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Fabry disease is an X-linked hereditary disorder caused by deficient α-galactosidase A (GLA) activity. Patients with Fabry disease are often treated with enzyme replacement therapy (ERT). However, ERT often induces the formation of neutralizing antidrug antibodies (ADAs), which may impair the therapeutic efficacy. Here, we report the case of a 32-year-old man with Fabry disease and resultant neutralizing ADAs who was treated by switching from agalsidase-α to agalsidase-β. We monitored biomarkers, such as plasma globotriaosylsphingosine (lyso-Gb3), urinary globotriaosylceramide (Gb3), urinary mulberry bodies, renal and cardiac parameters, and disease severity during the treatment period. Although plasma lyso-Gb3 and urinary Gb3 levels quickly decreased within two months after the initiation of ERT with agalsidase-α, they gradually increased thereafter. The urinary mulberry bodies continued to appear. Both the ADA titer and serum mediated GLA inhibition rates started to increase after two months. Moreover, 3.5 years after ERT, the vacuolated podocyte area in the renal biopsy decreased slightly from 23.1 to 18.9%. However, plasma lyso-Gb3 levels increased, and urinary Gb3, mulberry body levels, and ADA titers remained high. Therefore, we switched to agalsidase-β which reduced, but did not normalize, plasma lyso-Gb3 levels and stabilized renal and cardiac parameters. Disease severity was attenuated. However, urinary Gb3 and mulberry body levels did not decrease noticeably in the presence of high ADA titers. The kidneys take up a small amount of the administered recombinant enzyme, and the clearance of Gb3 that has accumulated in the kidney may be limited despite the switching from agalsidase-α to agalsidase-β., (© 2023. The Author(s), under exclusive licence to Japanese Society of Nephrology.)

Background: Fabry disease (FD) is characterized by deficient activity of α-galactosidase A (GLA). Consequently, globotriaosylceramide (Gb3) accumulates in various organs, causing cardiac, renal, and cerebrovascular damage. Gene therapies for FD have been investigated in humans. Strong conditioning is required for hematopoietic stem cell-targeted gene therapy (HSC-GT). However, strong conditioning leads to various side effects and should be avoided. In this study, we tested antibody-based conditioning for HSC-GT in wild-type and FD model mice., Methods: After preconditioning with an antibody-drug conjugate, HSC-GT using a lentiviral vector was performed in wild-type and Fabry model mice. In the wild-type experiment, the EGFP gene was introduced into HSCs and transplanted into preconditioned mice, and donor chimerism and EGFP expression were analyzed. In the FD mouse model, the GLA gene was introduced into HSCs and transplanted into preconditioned Fabry mice. GLA activity and Gb3 accumulation in the organs were analyzed., Results: In the wild-type mouse experiment, when anti-CD45 antibody-drug conjugate was used, the percentage of donor cells at 6 months was 64.5%, and 69.6% of engrafted donor peripheral blood expressed EGFP. When anti-CD117 antibody-drug conjugate and ATG were used, the percentage of donor cells at 6 months was 80.7%, and 73.4% of engrafted donor peripheral blood expressed EGFP. Although large variations in GLA activity among mice were observed in the FD mouse experiment for both preconditioning regimens, Gb3 was significantly reduced in many organs., Conclusions: Antibody-based preconditioning may be an alternative preconditioning strategy for HSC-GT for treating FD., Competing Interests: Declaration of competing interest H Kobayashi has received research grants from Amicus Therapeutics Inc. (Japan), Sumitomo Pharma Co., Ltd. (Japan), and JCR Pharmaceuticals Co., Ltd. (Japan). T Ohashi is an advisor for JCR Pharmaceuticals Co., Ltd. (Japan). The other authors declare no conflicting interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications., Competing Interests: Competing interests statement:The authors declare no competing interest.

