severity abscess score was recorded as: 1-2 visible lesions=severity score 1, 3-9 visible lesions=severity score 2, and >=10 visible lesions=severity score 3. Mice were monitored daily for health observations. Statistical analysis of both the lesion data and bacterial burden (CFU) data was performed by calculating p-values using the Dunn's Method. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO:104 HC plus SEQ ID NO:105 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO:66 HC plus SEQ ID NO:67 LC), mAb 5133 PRASA A6 HC-L4-AB (Construct 6 in Table 1; SEQ ID NO:70 HC plus SEQ ID NO:71 LC), mAb 5133 PRASA A6 HC-L4-E (Construct 15 in Table 1; SEQ ID NO: 848 HC plus SEQ ID NO:71 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO:856 HC plus SEQ ID NO:67 LC). All test articles were administered IP at a dose of 250 μg per animal, alone or in combination, 24 hours prior to infection.
Results.
Summary.
These data show that mAb 5133-FN3 fusion proteins bearing a tandem appendage of FN3 domains targeting both the leukotoxins ED and AB confer efficacy in this mouse model of kidney infection that is enhanced over mAb 5133-FN3 fusion proteins individually targeting leukotoxins ED and AB. Further, the efficacy of mAb 5133 PRASA A6 HC-L4-E-L4-AB dosed at 250 μg/animal is enhanced over the efficacy of mAb 5133 PRASA A6 HC-L4-AB dosed in combination with mAb 5133 PRASA A6 HC-L4-E (each dosed at 250 μg/animal) in support of the notion that the efficacy of mAb 5133 PRASA A6 HC-L4-E-L4-AB is improved over the sum of its composite parts (mAb 5133 PRASA A6 HC-L4-AB and mAb 5133 PRASA A6 HC-L4-E).
In understanding the relative contributions of the V-region and/or the anti-toxin FN3 components of mAb 5133-based FN3 fusion proteins with regard to efficacy in animal models of human S. aureus infections, a series of test articles were compared for their relative efficacy in a mouse model of skin infection. Specifically, a series of test articles were evaluated “head-to-head” that bear the same mAb 5133-derived V-region (targeting glycosylated forms of the SDR family of adhesins) but differ in their FN3 domain composition. As an isotype IgG1 control, non-antistaphylococcal antibody, CNTO3930 (SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC) was employed that targets the respiratory syncytial virus F (RSV-F) protein.
Procedure.
These studies employed SKH-1 Elite mice (Charles River Laboratories, Wilmington, Mass.) which are an outbred hairless, immunocompetent strain. Female 8-10 weeks old mice were administered test articles (8 per group) via intra-peritoneal (IP) injection in a fixed dose volume of 200 μL/mouse 4 hours before or after infection. Mice were subsequently infected under isoflurane anesthesia with a pre-determined fixed concentration (˜6.6×106 Log 10 CFU per mouse) of S. aureus MRSA strain JE2 (Fey et al., “A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes,” mBio 4(1):e00537-12 (2013), which is hereby incorporated by reference in its entirety) as a 0.1% suspension of dextrin microcarrier beads (Cytodex 1®, Sigma-Aldrich Chemical Company, St. Louis, Mo.) in a 200 μL volume by subcutaneous (SC) injection with a 27 G, ½ inch needle. Three days after infection, animals were euthanized by CO2 asphyxiation. Skin lesions were measured (length and width) by electronic digital caliper (Mitutoyo Corporation, Aurora, Ill.) and the corresponding skin was aseptically collected, serially diluted in sterile saline, and plated on TSA plates to determine bacterial burden. A lesion volume score was calculated from the following equation: LV=(π/6)(L×W2), where LV=lesion volume, L=length of the lesion in mm, and W=width of the lesion in mm (Bunce et al., Infect and Immunity, 60:2636-2640 (1992), which is hereby incorporated by reference in its entirety). Statistical analysis performed by unpaired t-test using GraphPad Prism, version 5.0. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO:66 HC plus SEQ ID NO:67 LC), mAb 5133 PRASA A6 HC-L4-E-L1-AB (Construct 12 in Table 1; SEQ ID NO:952 HC plus SEQ ID NO:67 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO:856 HC plus SEQ ID NO:67 LC). All test articles were administered IP at a dose of 500 μg per animal.
Results.
Summary.
These data indicate that mAb 5133-FN3 fusion proteins that target glycosylated forms of the SDR family of adhesin proteins and neutralize leukotoxins LukAB and LukED in exposed skin tissue, indicating therapeutic utility in the treatment of S. aureus mediated skin infections.
Leukotoxins are capable of killing key classes of human immune cells, including neutrophils both from the outside of the cell via a process triggered by engagement with specific receptors on the immune cell surface and are also capable of killing from the inside of the cell through leukotoxin-mediated escape from the phagolysosome (Alonzo and Torres, “The Bicomponent Pore-forming Leucocidins of Staphylococcus aureus,” Microbiol Mol Biol Rev. 78(2):199-230 (2014), which is hereby incorporated by reference in its entirety). The studies described herein exemplify the ability of mAb5133-FN3 fusion proteins that bind and neutralize leukotoxins LukED and LukAB to afford protection of primary human neutrophils from extracellular killing mediated by a S. aureus USA 300 CA-MRSA strain BK18807 (Kennedy et al., “Epidemic Community-associated Methicillin-resistant Staphylococcus aureus: Recent Clonal Expansion and Diversification,” Proc. Natl. Acad. Sci. U.S.A 105:1327-1332 (2008), which is hereby incorporated by reference in its entirety). As an isotype IgG1 control, non-antistaphylococcal antibody, CNTO3930 (SEQ ID No: 104 HC plus SEQ DI NO: 105 LC) was employed that targets the respiratory syncytial virus F (RSV-F) protein
Procedure.
S. aureus BK18807 was grown overnight in RPMI+CAS medium and then sub-cultured 1:100 in RPMI+CAS and grown for a further 5 hours. The culture was thereafter normalized to 1×109 CFU/mL with RPMI+CAS. The bacterial suspension (85 μL) (˜8.5×107 CFU) was then mixed with 180 μL of a 2.5 mg/mL stock concentration of each test article (450 μg each) and 455 μL of RPMI+10 mM HEPES added to yield a final volume of 720 μL. 96-well plates were coated with 20% human serum for 20 mins at 37 C.°+5% CO2 and then washed twice with RPMI plus 10 mM HEPES and 0.1% HSA (RPMI-HH). 80 μl of a freshly purified human primary polymorphonuclear leukocytes (PMNs) were then added to each test well corresponding to ˜250,000 cells per well and the plate incubated at room temperature (RT) for 30 mins. 20 μl of the opsonized suspension of bacteria were then added to each PMN containing well to yield a multiplicity of infection (MOI) of ˜10, and the plate incubated for 120 mins at 37 C ° in 5% CO2. Plates were then centrifuged for 5 mins at 1,500 RPM. For assessment of cytolysis by lactate dehydrogenase (LDH) release, 25 μL of the supernatants from each test well were transferred into a new black, clear bottom 96-well plate. 25 μL of CytoTox-ONE™ Assay reagent (Promega) was added and the plate incubated in the dark for 15 mins. CytoTox-ONE™ Assay reagent measures the release of LDH from cells with a damaged membrane via a coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product detected via plate reading in a spectrometer. These experiments were performed with purified human primary polymorphonuclear leukocytes (PMNs) from six independent donors. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO: 66 HC plus SEQ ID NO: 67 LC), mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO: 856 HC plus SEQ ID NO: 67 LC), mAb 5133 PRASA A6 HC-L4-E-L4-AB-FLAG (Construct 24 in Table 1; SEQ ID NO: 918 HC plus SEQ ID NO: 67 LC), c-Myc-mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 25 in Table 1; SEQ ID NO: 919 HC plus SEQ ID NO: 67 LC) and c-Myc-mAb 5133 PRASA A6 HC-L4-E-L4-AB-FLAG (Construct 26 in Table 1; SEQ ID NO: 920 HC plus SEQ ID NO: 67 LC).
Results.
Summary.
These data indicate that mAb 5133-FN3 fusion proteins that bind and neutralize the cytolytic activity of leukotoxins LukAB and LukED are capable of protecting human primary neutrophils from leukotoxin-mediated killing. This activity of mAb 5133-FN3 fusion protein targeting LukED and/or LukAB may be of therapeutic utility in the context of S. aureus mediated human infections.
Leukotoxins are capable of killing key classes of human immune cells, including neutrophils both from the outside of the cell via a process triggered by engagement with specific receptors on the immune cell surface and are also capable of killing from the inside of the cell through leukotoxin-mediated escape from the phagolysosome (Alonzo and Torres, “The Bicomponent Pore-forming Leucocidins of Staphylococcus aureus,” Microbiol Mol Biol Rev. 78(2):199-230 (2014), which is hereby incorporated by reference in its entirety). The studies described herein exemplify the ability of mAb5133-FN3 fusion proteins that bind and neutralize leukotoxins LukED and LukAB to afford protection to primary human neutrophils from intracellular killing mediated by S. aureus USA 300 MRSA strain LAC (Chambers, H. F., “Community-associated MRSA-resistance and Virulence Converge,” N. Engl. J. Med. 352:1485-1487 (2005), which is hereby incorporated by reference in its entirety). As an isotype IgG1 control, non-antistaphylococcal antibody, CNTO3930 (SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC) was employed that targets the respiratory syncytial virus F (RSV-F) protein.
Procedure.
