Functional Deimmunization of Botulinum Neurotoxin Protease Domain via Computationally Driven Library Design and Ultrahigh-Throughput Screening.

Botulinum neurotoxin serotype A (BoNT/A) is a widely used cosmetic agent that also has diverse therapeutic applications; however, adverse antidrug immune responses and associated loss of efficacy have been reported in clinical uses. Here, we describe computational design and ultrahigh-throughput screening of a massive BoNT/A light-chain (BoNT/A-LC) library optimized for reduced T cell epitope content and thereby dampened immunogenicity. We developed a functional assay based on bacterial co-expression of BoNT/A-LC library members with a Förster resonance energy transfer (FRET) sensor for BoNT/A-LC enzymatic activity, and we employed high-speed fluorescence-activated cell sorting (FACS) to identify numerous computationally designed variants having wild-type-like enzyme kinetics. Many of these variants exhibited decreased immunogenicity in humanized HLA transgenic mice and manifested in vivo paralytic activity when incorporated into full-length toxin. One variant achieved near-wild-type paralytic potency and a 300% reduction in antidrug antibody response in vivo. Thus, we have achieved a striking level of BoNT/A-LC functional deimmunization by combining computational library design and ultrahigh-throughput screening. This strategy holds promise for deimmunizing other biologics with complex superstructures and mechanisms of action.

CXCR6-based immunotherapy in autoimmune, cancer and inflammatory infliction.

T cells, including both CD4+ and CD8+ T cells, play a pivotal role in mediating various inflammation and immune disorders. A long-standing challenge in T cell-based immunotherapy is to precisely inactivate or delete the pathogenic T cells in inflammation and autoimmune diseases, or to selectively expand the immunocompetent T cell in tumor or other immune compromised situations, without inducing global immunosuppression or zealous immune activation respectively. To achieve this, a specific marker is needed to differentiate the pathogenic or immunocompetent T cell among the rests. Indeed, recent progress of immunology strongly suggests that CXC chemokine receptor 6 (CXCR6, CD186) is such a kind of marker. Here, we review the emerging role of CXCR6 as a novel target for immunotherapy and discuss the underlying mechanism. We propose that CXCR6-based immunotherapy will play a significant role in autoimmune, nonalcoholic steatohepatitis (NASH), tumor, coronavirus disease 2019 (COVID-19) and even ageing-related inflammatory infliction.

Development of a Humanized VHH Based Recombinant Antibody Targeting Claudin 18.2 Positive Cancers.

Claudin 18.2 (CLDN18.2), a tight junction (TJ) family protein controlling molecule exchange between cells, is frequently over-expressed in gastric cancer, pancreatic adenocarcinomas and in a fraction of non-small cell lung cancer cases. The tumor properties indicate that CLDN18.2 could be an attractive drug target for gastric and pancreatic cancers. In this study, we present effective strategies for developing anti-CLDN18.2 therapeutic candidates, based on variable domain of heavy chain of heavy chain antibodies (VHHs). CLDN18.2-specific VHHs were isolated by panning a phage display library from an alpaca immunized with a stable cell line highly expressing CLDN18.2. Humanized VHHs fused with human IgG1 Fc, as potential therapeutic candidates, exhibited desirable binding specificity and affinity to CLDN18.2. In vitro experiments showed that hu7v3-Fc was capable of eliciting both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) on CLDN18.2 positive tumor cells. In the mouse xenograft model, the anti-tumor efficacy of hu7v3-Fc was significantly more potent than Zolbetuximab, the benchmark anti-CLDN18.2 monoclonal antibody. Moreover, in vivo biodistribution using zirconium-89 (89Zr) labeled antibodies demonstrated that hu7v3-Fc (89Zr-hu7v3-Fc) exhibited a better tumor penetration and a faster tumor uptake than Zolbetuximab (89Zr-Zolbetuximab), which might be attributed to its smaller size and higher affinity. Taken together, anti-CDLN18.2 hu7v3-Fc is a promising therapeutic agent for human CLDN18.2 positive cancers. Furthermore, hu7v3 has emerged as a potential module for novel CLDN18.2 related therapeutics.