In clinical practice, the capacity for producing anti-carbohydrate antibodies is regarded as an entity, but supportive evidence is lacking. We hypothesized that the outcome of the gold standard test for clinical assessment of this capacity, antibody response to polysaccharide vaccination, correlated with the level of the abundant naturally occurring anti-carbohydrate antibody, anti-αGal. To perform an exploratory study, 47 HIV-infected adults were recruited from a vaccine trial. Participants received a 23-valent pneumococcal capsular polysaccharide vaccine. Plasma samples obtained just before and median 4 weeks after the vaccination were quantified for IgG anti-αGal antibody and IgG antibodies to polysaccharides present in the vaccine (serotypes 1, 7F and 19A) by solid-phase type immunoassays. The vaccination responses were assessed as a categorical variable (based on criteria defined by The American Academy of Allergy, Asthma & Immunology and the American College of Allergy, Asthma & Immunology) and as three different continuous variables (antibody increment, geometrical average and standard normal deviates of the achieved antibody concentrations). The baseline anti-αGal level predicted the vaccine response as a categorical variable (ROC-curve analysis, AUC = 0.71; 95%CI: 0.55-0.86) and as the three continuous variables (eg slope of linear regression of geometrical average = 0.37; 95%CI: 0.15-0.59). The correlation between the anti-αGal level and antibody responses to polysaccharide vaccination fits with a shared underlying capacity. Thus, the present study supports the notion of a measurable capacity for the production of anti-carbohydrate antibodies in each individual. Firm conclusions on the generalizability and clinical utility require further studies., (© 2020 The Scandinavian Foundation for Immunology.)

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-β and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r 2 = 0.9063 and r 2 = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-β and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-β and Moss-AGAL.

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from h FUT2 ×h GLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA - ) and positive (TSA + ) control groups. For both h FUT2 ×h GLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA + cells were more than twofold higher in comparison to TSA - cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA - h FUT2 ×h GLA cells as compared to the TSA - nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated h FUT2 ×h GLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of h FUT2 and h GLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.

Fabry disease (FD) is an X-linked lysosomal disorder caused by mutations in GLA gene resulting in lack of or faulty α-galactosidase A (α-GalA) enzyme. Enzyme replacement therapy (ERT) with recombinant human α-GalA enzyme (agalsidase) is the standard treatment option for FD. Infusion-related reactions (IRRs), with symptoms ranging from rigors, to fever, pain, vomiting, angioedema and diarrhea, are often seen due to immune response against the exogenous enzyme. To elucidate the mechanisms causing the IRRs in FD, eight patients who developed IRRs were investigated. All, except one, tested negative for agalsidase-specific IgE and had normal tryptase levels. Circulating dendritic cells were drastically reduced during IRRs, suggesting possible sequestration to the sites of inflammation. An increase in NK cells and a decrease in T cells were also observed. Cytokines IL-4, IL-8 and TNF-α showed a significant increase, indicating nonspecific degranulation of mast cells. All IRRs were managed successfully using a combination of standard premedications and mast cell stabilizers without any interruption of therapy. Taken together, the results indicate crosstalk between immune cells resulting in IgE-independent mast-cell-specific allergic inflammation. Mast cell stabilizers could be used to control IRRs and for safe reintroduction of agalsidase in patients previously treated with ERT.