S. aureus LAC and a variant deleted for the lukAB genes (LAC ALukAB) were grown overnight in RPMI+CAS medium and then sub-cultured 1:100 in RPMI+CAS and grown for a further 5 hours. The cultures were thereafter normalized to 1×109 CFU/mL with phosphate buffered saline (PBS) and further diluted in PBS in accord with the target multiplicity of infection (MOI). 96-well plates were coated with 20% human serum at 37° C. in 5% CO2 and then washed twice with RPMI+10 mM HEPES. Freshly purified human primary polymorphonuclear leukocytes (PMNs) were re-suspended in RPMI plus 10 mM HEPES and 0.1% HSA (RPMI-HH), was added to each test well corresponding to ˜250,000 per well. The plate was incubated at room temperature (RT) for 30 mins. Leukotoxin containing supernatants were prepared as a filtrate of culture supernatant from cultures of S. aureus LAC WT and AlukAB grown for 5 hours at 37° C. in RPMI+CAS following a 1:100 dilution of an overnight culture. Opsonization of S. aureus LAC was initiated by combining bacteria at a target MOI of 1.0 (5×106 CFU/mL in 23.6 μL), 47.25 μL of filtered S. aureus LAC culture supernatant (as an exogenous source of leukotoxins), 945 μL of 2.5 mg/mL stocks of test article plus 3709.15 mL of RPMI plus 10 mM HEPES to yield a final volume of 4725 μL. Per well, 50 μL of the opsonized bacteria mixture was combined with 50 μL of the fresh PMN suspension and synchronization effected by centrifuging the plates for 1,500 RPM for 7 minutes. Plates were incubated for 120 minutes at 37° C. in 5% CO2. Following incubation, plates were then spun at 1,400 RPM for 7 minutes. For assessment of cytolysis by lactate dehydrogenase (LDH) release, 25 μL of the supernatants from each test well were transferred into a new black, clear bottom 96-well plate. 25 μL of CytoTox-ONE™ Assay reagent (Promega) was added and the plate was incubated in the dark for 15 mins. CytoTox-ONE™ Assay reagent measures the release of LDH from cells with a damaged membrane via a coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product detected via plate reading in a spectrometer. These experiments were performed with purified human primary polymorphonuclear leukocytes (PMNs) from six independent donors. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO: 66 HC plus SEQ ID NO: 67 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO: 856 HC plus SEQ ID NO: 67 LC).
Results.
Summary.
These data indicate that mAb 5133-FN3 fusion proteins that bind and neutralize the cytolytic activity of leukotoxins LukAB are capable of protecting human primary neutrophils from leukotoxin-mediated killing following engulfment into the intracellular environment. This activity of mAb 5133-FN3 fusion protein targeting LukAB may be of therapeutic utility in the context of S. aureus mediated human infections.
Key classes of human immune cells including neutrophils kill S. aureus through opsonophagocytosis, a process where bacteria are engulfed into phagolysosome bodies and killed through the action of lytic and digestive enzymes. In counteracting this, S. aureus senses the acidic pH and the content of the phagolysosome environment, up-regulates the expression of key virulence determinants including leukotoxin AB, and kills the phagocyte from within following LukAB-mediated escape from the phagolysosome (Alonzo and Torres, “The Bicomponent Pore-forming Leucocidins of Staphylococcus aureus,” Microbiol Mol Biol Rev. 78(2): 199-230 (2014), which is hereby incorporated by reference in its entirety). The studies described herein exemplify the ability of mAb5133-FN3 fusion proteins that bind and neutralize leukotoxins LukED and LukAB to enhance opsonophagocytotic killing by primary human neutrophils. As a negative control, RSV PRASA A6 HC-L4-wtTENCON fusion protein (Construct 27 in Table 1; SEQ ID NO: 977 HC plus SEQ ID NO: 843 LC) was used. This construct targets the respiratory syncytial virus F (RSV-F) protein via its V-region and bears a parental (wild-type consensus) FN3 domain that exhibits no leukotoxin binding or neutralization activities.
Procedure.
These studies employed a variety of MRSA of the USA300 lineage (LAC, FPR, 18807, 18808 and 18809), a USA500 lineage strain (BK2395), and a MSSA strain (BK4645b). S. aureus strains were grown overnight in RPMI+CAS medium and then sub-cultured 1:100 in RPMI+CAS and grown for a further 5 hours. The cultures were thereafter normalized to 1×109 CFU/mL with RPMI+CAS. Leukotoxin containing supernatants were prepared as filtrates of culture supernatants from cultures of S. aureus μgrown for 5 hours at 37° C. in RPMI+CAS following a 1:100 dilution of an overnight culture. To effect opsonization, pre-determined aliquots of bacteria necessary to achieve the desired MOI were combined with test articles plus or minus the addition of culture supernatant (11 μL) and RPMI-HEPES added to bring the volume to 440 μL (with each test article at a final concentration of 1.25 mg/mL). 96-well plates were coated with 20% human serum at 37° C. in 5% CO2 and then washed twice with RPMI plus 10 mM HEPES and 0.1% HSA (RPMI-HH). A fresh preparation of purified human primary polymorphonuclear leukocytes (PMNs), re-suspended in RPMI plus 10 mM HEPES and 0.1% HSA (RPMI-HH) was added to each test well corresponding to ˜250,000 per well. The plate was incubated at room temperature (RT) for 30 mins. 20 μL of the opsonized S. aureus mixture was added per well and synchronization effected by centrifuging the plates for 1,500 RPM for 7 minutes. Plates were incubated for 120 minutes at 37° C. in 5% CO2. Following incubation, 11 μL of 1% saponin was added to each well and the plate incubated on ice for 20 mins. Thereafter, an aliquot from each well was serially diluted in phosphate buffered saline and aliquots plated on TSA plates to determine the remaining viable S. aureus cells through determination of colony forming units (CFUs). These experiments were performed with purified human primary polymorphonuclear leukocytes (PMNs) from six independent donors. Test articles evaluated were RSV PRASA A6 HC-L4-wtTENCON (Construct 27 in Table 1; SEQ ID NO: 977 HC plus SEQ ID NO: 843 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO: 66 HC plus SEQ ID NO: 67 LC), mAb 5133 PRASA A6 HC-L4-E (Construct 15 in Table 1; SEQ ID NO: 848 HC plus SEQ ID NO: 71 LC), mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 28 in Table 1; SEQ ID NO: 858 HC plus SEQ ID NO: 67 LC) and mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO: 856 HC plus SEQ ID NO: 67 LC).
Results.
Summary.
These data indicate that mAb 5133-FN3 fusion proteins that bind and neutralize the cytolytic activity of both leukotoxins LukED and LukAB afford the best enhancement of opsonophagocytotic killing mediated by human neutrophils. This activity of mAb 5133-FN3 fusion protein targeting both leukotoxins LukED and LukAB may be of therapeutic utility in the context of S. aureus mediated human infections.
S. aureus protein A (SpA) is a key immune evasion factor which is either expressed and assembled on the cell surface or is secreted by the bacteria. Protein A binds the Fc (Fragment, crystallizable) domain of immunoglobulins as well as the fragment antigen-binding (Fab) domains of VH3 class IgGs and IgM (Forsgren A., “Protein A from Staphylococcus aureus VI. Reaction with Subunits from Guinea Pig γ1- and γ2-globulin”, J. Immunol. 100: 927-30 (1968); Silverman G. J. and Goodyear, C. S., “Confounding B-cell Defences: Lessons from a Staphylococcal Superantigen”, Nat. Rev. Immunol. 6: 465-75 (2006), which are hereby incorporated by reference in their entirety). This Fc binding activity of Protein A enables S. aureus to escape opsonophagocytic killing, whereas crosslinking of VH3-type IgM B cell receptors disrupts the development of adaptive immune responses (Falugi F., et al. “The Role of Protein A in the Evasion of Host Adaptive Immune Responses by Staphylococcus aureus”, mBio 4: e00575-613 (2013), which is hereby incorporated by reference in its entirety). In exploring the relative contribution(s) of Fc-mediated binding by Protein A in limiting opsonophagocytic killing mediated by antibodies that target other cell surface localized S. aureus antigens, a series of monoclonal antibodies were identified that bind Protein A specifically via their Fab domains and these were subsequently engineered to remove Protein A binding mediated by Fc interactions.
Procedure.
Fab domains with high affinity for recombinant Protein A (ProSpec-TanyTechnoGene Ltd.) were identified via phage display and converted to human IgG1 mAbs. In the screening process, VH3 class Fabs were de-selected by selective restriction digestion. Three Protein A specific mAbs were selected for further characterization: ProA3 (SEQ ID NO: 1001 HC plus SEQ ID NO: 841 LC), SM1F5 (SEQ ID NO: 1007 HC plus SEQ ID NO: 1016 LC) and SM1F9 (SEQ ID NO: 1012 HC plus SEQ ID NO: 842 LC) and these were subsequently engineered to exhibit GluV8 protease resistance via introduction of the PRASA hinge region mutations (see Table 1) and/or Protein A binding via their Fc region via introduction of the A6 mutations (see Table 1). Characterization of Protein A binding was determined by ELISA. Briefly, plates were coated with streptavidin (5 μg/mL in PBS, 50 μL per well) and incubated overnight at 4° C. Plates were washed three times with ELISA wash buffer (0.15M NaCl, 0.02% Tween-20) and then coated with biotinylated Protein A at 2 μg/mL (50 L per well) in PBS for one hour at room temperature. Plates were then washed three times with ELISA wash buffer and then blocked with ELISA blocking buffer (3% BSA in PBS, 200 μL per well). Test articles were then added in a 3-fold dilution series starting at 10 μg/mL and the plates incubated at room temperature for one hour. Plates were then washed three times with ELISA wash buffer and 50 mL per well of HRP-conjugated goat anti-human kappa light chain (Millipore AP502P) added at a 1:15,000 dilution in 3% BSA in PBS and the plates incubated for one hour at room temperature. Plates were then washed five times with ELISA wash buffer and bound HRP detected using the 3,3′,5,5′-Tetramethylbenzidine (TMB; Fitzgerald) as a chromogenic substrate. Test Articles used were ProA3 PRASA A6 (Construct 29 in Table 1), ProA3 IgG1 (Construct 30 in Table 1), ProA3 PRASA (Construct 31 in Table 1), anti-LTA (Pagibaximab) IgG1 (Construct 32 in Table 1) and anti-LTA (Pagibaximab) PRASA A6 (Construct 33 in Table 1).
Results.
The binding of a monoclonal antibody to Protein A solely via Fc interaction is exemplified in
Summary.
While the reasons underlying the apparent higher binding affinity for plate-immobilized Protein A exhibited by the PRASA A6 variant of the anti-Protein A antibody ProA3 are unclear, these data exemplify the identification of IgG antibodies capable of binding S. aureus protein A via the Fab region that is distinct from normal Fc-mediated interactions.