Deimmunized Lysostaphin Synergizes with Small-Molecule Chemotherapies and Resensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

There is an urgent need for novel agents to treat drug-resistant bacterial infections, such as multidrug-resistant Staphylococcus aureus (MRSA). Desirable properties for new antibiotics include high potency, narrow species selectivity, low propensity to elicit new resistance phenotypes, and synergy with standard-of-care (SOC) chemotherapies. Here, we describe analysis of the antibacterial potential exhibited by F12, an innovative anti-MRSA lysin that has been genetically engineered to evade detrimental antidrug immune responses in human patients. F12 possesses high potency and rapid onset of action, it has narrow selectivity against pathogenic staphylococci, and it manifests synergy with numerous SOC antibiotics. Additionally, resistance to F12 and ß-lactam antibiotics appears mutually exclusive, and, importantly, we provide evidence that F12 resensitizes normally resistant MRSA strains to ß-lactams both in vitro and in vivo These results suggest that combinations of F12 and SOC antibiotics are a promising new approach to treating refractory S. aureus infections.

Globally deimmunized lysostaphin evades human immune surveillance and enables highly efficacious repeat dosing.

There is a critical need for novel therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant pathogens, and lysins are among the vanguard of innovative antibiotics under development. Unfortunately, lysins' own microbial origins can elicit detrimental antidrug antibodies (ADAs) that undermine efficacy and threaten patient safety. To create an enhanced anti-MRSA lysin, a novel variant of lysostaphin was engineered by T cell epitope deletion. This "deimmunized" lysostaphin dampened human T cell activation, mitigated ADA responses in human HLA transgenic mice, and enabled safe and efficacious repeated dosing during a 6-week longitudinal infection study. Furthermore, the deimmunized lysostaphin evaded established anti-wild-type immunity, thereby providing significant anti-MRSA protection for animals that were immune experienced to the wild-type enzyme. Last, the enzyme synergized with daptomycin to clear a stringent model of MRSA endocarditis. By mitigating T cell-driven antidrug immunity, deimmunized lysostaphin may enable safe, repeated dosing to treat refractory MRSA infections.

Evolutionary trade-offs associated with loss of PmrB function in host-adapted Pseudomonas aeruginosa.

Pseudomonas aeruginosa colonises the upper airway of cystic fibrosis (CF) patients, providing a reservoir of host-adapted genotypes that subsequently establish chronic lung infection. We previously experimentally-evolved P. aeruginosa in a murine model of respiratory tract infection and observed early-acquired mutations in pmrB, encoding the sensor kinase of a two-component system that promoted establishment and persistence of infection. Here, using proteomics, we show downregulation of proteins involved in LPS biosynthesis, antimicrobial resistance and phenazine production in pmrB mutants, and upregulation of proteins involved in adherence, lysozyme resistance and inhibition of the chloride ion channel CFTR, relative to wild-type strain LESB65. Accordingly, pmrB mutants are susceptible to antibiotic treatment but show enhanced adherence to airway epithelial cells, resistance to lysozyme treatment, and downregulate host CFTR expression. We propose that P. aeruginosa pmrB mutations in CF patients are subject to an evolutionary trade-off, leading to enhanced colonisation potential, CFTR inhibition, and resistance to host defences, but also to increased susceptibility to antibiotics.

Going native: Direct high throughput screening of secreted full-length IgG antibodies against cell membrane proteins.

Gel microdroplet - fluorescence activated cell sorting (GMD-FACS) is an innovative high throughput screening platform for recombinant protein libraries, and we show here that GMD-FACS can overcome many of the limitations associated with conventional screening methods for antibody libraries. For example, phage and cell surface display benefit from exceptionally high throughput, but generally require high quality, soluble antigen target and necessitate the use of anchored antibody fragments. In contrast, the GMD-FACS assay can screen for soluble, secreted, full-length IgGs at rates of several thousand clones per second, and the technique enables direct screening against membrane protein targets in their native cellular context. In proof-of-concept experiments, rare anti-EGFR antibody clones were efficiently enriched from a 10,000-fold excess of anti-CCR5 clones in just three days. Looking forward, GMD-FACS has the potential to contribute to antibody discovery and engineering for difficult targets, such as ion channels and G protein-coupled receptors.

