Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs

Phage Therapy: Combating Infections with Potential for Evolving from Merely a Treatment for Complications to Targeting Diseases

Antimicrobial resistance is considered to be one of the greatest challenges of medicine and our civilization. Lack of progress in developing new anti-bacterial agents has greatly revived interest in using phage therapy to combat antibiotic-resistant infections. Although a number of clinical trials are underway and more are planned, the realistic perspective of registration of phage preparations and their entering the health market and significantly contributing to the current antimicrobial crisis is rather remote. Therefore, in addition to planning further clinical trials, our present approach of phage treatment carried out as experimental therapy (compassionate use) should be expanded to address the growing and urgent needs of increasing cohorts of patients for whom no alternative treatment is currently available. During the past eleven years of our phage therapy center`s operation we have obtained relevant clinical and laboratory data which not only confirm the safety of the therapy but also provide important information shedding more light on many aspects of the therapy, contributing to its optimization and allowing for construction of the most appropriate clinical trials. New data on phage biology and interactions with the immune system suggest that in the future phage therapy may evolve from dealing with complications to targeting diseases. However, further studies are necessary to confirm this promising trend

“Endothelial Antibody Factory” at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases

The failures of anti-β-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-β-amyloid and anti-TDP43 Fabs turning them into an “antibody fragment factory”. When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect

DataSheet_1_Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses.docx

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs.</p

Macrophages Mediate a Switch between Canonical and Non-Canonical Wnt Pathways in Canine Mammary Tumors

<div><p>Objective</p><p>According to the current hypothesis, tumor-associated macrophages (TAMs) are “corrupted” by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell–TAM interactions are complicated and controversial we aimed to better define this phenomenon.</p><p>Methods and Results</p><p>Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial–mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs.</p><p>Conclusions</p><p>We demonstrated that TAMs mediate a “switch” between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis.</p><p>Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being “corrupted” by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis.</p><p>These data challenge the conventional understanding of TAM–cancer cell interactions.</p></div

Canonical and non-canonical Wnt pathways.

<p>Scheme of the interactions observed between canonical and non-canonical Wnt pathways.</p

The β-catenin staining pattern, number of tumor associated macrophages (TAMs) and expression (IOD, Integrated Optical Density) of Wnt-2, Wnt-5a, Dkk-1 and ROR-2 in canine mammary metastatic and non-metastatic tumors.

<p>The β-catenin staining pattern, number of tumor associated macrophages (TAMs) and expression (IOD, Integrated Optical Density) of Wnt-2, Wnt-5a, Dkk-1 and ROR-2 in canine mammary metastatic and non-metastatic tumors.</p

Primer's sequences used in this study and their annealing optimal temperature and time.

<p>The mRNA sequences of key genes were obtained from NCBI database. Primers were designed using PRIMER3 software (free on-line access) and checked using Oligo Calculator (free on-line access) and Primer-Blast (NCBI database).</p

Confocal and immunohistochemical analysis of canine mammary tumor cell lines grown in co-culture with macrophages.

<p>Confocal images show expression, distribution, and co-localization of actin and p-FSCN1 and formation of stress fibers in control (<b>A</b>), conditioned medium-treated (<b>B</b>), and co-cultured (<b>C</b>) CMT-U27 canine mammary cancer cells. Graph (<b>D</b>) and representative pictures (<b>E</b>) of cytokeratin and vimentin expression in control canine mammary cancer cells (CMT-U27) and after culture in macrophage-conditioned medium (MC-medium) or co-culture with macrophages. Monocultured neoplastic cells showed strong cytokeratin expression and weak vimentin expression. Cytokeratin expression was significantly weaker due to culture in macrophage-conditioned medium or co-culture with macrophages. On the other hand, vimentin expression was found to be significantly stronger due to neoplastic cell culture in macrophage-conditioned medium or co-cultured with macrophages. Cytokeratin (CK) and vimentin (VIM) expression was examined in control macrophages (<b>F</b>; graph and representative pictures). They showed no expression of cytokeratin whereas they showed vimentin expression at the same level as neoplastic cells grown in macrophage-conditioned medium (no significant difference). Images were generated using an Olympus BX60 microscope (200×); Cytokeratin and vimentin are indicated by brown precipitates.</p