Leukotoxins are capable of killing key classes of human immune cells, including neutrophils both from the outside of the cell via a process triggered by engagement with specific receptors on the immune cell surface and are also capable of killing from the inside of the cell through leukotoxin-mediated escape from the phagolysosome (Alonzo and Torres, “The bicomponent pore-forming leucocidins of Staphylococcus aureus,” Microbiol. Mol. Biol. Rev. 78(2):199-230 (2014), which is hereby incorporated by reference in its entirety). The studies described herein exemplify the ability of mAb5133-FN3 fusion proteins that bind and neutralize leukotoxins LukED and LukAB to afford protection of primary human neutrophils from extracellular killing mediated by a number of S. aureus strains and the dependence of this phenomenon on the expression of LukAB. These studies employed otherwise-isogenic pairs of strains that either produce LukAB or fail to do so due to an engineered deletion of the lukAB operon; specifically (i) S. aureus strain Newman (Baba et al., “Genome Sequence of Staphylococcus aureus Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands,” J. Bacteriol. 190(1):300-310 (2008), which is hereby incorporated by reference in its entirety) labeled ‘Newman-WT’ and a lukAB deletion derivative thereof (‘Newman−ΔAB’), (ii) S. aureus USA 300 MRSA strain LAC (Chambers, H. F., “Community-associated MRSA-resistance and virulence converge,” N. Engl. J. Med. 352:1485-1487 (2005), which is hereby incorporated by reference in its entirety) labeled ‘LAC-WT’ and a lukAB deletion derivative thereof (‘LAC-ΔAB’), (iii) S. aureus USA 300 MRSA strain BK18807, a 2005 isolate from a bacteremia patient (Kennedy et al., “Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification,” Proc. Natl. Acad. Sci. U.S.A 105:1327-1332 (2008), which is hereby incorporated by reference in its entirety) labeled ‘18807-WT’ and a lukAB deletion derivative thereof (‘18807-ΔAB’), (iv) S. aureus USA 300 MRSA strain BK18808, a 2005 isolate from a patient with endocarditis (Kennedy et al., “Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification,” Proc. Natl. Acad. Sci. U.S.A 105:1327-1332 (2008), which is hereby incorporated by reference in its entirety) labeled ‘18808-WT’ and a lukAB deletion derivative thereof (‘18808-ΔAB’), and (v) S. aureus USA 300 MRSA strain BK18809, a 2005 isolate from a bacteremia patient (Kennedy et al., “Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification,” Proc. Natl. Acad. Sci. U.S.A 105:1327-1332 (2008), which is hereby incorporated by reference in its entirety) labeled ‘18809-WT’ and a lukAB deletion derivative thereof (‘18809-ΔAB’).
Procedure.
S. aureus strains were grown overnight in RPMI+CAS medium and then sub-cultured 1:100 in RPMI+CAS and grown for a further 5 hours. The culture was thereafter normalized to 1×109 CFU/mL with PBS. 96-well plates were coated with 20% human serum for 20 mins at 37° C.+5% CO2 and then washed twice with RPMI plus 10 mM HEPES (RPMI-H). 75 μl of a freshly prepared preparation of purified human primary polymorphonuclear leukocytes (PMNs) was then added to each test well corresponding to ˜200,000 cells per well and the plate incubated at room temperature (RT) for 30 mins. 10 μL of Test Articles (at 1.25 mg/mL) were added to appropriate wells to achieve a final concentration of 125 μg/mL. 20 μL of pre-diluted bacteria were then added per well to yield a multiplicity of infection (MOI) of ˜25. Following a two hour incubation at 37° C. in 5% CO2, the plates were centrifuged for 5 mins at 1,500 RPM at 4° C. and assessment of cytolysis determined by lactate dehydrogenase (LDH) release. For this, 25 μL of the supernatants from each test well were transferred into a new black, clear bottom 96-well plate and 25 μL of CytoTox-ONE™ Assay reagent (Promega) was added and the plate incubated in the dark for 15 mins. CytoTox-ONE™ Assay reagent measures the release of LDH from cells with a damaged membrane via a coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product detected via plate reading in a spectrometer. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC), mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ ID NO: 66 HC plus SEQ ID NO: 67 LC), mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO: 856 HC plus SEQ ID NO: 67 LC) and mAb 5133 PRASA A6 HC-L4-E-L1-AB (Construct 12 in Table 1; SEQ ID NO: 952 HC plus SEQ ID NO: 67 LC).
Results.
In these studies, CNTO3930 and mAb 5133 PRASA A6 afforded the least protection of primary human neutrophils from killing mediated by all LukAB producing strains as indicated by the maximal LDH release observed. That this killing is dependent on LukAB production is supported by the observation that no LDH release is observed in the presence of either CNTO3930 or mAb 5133 PRASA A6 when strains lacking LukAB expression are employed. In contrast, both mAb5133-FN3 fusion proteins studied that bind and neutralize LukAB, i.e., mAb 5133 PRASA A6 HC-L4-E-L4-AB and mAb 5133 PRASA A6 HC-L4-E-L1-AB, afforded protection of primary human neutrophils from killing mediated by all LukAB producing strains as indicated by reduced LDH release with mAb 5133 PRASA A6 HC-L4-E-L4-AB typically affording somewhat enhanced protection over mAb 5133 PRASA A6 HC-L4-E-L1-AB. As expected, killing in the presence of mAb 5133 PRASA A6 HC-L4-E-L4-AB and mAb 5133 PRASA A6 HC-L4-E-L1-AB was observed to be dependent on the production of LukAB.
Summary.
These data indicate that mAb 5133-FN3 fusion proteins that bind and neutralize the cytolytic activity of leukotoxins LukAB are capable of protecting human primary neutrophils from leukotoxin-mediated killing. This activity of mAb 5133-FN3 fusion proteins targeting LukAB may be of therapeutic utility in the context of S. aureus mediated human infections.
Based on the genetic arrangements of genes encoding leukocidins and the co-regulation of loci encoding S and F subunits and the purification and characterization of native proteins, five cognate leukocidins have been identified in S. aureus: LukAB, LukS-PV/LukF-PV, LukED, HlgAB and HlgCB (Alonzo and Torres, “The bicomponent pore-forming leucocidins of Staphylococcus aureus,” Microbiol. Mol. Biol. Rev. 78(2): 199-230 (2014), which is hereby incorporated by reference in its entirety). However, with the exception of LukAB that is produced as a native heterodimer, a number of studies have reported that active leukocidins can be prepared through combination of non-cognate pairs of recombinant S and F subunits (Gravet et al., “Characterization of a novel structural member, LukE-LukD, of the bi-component staphylococcal leukotoxins family,” FEBS Letters 436: 202-208 (1998); Morinaga et al., “Purification, Cloning and Characterization of Variant LukE-LukD with Strong Leukocidal Activity of Staphylococcal Bi-Component Leukotoxin Family,” Microbiol. Immunol. 47(1): 81-90 (2003); Rouha et al., “Five birds, one stone: Neutralization of alpha-hemolysin and four bi-component leukocidins of Staphylococcus aureus with a single monoclonal antibody,” MAbs. 7(1): 243-54 (2015), which are hereby incorporated by reference in their entirety) and include the non-cognate pairs LukE/LukF-PV and LukE/HlgB. The studies described herein exemplify the ability of mAb5133-FN3 fusion proteins that bind and neutralize leukotoxins LukED (and LukAB) to afford protection of primary human neutrophils from extracellular killing mediated by LukE/LukF-PV and LukE/HlgB.
Procedure.
Recombinant variants of LukE, LukF-PV and HlgB subunits were individually prepared from S. aureus. To demonstrate the leukocidal activity of the non-cognate leukocidins LukE/LukF-PV and LukE/HlgB, individual subunits were combined on an equal weight basis with 200,000 freshly purified human primary polymorphonuclear leukocytes (PMNs) in RPMI+10 mM HEPES in a total volume of 100 μL and incubated for one hour at 37° C. in a CO2 incubator. 25 μl of supernatant was then carefully transferred to a new plate after spinning the plate down at 1500 RPM for 10 mins and cell lysis determined using the Cell Titer reagent (Promega) based on quantitation of the ATP present, an indicator of metabolically active cells. To determine the relative activity of mAb5133-FN3 fusion proteins in neutralizing the cytolytic activity of LukE/LukF-PV and LukE/HlgB leukotoxins against human PBMCs, 2.5 μg of LukE and 2.5 μg of HlgB, or 5 μg of LukE plus 5 μg of HlgLukF-PV, were combined with increasing concentrations of each test article and incubated on ice for 20 minutes. Freshly isolated primary human neutrophils (hPMNs, 200,000 cells in 70 μl of RPMI+10 mM HEPES+0.1% HSA) were then added and the mixtures incubated for 1-hour at 37° C. in a 5% CO2 incubator. The reaction plates were then centrifuged for 5 mins at 1,500 RPM at 4° C. and assessment of cytolysis determined by lactate dehydrogenase (LDH) release. For this, 25 μL of the supernatants from each test well were transferred into a new black, clear bottom 96-well plate, 25 μL of CytoTox-ONE™ Assay reagent (Promega) was added, and the plate incubated in the dark for 15 mins. CytoTox-ONE™ Assay reagent measures the release of LDH from cells with a damaged membrane via a coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product detected via plate reading in a spectrometer. Test articles evaluated were CNTO3930 (Construct 21 in Table 1; SEQ ID NO: 104 HC plus SEQ ID NO: 105 LC), mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ ID NO: 856 HC plus SEQ ID NO: 67 LC) and mAb 5133 PRASA A6 HC-L4-E-L1-AB (Construct 12 in Table 1; SEQ ID NO: 952 HC plus SEQ ID NO: 67 LC). As additional controls, purified LukED toxin (at a final concentration of 2.5 μg/mL) was added to reactions containing either CNTO3930 or mAb 5133 PRASA A6 HC-L4-E-L1-AB.
Results.
Summary.
These data indicate that mAb 5133-FN3 fusion proteins that bind LukE and neutralize the cytolytic activity of the LukED leukocidin are also capable of protecting human primary neutrophils from cytolytic killing mediated by two non-cognate leukocidins bearing the LukE subunit—namely, LukE/LukF-PV and LukE/HlgB. This broader leukocidin-neutralizing activity of mAb 5133-FN3 fusion proteins targeting LukE may be of therapeutic utility in the context of S. aureus mediated human infections.