Background: Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralizing anti-drug antibodies (ADA) inhibiting AGAL activity is associated with disease progression in affected male patients. In the current study, we performed a detailed epitope mapping of ADAs from antibody-positive males against infused AGAL., Methods: A detailed epitope mapping for 34 male FD patients with neutralizing ADAs against AGAL was performed. Based on this data, in silico analyses were used to identify potential epitope clusters and mapped surface-located or buried epitopes. ELISA-based assays against α-galactosidase B (NAGA) were performed to identify ADAs that potentially recognize shared epitopes of AGAL and NAGA. A subset of 20 patients was analyzed to assess if NAGA-recognizing ADAs against AGAL might affect long-term outcomes under ERT., Results: Thirty percent of the AGAL active site was recognized by patients' ADAs. No differences between buried and surface-located epitopes were observed. Dependent on the epitopes, ADAs against AGAL were also able to recognize human NAGA. Patients with NAGA recognizing anti-AGAL antibodies presented with lower plasma NAGA activities. The presence of NAGA-recognizing ADAs had no effect on disease progression., Conclusion: In conclusion, our current data underline previous reports demonstrating a large variation of antibody epitopes against AGAL. Detailed epitope mapping in affected patients might be the first step for the generation of patient-specific blocking peptides and/or immune adsorption columns for an individually tailored anti-antibody strategy., Competing Interests: Declaration of Competing Interest Malte Lenders received speaker honoraria, travel funding and research grants from Sanofi Genzyme, Shire Corporation/Takeda, and Amicus Therapeutics. Eva Brand received research grants and speaker honoraria from Sanofi Genzyme, Shire Corporation/Takeda, Amicus Therapeutics, and Greenovation. Franciska Stappers and David Scharnetzki have nothing to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fabry Disease (FD) is a rare, X-linked, lysosomal storage disease that mainly causes renal, cardiac and cerebral complications. Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A is available, but approximately 50% of male patients with classical FD develop inhibiting anti-drug antibodies (iADAs) that lead to reduced biochemical responses and an accelerated loss of renal function. Once immunization has occurred, iADAs tend to persist and tolerization is hard to achieve. Here we developed a pre-treatment prediction model for iADA development in FD using existing data from 120 classical male FD patients from three European centers, treated with ERT. We found that nonsense and frameshift mutations in the α-galactosidase A gene ( p = 0.05), higher plasma lysoGb3 at baseline ( p < 0.001) and agalsidase beta as first treatment ( p = 0.006) were significantly associated with iADA development. Prediction performance of a Random Forest model, using multiple variables (AUC-ROC: 0.77) was compared to a logistic regression (LR) model using the three significantly associated variables (AUC-ROC: 0.77). The LR model can be used to determine iADA risk in individual FD patients prior to treatment initiation. This helps to determine in which patients adjusted treatment and/or immunomodulatory regimes may be considered to minimize iADA development risk.

Background: Animal tissues and tissue-derived biomaterials are widely used in the field of xenotransplantation and regenerative medicine. A potential immunogenic risk that affects the safety and effectiveness of xenografts is the presence of remnant α -Gal antigen (synthesized by GGTA1 or/and iGb3S ). GGTA1 knockout mice have been developed as a suitable model for the analysis of anti-Gal antibody-mediated immunogenicity. However, we are yet to establish whether GGTA1/iGb3S double knockout (G/i DKO) mice are sensitive to Gal antigen-positive xenoimplants., Methods: α -Gal antigen expression in the main organs of G/i DKO mice or bovine bone substitutes was detected via a standardized ELISA inhibition assay. Serum anti- α -Gal antibody titers of G/i DKO mice after immunization with rabbit red blood cells (RRBC) and implantation of raw lyophilized bone substitutes (Gal antigen content was 8.14 ± 3.17 × 10 12 /mg) or Guanhao Biotech bone substitutes (50% decrease in Gal antigen relative to the raw material) were assessed. The evaluation of total serum antibody, inflammatory cytokine, and splenic lymphocyte subtype populations and the histological analysis of implants and thymus were performed to systematically assess the immune response caused by bovine bone substitutes and bone substitute grafts in G/i DKO mice., Results: α -Gal epitope expression was reduced by 100% in the main organs of G/i DKO mice, compared with their wild-type counterparts. Following immunization with RRBC, serum anti-Gal antibody titers of G/i DKO mice increased from 80- to 180-fold. After subcutaneous implantation of raw lyophilized bone substitutes and Guanhao Biotech bone substitutes into G/i DKO mice, specific anti- α -Gal IgG, anti- α -Gal IgM, and related inflammatory factors (IFN- γ and IL-6) were significantly increased in the raw lyophilized bone substitute group but showed limited changes in the Guanhao Biotech bone substitute group, compared with the control., Conclusion: G/i DKO mice are sensitive to Gal antigen-positive xenogeneic grafts and can be effectively utilized for evaluating the α -Gal-mediated immunogenic risk of xenogeneic grafts., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2020 Anliang Shao et al.)