Genetically enhanced lysozyme evades a pathogen derived inhibitory protein.

The accelerating spread of drug-resistant bacteria is creating demand for novel antibiotics. Bactericidal enzymes, such as human lysozyme (hLYZ), are interesting drug candidates due to their inherent catalytic nature and lack of susceptibility to the resistance mechanisms typically directed toward chemotherapeutics. However, natural antibacterial enzymes have their own limitations. For example, hLYZ is susceptible to pathogen derived inhibitory proteins, such as Escherichia coli Ivy. Here, we describe proof of concept studies demonstrating that hLYZ can be effectively redesigned to evade this potent lysozyme inhibitor. Large combinatorial libraries of hLYZ were analyzed using an innovative screening platform based on microbial coculture in hydrogel microdroplets. Isolated hLYZ variants were orders of magnitude less susceptible to E. coli Ivy yet retained high catalytic proficiency and inherent antibacterial activity. Interestingly, the engineered escape variants showed a disadvantageous increase in susceptibility to the related Ivy ortholog from Pseudomonas aeruginosa as well as an unrelated E. coli inhibitory protein, MliC. Thus, while we have achieved our original objective with respect to escaping E. coli Ivy, engineering hLYZ for broad-spectrum evasion of proteinaceous inhibitors will require consideration of the complex and varied determinants that underlie molecular recognition by these emerging virulence factors.

Inhibitor discovery of full-length New Delhi metallo-ß-lactamase-1 (NDM-1).

New Delhi metallo-ß-lactmase-1 (NDM-1) has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of ß-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of ß-lactam antibiotics have been detected. It could effectively hydrolyze most ß-lactams (k cat/K m ratios between 0.03 to 1.28 µmol⁻¹.s⁻¹), except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM) study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.

Separation of structurally similar nocathiacin analogues by reversed phase chromatography.

The complete separation of structurally similar compounds has been a challenge due mainly to their similarity on physical and chemical properties. In the present study, a simple and effective chromatographic method to separate and purify nocathiacin acid from its structural analogue nocathiacin I was developed. After evaluating mobile phase compositions on the retention characteristics by reversed phase high-performance liquid chromatography (HPLC), the elution order of nocathiacin I and nocathiacin acid was completely reversed, and the resolution value between the two analogues was improved, by varying pH value and ionic strength, to greater than 10 from merged peaks under initial conditions. In addition, a preparative isolation of nocathiacin acid was performed by reversed phase column chromatography under the guidance of the HPLC study. This chromatographic method resulted in an efficient process to obtain pure nocathiacin acid with a recovery rate of 83%. The present approach offers a new methodology for the separation of structurally closely related secondary metabolites.

Microbial generation of nocathiacin acid from nocathiacin I.

Microbial generation of nocathiacin acid from nocathiacin I by manipulating enzymatic activities related to C-terminal dehydroalanine hydrolysis in Amycolatopsis fastidiosa LCB1001 was investigated by comparing the effects of various factors including metal ions, amino acid precursors and starting pH on the transformation. CuCl(2) and cysteine significantly increased bioconversion yields, and starting pH and time were also major factors affecting the product yield. Under optimal conditions, the yield of nocathiacin acid was increased from 10.4mg/L to greater than 45.0mg/L after 6days of fermentation. The use of protease inhibitors indicated that enzymes such as peptidylglycine monooxygenase and alcylase I are likely involved in the bioconversion. This is the first report on the generation of nocathiacin acid through microbial conversion in situ. Compared to chemical methods, the present approach is more effective and could be broadly applied in the production of novel derivatives of microbial secondary metabolites.