A key feature of the pathogenesis of S. aureus in the bloodstream is the scavenging of iron through the production of toxins that lyse erythrocytes, releasing hemoglobin, the most abundant iron source in mammals. In recent studies the Duffy antigen receptor for chemokines (DARC) was identified as the receptor for the S. aureus hemolytic leukocidins LukED and HlgAB (Spaan et al., “Staphylococcus aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes,” Cell Host &Microbe 18(3): p. 363-370 (2015), which is hereby incorporated by reference in its entirety). Herein it is demonstrated that a mAb5133-FN3 fusion protein that exhibits LukED toxin neutralization activity as determined in assays employing freshly isolated hPMNs (see EXAMPLE 4), also protects human erythrocytes (red blood cells) from LukED-mediated hemolysis.
Procedure.
A constant concentration of 4.8 μg/mL per subunit of recombinant LukED (corresponding to the mouse LD90 concentration) in a volume of 40 μL was incubated with increasing concentrations of either mAb 5133 PRASA A6 (Construct 4 in Table 1; SEQ IDs #66 HC plus 67 LC) or mAb 5133 PRASA A6 HC-L4-E-L4-AB (Construct 11 in Table 1; SEQ IDs #856 HC plus 67 LC) in a total volume of 80 μL for 30 mins on ice. Blood from fresh human leukopaks was washed three times in 0.9% saline and red blood cells (RBCs) at 8×107 cells in 0.9% saline in a volume of 80 μL were added to the toxin-test article mixture in a 96-well plate. Two control reactions were also run in parallel: (i) LukED and RBCs alone with no test articles, and (ii) RBCs alone with 0.2% Triton-X100 with no test articles or LukED. After 30 mins of intoxication/incubation at 37° C. in the presence of 5% CO2, plates were spun down for 10 minutes at 1780 g at 4° C. 100 μL of the cell free lysates were then transferred to a new 96-well plate and the OD405 nm was measured as a measure of hemolysis. The data shown in
Results.
Summary.
These data indicate that mAb 5133-FN3 fusion proteins that bind LukE and neutralize the cytolytic activity of the LukED leucocidin versus hPMNs also neutralize the hemolytic activity of the LukED leucocidin versus human erythrocytes. LukED cytolysis of hPMNs is thought to be mediated following target cell engagement via the CXCR1/CXCR2 receptors whereas the hemolysis of erythrocytes is mediated following target cell engagement via the DARC receptor. Hence, these data support the notion that neutralization of the cytolytic activities of LukED mediated by mAb 5133-FN3 fusion proteins can be independent of the nature of target cell engagement as conferred by specific target cell receptors.
For higher order structural studies, hydrogen-deuterium exchange coupled with mass spectrometry analysis, referred to herein as HDX-MS, has proven a valid method in the identification of binding surfaces between interacting proteins (Hamuro et al., “Rapid Analysis of Protein Structure and Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry,” J. of Biomolecular Techniques 14: 171-182 (2003) and Hom et al., “The Role of Protein Dynamics in Increasing Binding Affinity for an Engineered Protein-Protein Interaction Established by H/D Exchange Mass Spectrometry,” Biochemistry 45: 8488-8498 (2006), which are hereby incorporated by reference in their entirety). Herein are described studies to identify the potential binding epitope(s) for the Luk17 FN3 protein (SEQ ID NO: 14) on the LukAB heterodimer by solution phase HDX-MS methods using a recombinant toxoid variant (LukA E323A) of LukAB protein produced from Staphylococcus aureus (SEQ ID NOs: 10 & 11). Recombinant LukAB heterodimer in the absence or presence of Luk17 FN3 was incubated in a deuterated water solution for predetermined times resulting in deuterium incorporation at exchangeable hydrogen atoms. Regions bound to the Luk17 FN3 protein were inferred to be those sites relatively protected from hydrogen-deuterium exchange and thus contain a lower fraction of deuterium than the reference LukAB protein in studies in which more than 99% of the LukAB protein was mapped to specific peptides.
Procedures.
Pepsin/protease type XIII digestion and LC-MS: for pepsin/protease type XIII digestion, 5 μg of LukAB in 125 μL control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) was denatured by adding 63 μL of 5 M guanidine hydrochloride (final pH 2.5) and incubating the mixture for 3 min. The mixture was then subjected to on-column pepsin/protease type XIII digestion and the resultant peptides were analyzed using an Ultra Performance Liquid Chromatography Mass Spectrometry (UPLC-MS) system comprised of a Waters Acquity UPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo). The peptides were separated on a 50 mm×1 mm C8 column with a 16.5 min gradient from 2-32% solvent B (0.1% formic acid in acetonitrile). Solvent A was 0.02% trifluoroacetic acid and 0.08% formic acid in water. The injection valve and pepsin/protease type XIII column and their related connecting tubes were housed inside a cooling box maintained at 11° C. And the second switching valve, C8 column and their related connecting stainless steel tubes were housed inside a chilled circulating box maintained at 0° C. Peptide identification was done through searching MS/MS data against the LukAB sequence using the Mascot software package (Koenig et al., “Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics,” J. Proteome Res. 7 (9): 3708-17 (2008), which is hereby incorporated by reference in its entirety). The mass tolerance for the precursor and product ions was 20 ppm and 0.05 Da, respectively.
H/D Exchange: 5 μL LukAB (10 μg) or 5 μL of the complex of LukAB and Luk17 FN3 protein (10 & 7.35 rig, respectively) were incubated with 120 μL deuterium oxide labeling buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) for 0 sec, 60 sec, 300 sec, 1800 sec, 7200 sec, and 14400 sec. Deuterium exchange was quenched by adding 63 μL of 5 M guanidine hydrochloride (final pH is 2.5) and the quenched sample was then subjected to on-column pepsin/protease type XIII digestion and LC-MS analysis as described above. The mass spectra were recorded in MS only mode. Raw MS data was processed using HDX WorkBench software for the analysis of H/D exchange MS data (Pascal et al., “Software for the Analysis of H/D Exchange MS Data,” J Am. Soc. Mass Spectrometry 23 (9), 1512-1521 (2012), which is hereby incorporated by reference in its entirety). The deuterium levels were calculated using the average mass difference between the deuterated peptide and its native form (to).
Results.
To establish at the peptide level interaction site(s) on LukAB targeted by the Luk17 FN3 protein, LukAB was incubated in deuterium oxide either alone or in complex with the Luk17 FN3 protein. The deuterium exchange was carried at room temperature for 0 s, 60 sec, 300 sec, 1800 sec, 7200 sec, and 14400 sec. The deuterium levels of the identified peptides were monitored from the mass shift on LC/MS. LukA did not show any significant reduction in deuterium uptakes upon binding to the Luk17 FN3 protein. In contrast, while most of the LukB peptides displayed identical or similar deuterium levels with and without the Luk17 FN3 protein molecule, two discrete peptides of LukB had significant decreased deuterium incorporation upon Luk17 FN3 protein binding. Specifically, the LukB peptide region 260IDWNRHGFWG269 (amino acid residue 260-269 of SEQ ID NO: 11) experienced strong deuterium protection. This strongly protected region is thus assigned as a potential component of the epitope for the Luk17 FN3 protein. In addition, the HDX-MS analysis also showed one marginally protected segment, 201LTRNGNLWAKDNFTPKDKMPVTVS224 (amino acid residues 201-224 of SEQ ID NO: 11). These two regions protected by solution phase interaction of the Luk17 FN3 protein with LukB (SEQ ID NO: 11) are highlighted in black (260IDWNRHGFWG269; peptide #1) and dark grey (201LTRNGNLWAKDNFTPKDKMPVTVS224; peptide #2) in the differential LukB heat map schematic shown in
These two putative LukAB/Luk17 FN3 protein interaction sites were mapped onto the published octameric crystal structure of the bi-component toxin LukAB (aka, LukGH) (Badarau et al., “Structure-Function Analysis of Heterodimer Formation, Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH,” J. Biol. Chem. 290, 142-156 (2015), which is hereby incorporated by reference in its entirety) and are shown in
Summary.
These data indicate that the Luk17 FN3 protein binds LukAB via specific interaction(s) with the LukB subunit. Based on models for the association of LukAB with the target cell receptor CD11b (Badarau et al., “Structure-Function Analysis of Heterodimer Formation, Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH,” J. Biol. Chem. 290, 142-156 (2015), which is hereby incorporated by reference in its entirety) (see EXAMPLE 21) and subsequent association at the target cell membrane in forming an octameric pore, it is assumed that Luk17 FN3 protein mediated neutralization of the cytolytic activity of LukAB occurs through perturbation of LukAB binding to target cells and/or the conformational transitions of LukAB associated with octameric pore formation in target cell membranes.
Herein are described studies to identify the potential binding epitope(s) for the Luk26 FN3 protein (SEQ ID NO: 25) on the LukE subunit structure by solution phase HDX-MS methods using recombinant LukE protein produced from Staphylococcus aureus (SEQ ID NO: 1055). In these studies, recombinant LukE protein in the absence or presence of the Luk26 FN3 protein was incubated in a deuterated water solution for predetermined times resulting in deuterium incorporation at exchangeable hydrogen atoms. Regions bound to the Luk26 FN3 protein were inferred to be those sites relatively protected from exchange and thus contain a lower fraction of deuterium than the reference LukE protein in studies in which more than 99% of the LukE protein was mapped to specific peptides.
Procedures.
Pepsin/protease type XIII digestion and LC-MS: for pepsin/protease type XIII digestion, 5 μg of LukE protein in 125 μL control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) was denatured by adding 63 μL of 5 M guanidine hydrochloride (final pH 2.5) and incubating the mixture for 3 min. The mixture was then subjected to on-column pepsin/protease type XIII digestion and the resultant peptides were analyzed using an Ultra Performance Liquid Chromatography Mass Spectrometry (UPLC-MS) system comprised of a Waters Acquity UPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo). The peptides were separated on a 50 mm×1 mm C8 column with a 16.5 min gradient from 2-32% solvent B (0.1% formic acid in acetonitrile). Solvent A was 0.02% trifluoroacetic acid and 0.08% formic acid in water. The injection valve and pepsin/protease type XIII column and their related connecting tubes were housed inside a cooling box maintained at 11° C. And the second switching valve, C8 column and their related connecting stainless steel tubes were housed inside a chilled circulating box maintained at 0° C. Peptide identification was done through searching MS/MS data against the LukAB sequence using the Mascot software package (Koenig et al., “Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics,”, J. Proteome Res. 7 (9): 3708-17 (2008), which is hereby incorporated by reference in its entirety). The mass tolerance for the precursor and product ions was 20 ppm and 0.05 Da, respectively.