Introduction and Objectives: Allergy to galactose-α-1,3-galactose (alpha-gal) is a peculiar form of food allergy generally manifesting as an anaphylactic reaction hours after mammalian meat consumption, due to the presence of specific IgE against this oligosaccharide. In addition, immediate anaphylaxis may develop after exposure to other sources of alpha-gal, such as monoclonal antibody cetuximab, vaccines, plasma expanders or anti-snake venoms. Sensitization to alpha-gal has also been implicated in the rapid degeneration of biological valve implants, and recognized as a cause of occupational disease in cattle raisers. The implication of tick bites in this type of sensitization has been accepted by all the research groups dedicated to this disease., Patients and Method: The present study describes the clinical and sensitization characteristics of 39 patients diagnosed with alpha-gal allergy in the hospitals of our province (Lugo, Monforte de Lemos and Burela, Spain)., Results: Most patients were middle-age males. Of note, is the fact that the series includes the first pediatric patient reported in Spain to date. The predominant clinical manifestations were urticaria or delayed anaphylaxis after consumption of mammalian meat. Seventy-four percent of the patients reported having suffered a previous tick bite, and the clinical presentation of anaphylaxis was significantly more prevalent in those with a persistent local reaction following the bite than in those with no such reaction (p = 0.032)., Conclusions: A review is also made of the disorder which, due to its variable clinical expression, is referred to as alpha-gal syndrome. The study concludes that a diagnosis of alpha-gal allergy should be considered in patients with urticaria-anaphylaxis of uncertain origin or manifesting after the administration of vaccines or products of bovine/porcine origin., (Copyright © 2019 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

The mammalian meat allergy known as the "α-Gal syndrome" relates to IgE specific for galactose-α-1,3-galactose (α-Gal), an oligosaccharide that is present in cells and tissues of nonprimate mammals. The recognition of delayed reactions to food derived from mammals in patients with IgE to α-Gal and also the association with tick bites have been increasing worldwide. In 2018, the National Institute of Allergy and Infectious Diseases, Division of Allergy, Immunology and Transplantation, sponsored a workshop on this emerging tick-related disease. International experts from the fields of tick biology, allergy, immunology, infectious disease, and dermatology discussed the current state of our understanding of this emerging medical condition. The participants provided suggestions for specific research priorities and for the development of resources to advance our knowledge of the mechanisms, diagnosis, management, and prevention of this allergic disease. This publication is a summary of the workshop and the panel's recommendations are presented herein., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Mice have been used as accepted tools for investigating complex human diseases and new drug therapies because of their shared genetics and anatomical characteristics with humans. However, the tissues in mice are different from humans in that human cells have a natural mutation in the α1,3 galactosyltransferase (α1,3GT) gene and lack α-Gal epitopes on glycosylated proteins, whereas mice and other nonprimate mammals express this epitope. The lack of α-Gal epitopes in humans results in the loss of immune tolerance to this epitope and production of abundant natural anti-Gal Abs. These natural anti-Gal Abs can be used as an adjuvant to enhance processing of vaccine epitopes to APCs. However, wild-type mice and all existing humanized mouse models cannot be used to test the efficacy of vaccines expressing α-Gal epitopes because they express α-Gal epitopes and lack anti-Gal Abs. Therefore, in an effort to bridge the gap between the mouse models and humans, we developed a new humanized mouse model that mimics humans in that it lacks α-Gal epitopes and secretes human anti-Gal Abs. The new humanized mouse model (Hu-NSG/α-Gal null ) is designed to be used for preclinical evaluations of viral and tumor vaccines based on α-Gal epitopes, human-specific immune responses, xenotransplantation studies, and in vivo biomaterials evaluation. To our knowledge, our new Hu-NSG/α-Gal null is the first available humanized mouse model with such features., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralising anti-drug antibodies (ADA) inhibiting AGAL activity during infusion is associated with disease progression in affected male patients. In this study we analysed if ADAs also inhibit endothelial enzyme uptake as well as intracellular enzyme activity. Therefore, fluorescence-labelled AGAL in combination with ADA-positive sera from FD patients (n = 8) was used to analyse enzyme uptake in endothelial and FD-specific cells. Furthermore, immune adsorption and a comprehensive ADA epitope mapping were performed. Pre-incubation of AGAL with ADAs significantly inhibited intracellular enzyme activity, which was rescued by immune adsorption (both P < .01). ADAs from some patients also inhibited enzyme uptake. ADA epitope mapping identified an epitope at position 121 to 140 aa potentially responsible for uptake inhibition for these patients. Further analyses revealed the presence of stable AGAL/ADA-immune complexes at pH 4.5 and decreased intracellular enzyme activity in endothelial cells (P < .001). Finally, the pre-incubation of AGAL with ADAs resulted in a reduced depletion of intracellular globotriaosylceramide in patient-derived AGAL-deficient cells, demonstrating a direct negative impact of ADAs on intracellular clearance. Neutralising ADAs may not only inhibit infused AGAL activity, but according to their epitopes can also inhibit endothelial AGAL uptake. Indeed, internalised AGAL/ADA-complexes may not dissociate, underlining the importance of novel therapeutic approaches for ADA reduction and prevention to increase therapy efficiency in affected patients., (© 2019 SSIEM.)

