SBIR-STTR Award

Antibiotic resistance complicates the majority of Staphylococcus aureus (S. aureus) infections, as a full two thirds of hospital-associated S. aureus infections and ~50% of those acquired in the community are now methicillin-resistant (MRSA). MRSA causes >450,000 infections in the US each year, and it is responsible for half of all deaths caused by drug-resistant bacteria. The increasing incidence of multi-drug resistance in S. aureus and other bacteria underscores the need for next-generation antibiotics capable of combating these dangerous pathogens. The majority of small molecule antibiotics inhibit genetically-encoded intracellular enzymes, and as a result they are subject to rapid evolution of bacterial resistance. An alternative therapeutic strategy leverages recombinant enzymes, such as Staphylococcus simulans lysostaphin (ssLST), which degrade cell wall peptidoglycan causing bacterial lysis and death. Due to peptidoglycan's conserved nature and complex biosynthesis, such lytic enzymes have proven less susceptible to evolved resistance. Unfortunately, as a bacterial protein itself, ssLST is known to drive a potent immune response in animals and humans. This immunogenicity and associated toxicity represent critical barriers to ssLST clinical translation. This project seeks to design and develop immunotolerant ssLST drug candidates. We will employ cutting- edge computational deimmunization algorithms and advanced biomolecular engineering and immunogenicity screening technologies to identify and silence immunogenic T cell epitopes within the ssLST sequence. Importantly, our innovative methods simultaneously optimize protein therapeutics for both low immunogenic potential and high level function. We have previously deimmunized the ssLST catalytic domain, and here we aim to develop immunotolerant ssLST cell wall binding domains so as to complete global deimmunization of the protein. In Aim 1, proprietary deimmunization algorithms will be applied to design functionally deimmunized variants of the ssLST cell wall binding domain. In Aim 2, selected cell wall binding domain variants will be fused to our existing deimmunized catalytic domains, and the full length proteins will be characterized by analysis of expression yield, thermostability, bacterial lysis kinetics, and antibacterial activity as measured by minimal inhibitory concentration. Aim 3 will assess the immunogenicity of lead candidates using ex vivo immunoassays with human peripheral blood mononuclear cells. Specifically, immunoreactive T cells in donor samples will be quantified following stimulation with either wild type or deimmunized ssLST. The computational design expertise of Stealth Biologics LLC offers powerful synergy with the experimental capabilities of the Dartmouth research laboratories, and the proposed partnership is built upon a proven 7-year collaboration that has opened new frontiers in the field of biotherapeutic deimmunization. Successfully achieving the project goals will ultimately yield potent anti-staphylococcal drugs that have been optimized so as to provide safe and highly efficacious treatment of MRSA and other staph infection in humans.

Public Health Relevance Statement:


Public Health Relevance:
Staphylococcus simulans lysostaphin (ssLST) is a highly effective anti-staphylococcal biocatalyst that efficiently kills Staphylococcus aureus pathogens, including methicillin-resistant S. aureus (MRSA). Unfortunately, as a bacterial protein itself, ssLST is known to drive a potent immune response that can result in loss of efficacy and toxicity. This proposal seeks to employ advanced protein design and engineering tools in order to develop modified ssLST proteins having wild-type stability and catalytic function but reduced immunogenicity. These designer enzymes could be powerful therapeutics for MRSA and other drug-resistant staph infections.

Project Terms:
Acute; Algorithms; Alleles; Anabolism; Animals; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Antigen-Antibody Complex; Bacteremia; Bacteria; Bacterial Proteins; bacterial resistance; bacteriocin; base; Binding (Molecular Function); Biological Response Modifier Therapy; Biotechnology; Blood; Catalytic Domain; Cause of Death; Cell Wall; Centers for Disease Control and Prevention (U.S.); Cessation of life; chemotherapy; Clinical; clinical application; Collaborations; combat; Communities; Complex; cost; Cytolysis; design; Development; drug candidate; drug development; drug discovery; Drug Formulations; Drug resistance; drug resistant bacteria; Economics; Engineering; engineering design; Ensure; Enzymes; Epitopes; Evolution; Exhibits; experience; Eye Infections; frontier; Funding; Future; Genus staphylococcus; Goals; Grant; Health; Hospitalization; Hospitals; Human; human disease; Immune response; Immunoassay; immunogenic; immunogenicity; in vivo; Incidence; indexing; Infection; innovation; Killings; Kinetics; Laboratory Research; Lead; Legal patent; Length; Length of Stay; Letters; Life; Lung; Lysostaphin; LytA enzyme; Lytic; Measures; Medical; Methicillin Resistance; methicillin resistant Staphylococcus aureus (organism); Methods; Modeling; Mortality Vital Statistics; mouse model; Multi-Drug Resistance; Nature; next generation; Operative Surgical Procedures; pathogen; patient population; Patients; Peptidoglycan; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Pharmacology and Toxicology; Phase; Phenotype; pre-clinical; programs; prophylactic; Proteins; public health relevance; Recombinants; Relative (related person); Research; Resistance; Resistance development; response; Safety; Sampling; screening; Severities; Site; Skin; small molecule; Solutions; Staphylococcus aureus; Stream; Structure; T-Lymphocyte; T-Lymphocyte Epitopes; Technology; Therapeutic; therapeutic protein; thermostability; tool; Toxic effect; Translations; United States National Institutes of Health; Variant