H/D Exchange: 5 μL of LukE (5 μg) protein alone or 5 μL of LukE plus the Luk26 FN3 protein (5 and 3.93 μg, respectively) were incubated with 120 μL deuterium oxide labeling buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) for 0 sec, 60 sec, 300 sec, 1800 sec, 7200 sec, and 14400 sec. Deuterium exchange was quenched by adding 63 μL of 5 M guanidine hydrochloride (final pH is 2.5) and the quenched sample was then subjected to on-column pepsin/protease type XIII digestion and LC-MS analysis as described above. The mass spectra were recorded in MS only mode. Raw MS data was processed using HDX WorkBench software for the analysis of H/D exchange MS data (Pascal et al., “Software for the Analysis of H/D Exchange MS Data,” J. Am. Soc. Mass Spectrometry 23 (9), 1512-1521 (2012), which is hereby incorporated by reference in its entirety). The deuterium levels were calculated using the average mass difference between the deuterated peptide and its native form (to).
Results.
To establish at the peptide level interaction site(s) on LukE targeted by the Luk26 FN3 protein, LukE was incubated in deuterium oxide either alone or in complex with the Luk26 FN3 protein. The deuterium exchange was carried at room temperature for 0 s, 60 sec, 300 sec, 1800 sec, 7200 sec, and 14400 sec. The deuterium levels of the identified peptides were monitored from the mass shift on LC/MS. While most of the LukE peptides displayed identical or similar deuterium levels with and without Luk26 Fn3 protein, two peptide segments showed significantly decreased deuterium incorporation upon binding. Specifically, LukE shows significant reduction in deuterium uptake upon binding to Luk26 Fn3 protein at peptide regions, 69TSFSDVKGSGYELT82 and 255LFPRTGIYAERKHNAFVNRNF275—as per the amino acid numbering used in SEQ ID No: 1055 (these regions correspond to amino acid residues 86-99 of SEQ ID NO: 1054 and 272-292 of SEQ ID NO: 1054, respectively). These two regions protected by the Luk26 FN3 protein are highlighted in black in the differential heat map schematic shown in
These putative LukE/Luk26 FN3 protein interaction sites were mapped on the published high-resolution crystal structure of Luk E (Nocadello et al., “Crystal structures of the components of the Staphylococcus aureus leukotoxin ED” Acta. Cryst. D72: 113-120 (2016) PDB entry 3roh, which is hereby incorporated by reference in its entirety). These interaction sites were found to lie in close proximity to each other in the three dimensional structure (see highlighted regions of
Summary.
These data indicate that the Luk26 FN3 protein binds LukE via specific interaction(s) in the rim domain region and this binding is sufficient to neutralize the cytolytic (EXAMPLE 4) and hemolytic activity (EXAMPLE 19) of LukED. As the rim domains of leukotoxins are thought to be important for interaction with the lipid bilayer of target cell membranes and for interaction with specific target cell membrane-bound receptors, it is possible that the binding of the Luk26 FN3 protein to LukE perturbs either receptor engagement, membrane binding and/or the formation of LukED heterodimers and/or higher oligomers at the surface of the membrane. In the case of LukED, Luk26 FN3 protein mediate blocking of receptor engagement could occur via perturbation of interaction with the Duffy antigen receptor for chemokines (DARC) on red blood and endothelial cells (Spaan et al., “Staphylococcus aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes,” Cell Host Microbe 18(3):363-70 (2015), which is hereby incorporated by reference in its entirety) or via perturbation of interactions with the chemokine receptors CXCR1 and CXCR2 on neutrophils (Spaan et al., “Staphylococcus aureus Leukotoxin ED Targets the Chemokine Receptors CXCR1 and CXCR2 to Kill Leukocytes and Promote Infection,”, Cell Host Microbe 14(4): 453-459 (2013), which is hereby incorporated by reference in its entirety).
Herein are described studies to identify the binding epitope for the Luk17 FN3 protein on leukotoxin LukAB by determination of a high resolution X-ray crystal structure of a ternary complex (1:1:1) comprised of the Luk17 FN3 protein, LukAB and a LukAB-specific Fab (fragment, antigen-binding).
Procedures.
Proteins. The proteins used for the structural studies described herein are (i) a recombinant toxoid variant (LukA E323A) of LukAB protein produced from Staphylococcus aureus (SEQ ID NOs: 10 & 11) referred to herein as ‘LukAB’, (ii) a poly-histidine tagged variants of the Luk17 FN3 protein (SEQ ID NO: 1053) referred to herein as ‘S17’, and (iii) a recombinant Fab (SEQ ID NOs: 1078 HC plus 1079 LC; construct 34 in Table 1) derived from a LukAB-specific mAb (SEQ ID NOs: 1080 HC plus 1081 LC; construct 35 in Table 1) and herein referred to as ‘214F’. LukAB was purified at a final concentration of 5 mg/mL in 10 mM Na acetate, pH 5.5. The Luk17 FN3 protein purified at a final concentration of 8.4 mg/mL in 25 mM Tris, pH 7.4, 50 mM NaCl. 214F was purified at a final concentration of 1.91 mg/mL in 20 mM MES, 200 mM NaCl, pH 6.0.
Complex Formation, Crystallization and X-Ray Data Collection.
A ternary complex consisting of LukAB, the Luk17 FN3 protein and 214F was prepared by incubation of LukAB with excess Luk17 FN3 and 214F. The complex was purified by cation exchange chromatography on a Mono S5/50 GL column (GE Healthcare) pre-equilibrated in 20 mM HEPES pH 7.5, 10% glycerol. After loading the sample on the column, the ternary complex was eluted with a linear gradient of 20 mM HEPES pH 7.5, 10% glycerol, 1 M NaCl. Fractions were analyzed by SDS-PAGE to confirm the presence of the ternary complex. Fractions containing the purified LukAB/Luk17 FN3/214F complex were pooled and concentrated to 13.42 mg/mL in 20 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol. Crystallization screening and optimization was performed using a Mosquito crystallization robot (TTP Labtech) with seeding using seeds of the ternary complex. A crystal was harvested from 0.1 M MES pH 6.5, 26% PEG 3350, 0.2M diammonium tartrate and mixed with cryoprotectant solution composed of 0.1 M MES pH 6.5, 25% PEG 3350, 0.2 M diammonium tartrate, 20% glycerol. The crystal was flash-cooled in liquid nitrogen. The diffraction data were collected at Advanced Photo Source (beamline 17-ID) at the Argonne National Laboratory. The X-ray data were processed with “XDS” (Kabsch, W., Acta. Crystallogr. D. Biol. Crystallogr. 66(2): 125-132 (2010), which is hereby incorporated by reference in its entirety) to a resolution of 3 Å. The details of the X-ray data are summarized in Table 2 below.
Structure Determination.
The structure of the ternary complex was solved by molecular replacement with Phaser (Read, “Pushing the boundaries of molecular replacement with maximum likelihood,” Acta. Crystallogr. D. Biol. Crystallogr. 57(10): 1373-1382 (2001), which is hereby incorporated by reference in its entirety) in several steps. The LukAB dimer from pdb ID 4tw1 (Badarau et al., “Structure-Function Analysis of Heterodimer Formation, Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH,” J. Biol. Chem. 290, 142-156 (2015), which is hereby incorporated by reference in its entirety) was used as a template after the pore forming segments were manually removed. The 214F Fab was located using a homology model of the Fv generated in MOE (CCG, Montreal) and the two constant domains from 3na9 (Luo et al., “Coevolution of antibody stability and Vkappa CDR-L3 canonical structure,” J. Mol. Biol. 402(4): 708-719 (2010), which is hereby incorporated by reference in its entirety). Finally, the S17 was located using a homology model based upon a monomer from pdb id 3tes (Jacobs et al., “Design of novel FN3 domains with high stability by a consensus sequence approach,” Protein Eng. Des. Sel. 25(3): 107-117 (2012), which is hereby incorporated by reference in its entirety). Refinement was carried out with Phenix (Adams et al., “Recent developments in the PHENIX software for automated crystallographic structure determination,” J. Synchrotron Radiat. 11(1): 53-55 (2004), which is hereby incorporated by reference in its entirety) and model fitting with COOT (Emsley et al., “Coot: model-building tools for molecular graphics,” Acta. Crystallogr. D. Biol. Crystallogr. 60(12 Pt 1): 2126-2132 (2004), which is hereby incorporated by reference in its entirety). The refinement statistics are summarized in Table 2. All graphics was generated with Pymol (Schrodinger LLC., www.pymol.org) and all other calculations were carried out in CCP4 (Collaborative Computational Project 1994).
Results.
The overall structure of the LukAB/S17/214F ternary complex is shown in
The LukAB in the ternary complex is a heterodimer with the LukA/LukB interface corresponding to interface 2 of the previously determined LukAB structure and is consistent with a published mutational analysis of the subunit interface (Badarau et al., “Structure-Function Analysis of Heterodimer Formation, Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH,” J. Biol. Chem. 290, 142-156 (2015, which is hereby incorporated by reference in its entirety). The LukA and LukB monomer structures are very similar to those in the octamer structure (rmsds of 0.36 Å for 223 LukA Ca atoms and 0.34 Å for 224 LukB Ca atoms, respectively) except for the loop segments involved in the octamer formation. As is shown in
The binding sites for S17 and Fab 214F on LukAB are distant from the switching segments;
S17 binds the so-called rim domain of the LukB subunit (
S17 binds LukB via amino acid residues exposed on its concave surface corresponding to residues that are variant with respect to the parental FN3 binding protein (SEQ ID NO: 1). Based on S17/LukB crystal contacts, the residues that define the S17 paratope for binding LukB are Ml, W33, T35, E37, K39, F40, Y41, R42, A45, V47, E67, K69, W71, V73, W82 & P83 and are highlighted in
Summary.