Objective: To summarize the current understanding of anaphylaxis, with an emphasis on major findings that have been reported within the last 10 years., Data Sources: Queries relating to anaphylaxis, immunoglobulin E (IgE), and mast cells were conducted with PubMed and Google Scholar, searching for primary articles and review papers., Study Selections: We focused on articles written in English and which were reported in major allergy and immunology journals., Results: Anaphylaxis represents an extreme manifestation of a form of allergic immunity that appears to have evolved to protect against "toxic" threats that present at skin and mucosal barriers. The factors that have contributed to a rise in anaphylaxis are increasingly appreciated to relate to changes in hygiene and microbial ecology that have occurred with industrialization. Induction of allergen-specific IgG4 is often part of the allergic response and is associated with protection against anaphylaxis. The recognition of the α-Gal syndrome suggests that carbohydrates can be epitopes that are relevant to anaphylaxis and that IgE-mediated reactions do not always occur "immediately.", Conclusion: Our understanding of anaphylaxis has advanced significantly over the past 10 years. It is anticipated that ongoing research will build on this foundation to further advance our knowledge of anaphylaxis and also translate into clinically meaningful therapies., (Copyright © 2019. Published by Elsevier Inc.)

Background: Xenografts are an attractive alternative to traditional bone grafts because of the large supply from donors with predictable morphology and biology as well as minimal risk of human disease transmission. Clinical series involving xenograft bone transplantation, most commonly from bovine sources, have reported poor results with frequent graft rejection and failure to integrate with host tissue. Failures have been attributed to residual alpha-Gal epitope in the xenograft which humans produce natural antibody against. To the authors' knowledge, there is currently no xenograft-derived bone graft substitute that has been adopted by orthopedic surgeons for routine clinical use., Methods: In the current study, a bone scaffold intended to serve as a bone graft substitute was derived from porcine cancellous bone using a tissue decellularization and chemical oxidation protocol. In vitro cytocompatibility, pathogen clearance, and alpha-Gal quantification tests were used to assess the safety of the bone scaffold intended for human use., Results: In vitro studies showed the scaffold was free of processing chemicals and biocompatible with mouse and human cell lines. When bacterial and viral pathogens were purposefully added to porcine donor tissue, processing successfully removed these pathogens to comply with sterility assurance levels established by allograft tissue providers. Critically, 98.5% of the alpha-Gal epitope was removed from donor tissue after decellularization as shown by ELISA inhibition assay and immunohistochemical staining., Conclusions: The current investigation supports the biologic safety of bone scaffolds derived from porcine donors using a decellularization protocol that meets current sterility assurance standards. The majority of the highly immunogenic xenograft carbohydrate was removed from donor tissue, and these findings support further in vivo investigation of xenograft-derived bone tissue for orthopedic clinical application., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Fabry disease is a rare inborn error of the enzyme α-galactosidase (Α-Gal) and results in lysosomal substrate accumulation in tissues with a wide range of clinical presentations. The disease has attracted a lot of interest over the last years and several issues has been discovered up to now leading to increasing knowledge and awareness of the disease. However, several aspects are still unclear and under investigation. Thus, the new challenges that physicians encounter are the discovering of the pathogenic mechanisms, the neutralising antibodies to ERT, the long-term efficacy of therapies. In this article, we summarise and review the latest developments in the science community regarding diagnosis, management and monitoring of Fabry disease concerning in particular its physiopathology, novel biomarkers, antibodies development and novel treatment options., (Copyright by Società Italiana di Nefrologia SIN, Rome, Italy.)