The increasing incidence of multi-drug resistance in Staphylococcus aureus and other bacteria represents a public health crisis. Two thirds of hospital-associated S. aureus infections and ~50% of those acquired in the community are now methicillin-resistant (MRSA). MRSA causes >450,000 infections in the US each year, and it is responsible for half of all US deaths caused by drug-resistant bacteria. This threat to public health is creating demand for new therapeutic agents, but traditional antibiotic development pipelines are not keeping pace with the escalating problem. Moreover, antibacterial chemotherapies have proven widely susceptible to rapid evolution of bacterial resistance. Bacteriolytic enzymes, such as Staphylococcus simulans lysostaphin, are an innovative new class of antibiotics that catalytically dismantle cell wall peptidoglycan causing bacterial lysis and death. Due to peptidoglycan’s conserved nature and complex biosynthesis, such enzymes have proven less susceptible to emergent resistance. Unfortunately, lysostaphin elicits anti-drug antibodies in vivo, and this immunogenicity and associated toxicity are barriers to clinical translation. Supported by a successful Phase I STTR grant, Stealth Biologics has re-engineered lysostaphin for reduced immunogenicity in humans. The pivotal outcomes of the Phase I STTR project were: 1) design and construction of F12, a globally deimmunized variant of lysostaphin; 2) in vitro validation of F12’s anti-MRSA potency; 3) preliminary quantification of F12 synergy with FDA approved antibiotics; 4) demonstration of reduced immunogenicity in human cellular immunoassays; and 5) confirmation of reduced in vivo immunogenicity and consequent enhanced therapeutic efficacy in humanized HLA transgenic mice. Collectively, these data suggest that F12 is a promising therapeutic for drug-resistant S. aureus infections. In the proposed Phase II STTR, we have designed a systematic and focused strategy for constructing an F12 target product profile (TPP). In Aim 1, F12 manufacturing and purification will be optimized and scaled up. In Aim 2, an initial clinical indication will be selected based on F12’s in vivo efficacy in two well-established models: rabbit bacteremia/endocarditis and murine skin infection. Efficacy studies will be supported by rigorous experimental analyses of in vitro potency, in vitro resistance susceptibility, in vivo maximum tolerated dose, and in vivo pharmacokinetics. Aim 3 will yield a preliminary toxicity and immunogenicity profile in rabbits and humanized mice that have received escalating single or repeated doses of F12. The resulting data package will enable construction of a TPP that will guide design and execution of comprehensive IND-enabling studies. We anticipate that F12-based antibacterial therapies will prove to be potent, safe, and amenable to repeated dosing. As such, F12 will benefit from competitive advantages relative to more immunogenic phage endolysins, and ultimately it may represent a breakthrough drug for life-threatening MRSA infections.

Public Health Relevance Statement:
Staphylococcus simulans lysostaphin is a promising therapeutic candidate for treating drug-resistant Staphylococcus aureus infections, including MRSA, but as a bacterial protein itself, lysostaphin elicits anti-drug immune responses that limit its clinical utility. Stealth Biologics has engineered a functionally deimmunized lysostaphin variant, and this proposal aims to conduct pre-clinical efficacy, safety, and manufacturing studies to position this drug candidate for a subsequent IND-enabling program. Our deimmunized lysostaphin could represent a breakthrough therapeutic option for addressing life-threatening MRSA infections.

Project Terms:
Achievement; Acute; Address; Anabolism; Animal Model; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Antibodies; arm; Bacteremia; Bacteria; Bacterial Infections; Bacterial Proteins; bacterial resistance; Bacteriophages; Biological; Biological Assay; Biological Response Modifier Therapy; Cause of Death; Cell Wall; Centers for Disease Control and Prevention (U.S.); Cessation of life; chemotherapy; Clinical; clinical efficacy; clinical translation; Communities; Community Hospitals; community setting; Complex; Contracts; Cytolysis; Dangerousness; Data; design; design and construction; Development; Dose; drug candidate; Drug Costs; Drug Kinetics; Drug resistance; drug resistant bacteria; efficacy study; Endocarditis; endolysin; Engineering; Enzymes; Evolution; exhibitions; Exhibits; Eye Infections; FarGo; FDA approved; Funding; Future; Genetic Engineering; Genus staphylococcus; Goals; Grant; Health; Hospitals; Human; humanized mouse; Immune response; Immunoassay; immunogenic; immunogenicity; improved; In Vitro; in vivo; Incidence; Infection; Infectious Skin Diseases; innovation; Killings; Life; Lung; Lysostaphin; LytA enzyme; Manufactured Materials; Manufacturer Name; Maximum Tolerated Dose; Measures; Methicillin Resistance; methicillin resistant Staphylococcus aureus; Microbial Biofilms; microbiome; Modeling; mouse model; Multi-Drug Resistance; multidisciplinary; Mus; Nature; Oryctolagus cuniculus; Osteomyelitis; Outcome; pathogen; Patients; Peptidoglycan; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Phase; Phenotype; Physicians; Pichia; Plankton; Plasma; Positioning Attribute; pre-clinical; Predisposition; Process; Production; programs; Public Health; Quality Control; Race; Resistance; resistant strain; Risk; Safety; Sampling; scale up; Scientist; Sepsis; Skin; skin lesion; Small Business Technology Transfer Research; Standardization; Staphylococcus aureus; stem; Structure; synergism; Therapeutic; Therapeutic Agents; therapeutic candidate; Toxic effect; Transgenic Mice; Treatment Efficacy; Validation; Vancomycin-resistant S. aureus; Variant