The structure of the LukAB/S17/214F ternary complex reveals a complete LukAB heterodimer with the octamer-forming segments in very different conformations from those observed in a previously published octameric LukAB structure (Badarau et al., “Structure-Function Analysis of Heterodimer Formation, Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH,” J. Biol. Chem. 290: 142-156 (2015), which is hereby incorporated by reference in its entirety). The structurally defined epitope for the S17 (Luk17 FN3) protein on LukB is consistent with data from solution phase HDX mapping studies of LukAB (EXAMPLE 19) and the characterization of site-directed variants of LukB (EXAMPLE 22). Finally, the structure may also indicate an interesting neutralization mechanism for S17 wherein the interaction of LukAB with the target cell receptor (CD11b) and/or cell membrane is sterically blocked. In addition, it is possible that S17 binding prevents the normal conformational changes LukAB undergoes in transitioning from a heterodimer to an octameric, membrane embedded pore.
Further to the characterization of site-directed mutants of the Luk17 FN3 protein in terms of LukAB binding and neutralization characteristics (EXAMPLE 5), herein are described equivalent studies to confirm specific Luk17 FN3/LukB interactions at the paratope/epitope interface as identified by determination of a high resolution X-ray crystal structure of a ternary complex (1:1:1) comprised of the Luk17 FN3 protein, LukAB and the ‘214F’ LukAB-specific Fab (see EXAMPLE 21).
Procedures.
The proteins used for the studies described herein are (i) site-directed LukB mutant variants of a recombinant toxoid variant of the LukAB protein (LukA E323A) that bear poly-histidine and streptavidin binding sequences at the amino-terminus of the LukA sequence and correspond to SEQ ID NOs: 1033-1052 (LukB variants) combined with SEQ ID NO: 1021 (LukA), and (ii) a variant of the Luk17 FN3 protein that bears a carboxyl-terminal poly-histidine sequence (SEQ ID NO: 1053). Recombinant LukAB variants were purified by nickel affinity chromatography as soluble heterodimers from an E. coli strain expressing biotin ligase. The Luk17 FN3 protein was purified from E. coli by nickel affinity chromatography.
Determination of Affinity (KD) and Dissociation (Kdis) Constants.
KD and Kdis values for the LukAB variants bearing site-directed mutations in LukB were determined by Bio-Layer Interferometry (BLI) using an OctetRED 384 instrument (forteBIO Inc.) running the basic kinetics protocol (“Biomolecular Binding Kinetics Assays on the Octet Platform”, at outlined in Application Note 14 from Pall forteBIO Corp. accessible via the ForteBIO websiste, which is hereby incorporated by reference in its entirety). Briefly, biotinylated LukAB mutant proteins were loaded onto Dip and Read™ Streptavidin (SA) Biosensor pins at a 10 μg/mL concentration in PBS pH7.2 for 10 minutes. A baseline was then set by washing with phosphate buffered saline (PBS, pH 7.2) for 1 minute. The association rate for the Luk17 FN3 protein was then measured by addition of a 10 ug/mL concentration in PBS (pH7.2) over a period of 10 minutes. The Luk17 FN3 protein dissociation rate was then measured following washing with PBS (pH7.2) for 5 minutes. Curve fitting to determine KD values used the initial 60 seconds of association and dissociation steps to analyze on and off rates.
Results.
Based on Luk17 FN3/LukB crystal contacts, the Luk17 FN3 binding epitope on LukAB is minimally composed of LukB residues: Y73, W74, N191, N192, R193, K195, N206, L207, W208, W262, N263, R264, H265, G266, F267, Y270 of SEQ ID NO: 11 (
Considering the extent of the of the Luk17 FN3/LukB interaction surface as identified in the LukAB/Luk17 FN3 (S17)/214F crystal structure (EXAMPLE 21), it was not anticipated that individual site-directed (substitution) mutants of either the LukB epitope or the Luk17 FN3 protein paratope would negate binding in total. However, it was anticipated that mutations that disrupt key paratope-epitope interactions would have a significant impact on the affinity of the interaction between LukAB and the Luk17 FN3 protein. Of the LukB site-directed mutants evaluated, Tyr73Ala, Trp74Ala, Arg264Glu and Trp268Ala were observed to have the most significant impact on Luk17 FN3 affinity (as reflected in KD values;
Summary.
Mutational analysis of the Luk17 FN3 (S17) protein binding characteristics of LukAB variants described herein substantiate the importance of LukB residues identified via X-ray crystallization studies of the LukAB/S17/214F ternary complex as key components of the LukAB neutralization epitope of the Luk17 FN3 (S17) protein (EXAMPLE 21). Of the LukB site-directed mutants evaluated, Tyr73Ala, Trp74Ala, Arg264Glu and Trp268Ala were observed to have the most significant impact on Luk17 FN3 affinity (as reflected in KD values) with Tyr73Ala and Arg264Glu exhibiting the most significant impact on dissociation characteristics (as reflected in Kdis values).
Further to the characterization of site-directed mutants of the Luk26 FN3 protein in terms of LukE binding and LukED neutralization characteristics (EXAMPLE 4,
Procedures.
The proteins used for the studies described herein are (i) site-directed mutant variants of a recombinant form of LukE that bears poly-histidine and streptavidin binding sequences at the amino-terminus of the LukE sequence (SEQ ID NO: 1056) and correspond to SEQ ID NOs: 1057-1076, and (ii) a variant of the Luk26 FN3 protein that bears a carboxyl-terminal poly-histidine sequence (SEQ ID NO: 1077). Recombinant LukE variants were purified by nickel affinity chromatography as soluble proteins from an E. coli strain expressing biotin ligase. The Luk26 FN3 protein was purified from E. coli by nickel affinity chromatography.
Determination of Affinity (KD) and Dissociation (Kdis) Constants.
KD and Kdis values for the LukED variants bearing site-directed mutations in LukE were determined by Bio-Layer Interferometry (BLI) using an OctetRED 384 instrument (forteBIO Inc.) running the basic kinetics protocol (“Biomolecular Binding Kinetics Assays on the Octet Platform”, at outlined in Application Note 14 from Pall forteBIO Corp. accessible via the ForteBIO website, which is hereby incorporated by reference in its entirety). Briefly, biotinylated LukE mutant proteins were loaded onto Dip and Read™ Streptavidin (SA) Biosensor pins at a 10 μg/mL concentration in PBS pH7.2 for 10 minutes. A baseline was then set by washing with phosphate buffered saline (PBS, pH 7.2) for 1 minute. The association rate for the Luk26 FN3 protein was then measured by addition of a 10 ug/mL concentration in PBS (pH7.2) over a period of 10 minutes. The Luk26 FN3 protein dissociation rate was then measured following washing with PBS (pH7.2) for 5 minutes. Curve fitting to determine KD values used the initial 60 seconds of association and dissociation steps to analyze on and off rates.
Results.
Based on the LukE residues identified as potential components of the Luk26 FN3 binding epitope by HDX mapping (
Localization of the specific residues inferred from these data to represent components of the LukE binding and LukED neutralization epitope for the Luk26 FN3 were mapped on the published crystal structure of LukE (Nocadello et al., “Crystal structures of the components of the Staphylococcus aureus leukotoxin ED,” Acta. Cryst. D72: 113-120 (2016) which is hereby incorporated by reference in its entirety; PDB entry 3ROH) and are highlighted in schematic from in
Summary.
Analysis of the Luk26 FN3 protein binding characteristics of the LukE variants described herein substantiate the importance of key residues in the rim domain of LukE in forming the LukE binding and LukED neutralization epitope for the Luk26 FN3 protein. Specifically, residues Ser89, Val91, Lys92, Gly93Arg, Tyr96, Leu98 and Thr99, Tyr269, Phe287 and Arg290 of SEQ ID NO: 1054 define a minimal epitope for the Luk26 FN3 protein. As the rim domains of leukotoxins are thought to be important for interaction with the lipid bilayer of target cell membranes and for interaction with specific target cell membrane-bound receptors, it is possible that the binding of the Luk26 FN3 protein to LukE perturbs either receptor engagement, membrane binding and/or the formation of LukED heterodimers and/or higher oligomers at the surface of the membrane. In the case of LukED, Luk26 FN3 protein mediate blocking of receptor engagement could occur via perturbation of interaction with the Duffy antigen receptor for chemokines (DARC) on red blood and endothelial cells (Spaan et al., “Staphylococcus aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes,” Cell Host Microbe 18(3):363-70 (2015), which is hereby incorporated by reference in its entirety) or via perturbation of interactions with the chemokine receptors CXCR1 and CXCR2 on neutrophils (Spaan et al., “Staphylococcus aureus Leukotoxin ED Targets the Chemokine Receptors CXCR1 and CXCR2 to Kill Leukocytes and Promote Infection,” Cell Host Microbe 14(4): 453-459 (2013), which is hereby incorporated by reference in its entirety).
The specificity of mAb 5133 for glycosylated forms of recombinant variants of the S. aureus SdrC protein, a member of the Serine-Aspartate Repeat (SDR) family, was demonstrated in WO2015089073 to Torres et al., which is hereby incorporated by reference in its entirety. Specifically, following incubation of purified, recombinant SdrC proteins with whole cell lysates prepared from S. aureus strain JE2 (Fey et al., “A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes”, mBio Volume 4 Issue 1 e00537-12 (2013), which is hereby incorporated by reference in its entirety), specific protein bands were detected via western blot in contrast to those detected following incubation with lysates prepared from S. aureus NE105, an otherwise-isogenic derivative of JE2 that lacks expression of the SdgB glycosyltransferase (see WO2015089073 at EXAMPLE 14, which is hereby incorporate by reference in its entirety). Further, incubation of purified, recombinant SdrC proteins with a recombinant form of the SdgB glycosyltransferase similarly yielded the mAb 5133 specific epitope in a manner that was dependent on the presence of uridine diphosphate N-acetylglucosamine (UDP-GlcNac) (PCT/US2014/069347; EXAMPLE 15).
In EXAMPLE 1 herein, the minimal epitope for mAb 5133 was further defined through studies of mAb5133 binding to a synthetic peptide in the presence or absence of in vitro glycosylation with recombinant S. aureus SdgB glycosyltransferase which indicated that (i) that the epitope target of mAb 5133 and mAb 5133-FN3 fusion proteins can be defined minimally as a peptide sequence containing as few as ten copies of the SD repeat sequence that has been modified by the S. aureus SdgB glycosyltransferase in the presence of UDP-GlcNac, and (ii) that no other sequences from the SdrC protein, or other S. aureus SDR family members [Clumping Factor A (ClfA), Clumping Factor B (ClfB), SdrD or SdrE], are necessary components of the minimal antigen epitope recognized by mAb 5133.