Introduction: Tralokinumab is a monoclonal antibody (mAb) that neutralizes interleukin (IL)-13, a cytokine involved in the pathogenesis of asthma., Objective: The objectives of this study were to characterize the potential immunogenic properties of tralokinumab and report data for anti-drug antibodies (ADAs) and hypersensitivity reactions from two phase III clinical trials., Methods: The oligosaccharide structure of tralokinumab, Fab-arm exchange, and ADAs were characterized by standard techniques. Hypersensitivity adverse events (AEs) were evaluated in two pivotal clinical trials of tralokinumab in severe, uncontrolled asthma: STRATOS 1 and 2 (NCT02161757 and NCT02194699)., Results: No galactose-α-1,3-galactose (α-Gal) epitopes were found in the Fab region of tralokinumab and only 4.5% of glycoforms contained α-Gal in the Fc region. Under non-reducing conditions, Fab-arm exchange did not take place with another immunoglobulin (Ig) G 4 mAb (mavrilimumab). However, following glutathione reduction, a hybrid antibody with monovalent bioactivity was detected. ADA incidences (titers) were as follows: STRATOS 1-every 2 weeks (Q2 W) 0.8% (26.0), every 4 weeks (Q4 W) 0.5% (26.0), placebo 0.8% (52.0); STRATOS 2-Q2 W 1.2% (39.0), placebo 0.8% (13.0). Participant-reported hypersensitivity AE rates were as follows: STRATOS 1-Q2 W 25.9%, Q4 W 25.0%, placebo 25.5%; STRATOS 2-Q2 W 13.2%, placebo 9.0%. External evaluation for anaphylaxis by Sampson criteria found no tralokinumab-related severe hypersensitivity or anaphylaxis reactions., Conclusion: Preclinical assessments suggested a low likelihood of immunogenicity for tralokinumab. In STRATOS 1 and 2, ADA incidence was low, no differences were found between tralokinumab-treated and placebo groups in reporting of hypersensitivity reactions, and there were no Sampson criteria-evaluated anaphylaxis events with tralokinumab treatment. Together, the results suggest that tralokinumab treatment would not increase the risk for severe hypersensitivity or anaphylactic reactions.

Background: Treatment of Fabry disease (FD) with recombinant alpha-galactosidase A (r-αGAL A) is complicated by the formation of anti-drug antibodies in the majority of male patients with the classical disease phenotype. Detailed information regarding antibody subtypes, onset and persistence of antibody development and their effect on treatment efficacy is sparse., Methods: A retrospective study was carried out in 39 male patients with classical FD, treated with either agalsidase-alfa or agalsidase-beta (mean follow up of 10 years). With six to twelve months intervals plasma-induced in vitro inhibition of enzyme activity, lysoglobotriaosylsphingosine (lysoGb3) levels and renal function were assessed. In a subset of 12 patients, additionally anti- r-αGAL A IgM, IgA and IgG1, 2, 3 and 4 levels were analyzed., Results: In 23 out of 39 patients, plasma-induced in vitro inhibition of r-αGAL A activity was observed (inhibition-positive). The inhibition titer was strongly negatively correlated to the decrease in lysoGb3: agalsidase-alfa (FE log10(inhibition)  = -10.3, P ≤.001), agalsidase-beta (FE log10(inhibition)  = -4.7, P ≤.001). Inhibition-positive patients had an accelerated decline in renal function (FE = 1.21, p = .042). During treatment IgG1 anti-r-αGAL A levels increased only in inhibition-positive patients (p = .0045). IgG4 anti-r-αGAL A antibodies developed in 7 out of 9 inhibition-positive patients. Other antibody subclasses were either not present or too low to quantify., Conclusion: Development of inhibiting antibodies against r-αGAL A negatively affects the biochemical response to ERT and resulted in an accelerated decline in renal function. The presence of IgG1 and IgG4 anti-r-αGAL A antibodies is associated with in vitro αGAL A activity inhibition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Use of enzyme replacement therapy (ERT) to treat Fabry disease, caused by deficient lysosomal α -galactosidase A activity, can lead to formation of neutralizing antidrug antibodies (ADAs). These antibodies are associated with increased accumulation of plasma globotriaosylceramide (Gb3) and disease progression. Because agalsidase ERT can saturate ADA-binding sites during infusions (achieving agalsidase/antibody equilibrium), we investigated in this open cohort study whether saturated patients (who have excess agalsidase after infusions) experience better clinical outcomes compared with not saturated patients (who have excess ADAs after infusions)., Methods: We isolated ADAs from sera of 26 men with Fabry disease receiving ERT (for a median of 94 months) and determined the amount of agalsidase necessary for antibody saturation. Clinical and biochemical outcomes included measurements of eGFR, interventricular septum thickness, and lyso-Gb3., Results: ADA titers decreased significantly in all patients during infusion. Agalsidase- α and agalsidase- β had similar ADA-binding capacity and comparable ADA saturation frequency. Fourteen patients with saturated ADAs presented with mild (but significant) loss of eGFR, stable septum thickness, and significantly decreased lyso-Gb3 levels. The 12 not saturated patients had a more pronounced and significant loss of eGFR, increased septum thickness, and a smaller, nonsignificant reduction in lyso-Gb3, over time. In three patients, dose escalation resulted in partially elevated ADA titers, but importantly, also in reduced lyso-Gb3 levels., Conclusions: A not saturated ADA status during infusion is associated with progressive loss of eGFR and ongoing cardiac hypertrophy. Dose escalation can result in saturation of ADAs and decreasing lyso-Gb3 levels, but may lead to increased ADA titers., (Copyright © 2018 by the American Society of Nephrology.)