As demonstrated herein, that a minimal epitope for mAb 5133 can be further defined as a single GlcNac modified Serine residue in the context of a peptide comprised often SD repeat units. Further, specifics of the interaction of mAb 5133 with the GlcNac moiety are apparent from a high-resolution structure of the variable region of mAb 5133 determined in the presence of GlcNac.
Procedures.
Peptides, Proteins & Reagents.
For peptide studies, a series of twenty eight (28) residue peptides were synthesized and purified (New England Peptide, Inc., Gardner, Mass.) with the following sequences:
Each peptide bears an eight residue poly-Histidine sequence and is additionally modified with an amino-terminal biotin moiety attached via a long chain (LC) linker. The ‘SD-GlcNac peptide’ was synthesized with a single GlcNac introduced on the carboxyl-terminal serine residue.
For in vitro glycosylation reactions, 100 g of either peptide was incubated with 4 μg of recombinant SdgB protein in 100 μL of 100 mM Tris pH 7.5 containing 10% glycerol and 30 μg of uridine diphosphate N-acetylglucosamine (UDP-GlcNac) at 37 C° for 1 hour. Analysis of the extent of in vitro glycosylation was determined by matrix-assisted laser desorption/ionization (MALDI) analysis. The binding of mAb 5133 to the SD, TD and SD-GlcNAc peptides (+/− in vitro SdgB mediated glycosylation) was determined using a plate-based ELISA format wherein the biotinylated peptides were captured on high binding 96-well ELISA plates (Nunc) coated with streptavidin at 5 μg/mL in PBS and incubated overnight at 4° C. Detection of bound test articles was performed using an HRP-conjugated F(ab′)2 fragment donkey anti-human IgG (H+L) (Jackson Immunoresearch 709-006-149 lot 112932) and detection of streptavidin plate-bound SD peptide (+/− glycosylation) by use of an HRP-conjugated anti-polyhistidine antibody (R&D Systems MAB050H polyhistidine HRP MAb Clone AD1.1.10). POD Chemiluminescence substrate (Roche-cat#11582950001) was then added to the plates and absorbance was read immediately on the Perkin Elmer EnVision Multilabel Reader at 405 nm. The data were analyzed using GraphPad Prism. Values were transformed to a log scale and fit using a non-linear regression sigmoidal dose-response equation resulting in an eleven point binding curve for each antibody against the SD peptides (+/− glycosylation) antigen.
A recombinant form of the SdgB glycosyltransferase (SEQ ID NO: 99) was purified as described in EXAMPLE 1. SM1B229 (SEQ ID NOs: 1082 HC and 1083 LC; construct 36 in Table 1), a Fab variant of mAb 5133, was produced by transient expression in HEK 293 cells, and purified by Ni-affinity chromatography, SEC, and ion exchange in a final buffer of 20 mM MES pH 6.5, 0.15 M NaCl. N-Acetylglucosamine (GlcNAc) was purchased from Sigma-Aldrich (Catalog number A8625). For co-crystallization, the SM1B229 Fab was mixed with concentrated GlcNAc to a final concentration of 100 mM. The final sample was 16 mg/mL SM1B229 Fab in 20 mM MES pH 6.5, 0.15 M NaCl, 100 mM GlcNAc.
Crystallization.
Crystallization screening was performed with seeding using crystal seeds of SM1B229 Fab in 20% PEG 3350, 0.1 M ammonium nitrate, 0.1 M potassium formate.
Data Collection and Processing.
A crystal was harvested from 20.2% PEG3350, 0.2 M sodium fluoride and mixed with cryo-protectant solution composed of 21.6% PEG 3350, 0.2 M sodium fluoride, 20% glycerol. The crystal was flash-cooled in liquid nitrogen. The SM1B229+GlcNAc X-ray diffraction data were collected at the Advanced Photon Source (APS, Argonne National Laboratory on beamline IMCA-CAT and detected with a Pilatus 6M detector. Diffraction intensities were processed with the X-ray Detector Software (XDS) software package.
Structure Determination and Analysis.
Molecular replacement was performed using Phaser (Read, “Pushing the boundaries of molecular replacement with maximum likelihood,” Acta. Crystallogr. D. Biol. Crystallogr. 57(10): 1373-1382 (2001), which is hereby incorporated by reference in its entirety) with a search model composed of the free SM1B229 Fab structure. Refinement was carried out with Phenix (Adams et al., “Recent developments in the PHENIX software for automated crystallographic structure determination”, J. Synchrotron Radiat. 11(1): 53-55 (2004), which is hereby incorporated by reference in its entirety) and model fitting with COOT (Emsley et al., “Coot: model-building tools for molecular graphics,” Acta. Crystallogr. D. Biol. Crystallogr. 60(12 Pt 1): 2126-2132 (2004), which is hereby incorporated by reference in its entirety). The refinement statistics are summarized in Table 3. All graphics was generated with Pymol (Schrodinger LLC., www.pymol.org) and all other calculations were carried out in CCP4 (Collaborative Computational project 1994). The SM1B229 Fab/GlcNac co-crystal structure was solved to 2.50 Å resolution.
Results.
To further define a minimal epitope for mAb 5133, a series of synthetic peptides with or without in vitro glycosylation with the SdgB glycosyltransferase were employed. As is shown in
The X-ray crystal structure of SM1B229 (a Fab variant of mAb 5133) in complex with N-Acetylglucosamine (GlcNAc) was determined at 2.50 Å resolution. Table 3 shows the X-ray data statistics and refinement statistics for a structure determined for SM1B229 (a Fab variant of mAb 5133) in the presence of GlcNac. There are four molecules per asymmetric unit (heavy chain ID's: H, A, C, E; light chain ID's: L, B, D, F). GlcNAc was found only in two copies: HL and AB, where the GlcNAc molecule binds to chain H and chain A. The variable regions of HL and AB superimpose with rmsd=0.27 and GlcNAc is in the same orientation for both copies. Structural analysis was performed with copy AB. As is shown in
GlcNAc modification of serine residues creates an antigen that is predicted to be highly polar and acidic. The electrostatic surface potential of SM1B229 reveals a basic pocket that is formed by CDR-H3, composed of the amino acids indicated in the variable heavy region sequence shown in
Summary.
The studies described herein further define a minimal binding epitope for mAb 5133 as a single GlcNac modified serine residue in the context of an SD repeat sequence. The structure of SM1B229 (a Fab variant of mAb 5133) in complex with GlcNAc reveals unique interactions with features of the GlcNac moiety. Finally, inspection of the antigen binding pocket reveals residues that are predicted to engage the acidic aspartate residues of the antigen and implies that multiple SerGlcNaCAsp units may be accommodated.
Herein is described the characterization of fibronectin type III (FN3) domain variants which bind the alpha hemolysin (Hla) protein of Staphylococcus aureus.
Procedures.
Protein Reagents.
Test articles included in these studies included recombinant, purified polyhistidine-tagged versions of Luk967 (SEQ ID NO: 1097), Luk969 (SEQ ID NO: 1099), Luk982 (SEQ ID NO: 1112), Luk1012 (SEQ ID NO: 1142), Luk970 (SEQ ID NO: 1100) that were selected as Hla binders and the parental control FN3 binding domain protein TENCON parent (SEQ ID NO: 1241); all were purified from E coli using standard methods by nickel affinity chromatography. A poly-histidine tagged variant of a toxoid (H35L) form of Hla (Menzies et al., “Site-directed mutagenesis of the alpha-toxin gene of Staphylococcus aureus: role of histidines in toxin activity in vitro and in a murine model,” Infect. Immun. 62:1843-47 (1994), which is hereby incorporated by reference in its entirety) was purified from E. coli by nickel affinity chromatography and corresponds to SED ID NO: 1086. For ELISA assays, HlaH35L was biotinylated in vitro using the SureLINK™ Chromophoric Biotin Labeling Kit (KPL, Inc.). Human serum albumin (HSA) conjugated with biotin (10-20 moles Biotin per mole of albumin) was purchased from Rockland Immunochemicals Inc. (Product #009-0633). A mouse monoclonal [8B7] specific to alpha-hemolysin was purchased from IBT Bioservices (Product #0210-001). Finally, an HRP-conjugated monoclonal antibody specific for the detection of poly-histidine sequences was purchased from R&D Systems (Product #MAB050H).
ELISA assays. The relative binding of the FN3 domain proteins and control test articles to purified, recombinant HlaH35L protein and HSA was determined by ELISA. Briefly, 100l of a 5 μg/mL solution of streptavidin (in PBS) was added per well of a 96 well White Maxisorp plate (Nunc-cat#436110) and incubated overnight at 4° C. Wells were then washed 3× with TBST (50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and blocked with 300 μL/well with StartingBlock T20 (Pierce cat#37543) and incubated 45-60 minutes at room temperature (RT). The plate was then washed 3 times with TBST and 0.2 μg of biotinylated versions of the target antigens (HSA or HlaH35L in 100 μL) were added to each test well and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was then washed 3 times with TBST. Test articles were diluted to 1 μM in StartingBlock T20 and 100 μL added to test wells and the plate incubated 45-60 minutes at RT with gentle shaking. The plate was then washed 3 times with TBST. For detection of bound FN3 domain test articles, 100 μL/well of a polyclonal anti-FN3-HRP antibody diluted 1:5000 in Starting block T20 was added and the plate incubated for 45-60 min at RT with gentle shaking. The plate was then washed 3 times with TBST. For detection of bound test articles, 100 μL/well of a peroxidase-conjugated, F(ab′)2 fragment of a goat anti-mouse IgG, FC-γ fragment specific antibody (Jackson Immuno Research product 115-036-071) diluted 1:5000 in Starting block T20 was added and the plate incubated for 45-60 min at RT with gentle shaking. The plate was then washed 3 times with TBST. To detect bound F(ab′)2 fragment of the goat anti-mouse antibody, 100 μL/well of the POD Chemiluminescence substrate (Roche-cat#11582950001) was added immediately prior to reading plates and the plates read using an Envision reader within 15 minutes of the substrate addition.
Results.
Summary.
These data indicate that FN3 domain variants can be identified that exhibit specific binding to the S. aureus Hla (alpha hemolysin) protein. Based on the past characterization of related FN3 domain variants that selectively bind other toxin proteins of S. aureus (see WO2015089073 to Torres, which is hereby incorporated by reference in its entirety), it is anticipated that a subset of the FN3 domain variants described herein will neutralize the cytolytic activity of Hla.
The so-called stem domains of the bi-component leukocidins of S. aureus are a key structural element necessary for the formation of the active oligomeric, pore forms of the toxins that pierce the host cell membrane and trigger cell death via osmotic lysis. Examination of the amino acid sequences of the S. aureus LukE and LukD sequences previously revealed glycine-rich motifs that localize to the stem domains of the monomer subunits of each leukocidin and leukocidin variants bearing in-frame deletions in these sequences and those of related leukocidins were found to be both inactive as toxins and exhibited a dominant-negative phenotype (Reyes-Robles et al., “Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis,” EMBO Reports 17(3): 428-40 (2016), which is hereby incorporated by reference in its entirety). Determination of a heterodimeric structure of LukAB in a pre-pore conformation (see EXAMPLE 21) led to the design of more extensive stem loop deletion variants and their characterization is described herein. Critically it is demonstrated herein that such mutants retain the LukAB neutralization epitope recognized by the Luk17 FN3 domain protein (see EXAMPLES 21 and 22) and therein have potential utility as protein antigens in vaccine compositions.
Procedures.
Proteins & Reagents.
Variants of LukA and LukB that bear in-frame deletions+/− insertions in the stem domain were designed and are described in Table 4. In each case, these stem mutants were created in the context of a toxoid variant of the LukAB protein (LukA E323A) (DuMont et al., “Identification of a Crucial Residue Required for Staphylococcus aureus LukAB Cytotoxicity and Receptor Recognition,” Infect Immun. 82(3):1268-76 (2014), which is hereby incorporated by reference in its entirety) and were further engineered to bear poly-histidine sequences at the amino-terminus of the LukA subunit. Recombinant LukAB stem mutant proteins were purified from E. coli cells co-expressing each subunit and purified by nickel affinity chromatography as soluble heterodimers from E. coli. For these studies, variants of the anti-LukAB Luk17 FN3 domain protein and the parental TENCON control FN3 domain protein were engineered that bear carboxyl-terminal extensions including both poly-histidine and streptavidin binding sequences and correspond to SEQ ID NOs: 1153 and 1152, respectively, and are herein referred to as Luk17-His-SA and TENCON-His-SA. Both proteins were purified by nickel affinity chromatography from an E. coli strain expressing biotin ligase.
Luk17 FN3 Domain Binding Studies.
Binding of the biotinylated variants of the Luk17-His-SA and TENCON-His-SA FN3 domain proteins to LukAB and the LukAB stem mutant variants was determined by Bio-Layer Interferometry (BLI) using an OctetRED 384 instrument (forteBIO Inc.). Briefly, the Luk17-His-SA and TENCON-His-SA proteins were loaded onto Dip and Read™ Streptavidin (SA) Biosensor pins at a 10 μg/mL concentration in PBS pH7.2 for 5 minutes. A baseline was then set by washing with phosphate buffered saline (PBS, pH 7.2) for 1 minute. Thereafter, either LukAB or stem domain LukAB variants were loaded at a 10 ug/mL concentration in PBS (pH7.2) and association measured over a period of 5 minutes. The baseline was then reset by washing with phosphate buffered saline (PBS, pH 7.2) for 1 minute.
Results.
Similarly,
Summary.
The studies described herein establish the potential of structure-based, designed variants of the LukAB leukotoxin as vaccine antigens. Specifically, LukAB variants that possess in-frame deletions in the loop sequences important for the formation of the active oligomeric pore forms necessary for localization in or at the host cell membrane retain a LukAB neutralization epitope for the Luk17 FN3 protein. Use of such LukAB variants as protein antigens in vaccine compositions is therein expected to elicit the generation antibodies that similarly bind and neutralize the cytolytic activity of LukAB.
FN3 domain proteins have molecular weights in the 10-11 kDa range and are rapidly cleared from the bloodstream via renal clearance as they are significantly below the glomerular filtration molecular weight cut-off of ˜70 kDa. Hence, the measured half-lives of FN3 domain proteins in serum are short (<1 hour). Herein, is described the characterization of designed fusion proteins wherein the parental FN3 domain proteins are genetically fused with partner protein domains that confer the property of extending the serum exposure of the fusion proteins. In one example, the anti-LukE FN3 domain protein Luk26 is fused with an FN3 domain protein that binds serum albumin. In a second example, the anti-LukE FN3 domain protein Luk26 is fused with an FN3 domain protein that binds transferrin. In a third example, the anti-LukE FN3 domain protein Luk26 is fused with a serum albumin binding domain protein. For each example, equivalent fusion proteins were also made to the parental FN3 domain protein, TENCON. In all cases, four copies of a G4S linker were employed at the fusion juncture and poly-histidine sequences were added at the carboxyl terminus to facilitate purification. Serum albumin and transferrin are two of the most highly abundant proteins in the blood and it was assumed that fusion of FN3 domain proteins to high affinity binding domains for each of these serum proteins would result in extension of the residence time (and therein exposure) in the serum of the fusion proteins when compared to the parental FN3 domain proteins.
Procedures.
Test Articles.
Table 5 provides details of the test articles employed in the studies described herein. In all cases, proteins were purified from E. coli by nickel affinity chromatography using standard methods and endotoxin contaminants removed using a commercial kit (Acrodisc® Units with Mustang® E Membrane, Pall Corp.).
Detection of Test Articles in Serum.
Test articles were formulated in phosphate buffered saline (pH 7.2) and 500 μs of each (in a volume of 100 μL) was administered to female, 5-6 week old Swiss Webster ND4 mice via the retro-orbital (RO) route with a group of three animals employed per test article. 50 μL of blood was collected from each animal two hours post-dosing via the tail and +24 hour blood samples obtained via terminal cardiac puncture. At each time-point, the blood was collected into serum separator tubes, allowed to sit for at least 30 minutes, and then centrifuged for 5 minutes at 4500 rpm. Serum samples were then collected and frozen at −80° C. for subsequent analysis. For analysis by western blot, an aliquot of each serum sample was thawed on ice and the serum pooled for each test article for the +2 and +24 hour samples by combination of sera from each of the three animals per group. Each sample was diluted 1:10 in PBS and 2 μL loaded per lane on an SDS-PAGE gel. Separated proteins were transferred to a nitrocellulose membrane and the test articles detected by use of a fluorescently conjugated (IRDye® 680LT Infrared Dye (LICOR)) preparation of a rabbit monoclonal antibody specific for the framework of the TENCON FN3 binding domain protein and that is cross-reactive with the Luk26 FN3 domain protein.
LukED Neutralization Studies.
The FN3 domain test articles, starting at 7.2 μM, were titrated against a constant dose (LD90) of purified LukED on freshly isolated human PMNs. Freshly isolated human PMNs (hPMNs, 200,000) from healthy donors were intoxicated for 1 hour in a final volume of 100 μl in RPMI+10 mM HEPES+0.1% human serum albumin. Following a 1 hour intoxication in a 37° C. CO2 incubator, 25 μl of supernatant was carefully transferred to a new plate after spinning the plate down at 1500 RPM for 10 mins. Cell Titer reagent (Promega) was added to the remaining cells and incubated for 1.5 hours. The 25 μl of supernatant were mixed with equal amounts of CytoTox-ONE™ Assay reagent (Promega) that rapidly measures the amount of released of lactate dehydrogenase (LDH) from cells with a damaged membrane. LDH released into the culture medium was measured with a 10-minute coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. For these ex vivo neutralization experiments, purified LukED was used at a final concentration of 72.5 nM (2.5 μg/mL per subunit).
LukED Intoxication Studies.
5 week old ND4 mice were treated with 130 μl of a 138. μM normalized stock of each test article via intravenous (IV) retroorbital administration in groups of 3 mice. After an hour, mice were intoxicated with purified toxin at 6 μg per subunit of LukED (lethal dose) IV and then monitored for signs of imminent death: labored breathing, ruffled fur, and paralysis/lack of movement. Mice that survived the first intoxication were then given a second lethal dose of LukED IV approximately 4.5 hours after the first lethal dose of LukED (for a total of 24 μg in 5 hrs). Mice were monitored for the same signs of imminent death as previously described. In a further study, mice were treated with SABD-LukE26 at doses corresponding to a 1×, 10× and 100× molar ratio to the administered LukED toxin and then challenged 5, 24 and 48 hours post-dosing.
Results.
Finally,
Summary.
The studies described herein establish that FN3 domain proteins that bind LukE and neutralize the cytolytic activity of LukED ex vivo are able to protect mice from lethal intoxication mediated by LukED toxin if they are fused to protein domains that bind serum proteins such as to extend their serum residence time and therein exposure. In the context of the design and development of protein biologics that neutralize the cytolytic activity of bacterial toxins, a number of fusion partners can be envisaged for toxin-targeting FN3 domain proteins that should extend serum residence time and exposure including appendage to immunoglobulins via the light and/or heavy chain sequences as exemplified elsewhere in this application.
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Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the disclosure and these are therefore considered to be within the scope of the disclosure as defined in the claims which follow.
This application is a national stage application under 35 U.S.C. § 371 of PCT Application No. PCT/US2016/035931, filed Jun. 4, 2016, which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/171,788, filed Jun. 5, 2015, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/035931 | 6/4/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/197071 | 12/8/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7608276 | Masignani et al. | Oct 2009 | B2 |
| 9783582 | Torres et al. | Oct 2017 | B2 |
| 10316067 | Torres et al. | Jun 2019 | B2 |
| 20100216708 | Jacobs et al. | Aug 2010 | A1 |
| 20110274693 | Torres et al. | Nov 2011 | A1 |
| 20190330284 | Torres et al. | Oct 2019 | A1 |
| Number | Date | Country |
|---|---|---|
| 02059148 | Aug 2002 | WO |
| 2006135912 | Dec 2006 | WO |
| 2009049351 | Apr 2009 | WO |
| 2010027828 | Mar 2010 | WO |
| 2010037041 | Apr 2010 | WO |
| 2010119343 | Oct 2010 | WO |
| 2012177658 | Dec 2012 | WO |
| 2013096948 | Jun 2013 | WO |
| 2013165613 | Nov 2013 | WO |
| 2015089073 | Jun 2015 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20190127444 A1 | May 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62171788 | Jun 2015 | US |