Background: Fabry disease (OMIM #301500) is an X-linked disorder caused by alpha-galactosidase A deficiency with two major clinical phenotypes: classic and non-classic of different prognosis. From 2001, enzyme replacement therapies (ERT) have been available. We aimed to determine the epidemiology and the functional characteristics of anti-drug antibodies. Patients from the French multicenter cohort FFABRY (n = 103 patients, 53 males) were prospectively screened for total anti-agalsidase IgG and IgG subclasses with a home-made enzyme-linked immunosorbent assay (ELISA), enzyme-inhibition assessed with neutralization assays and lysoGb3 plasma levels, and compared for clinical outcomes., Results: Among the patients exposed to agalsidase, 40% of men (n = 18/45) and 8% of women (n = 2/25) had antibodies with a complete cross-reactivity towards both ERTs. Antibodies developed preferentially in men with non-missense GLA mutations (relative risk 2.88, p = 0.006) and classic phenotype (58.6% (17/29) vs 6.7% (1/16), p = 0.0005). Specific anti-agalsidase IgG1 were the most frequently observed (16/18 men), but the highest concentrations were observed for IgG4 (median 1.89 μg/ml, interquartile range (IQR) [0.41-12.24]). In the men exposed to agalsidase, inhibition was correlated with the total IgG titer (r = 0.67, p < 0.0001), especially IgG4 (r = 0.75, p = 0.0005) and IgG2 (r = 0.72, p = 0.001). Inhibition was confirmed intracellularly in Fabry patient leucocytes cultured with IgG-positive versus negative serum (median: 42.0 vs 75.6%, p = 0.04), which was correlated with IgG2 (r = 0.67, p = 0.017, n = 12) and IgG4 levels (r = 0.59, p = 0.041, n = 12). Plasma LysoGb3 levels were correlated with total IgG (r = 0.66, p = 0.001), IgG2 (r = 0.72, p = 0.004), IgG4 (r = 0.58, p = 0.03) and IgG1 (r = 0.55, p = 0.04) titers. Within the classic group, no clinical difference was observed but lysoGb3 levels were higher in antibody-positive patients (median 33.2 ng/ml [IQR 20.6-55.6] vs 12.5 [10.1-24.0], p = 0.005)., Conclusion: Anti-agalsidase antibodies preferentially develop in the severe classic Fabry phenotype. They are frequently associated with enzyme inhibition and higher lysoGb3 levels. As such, they could be considered as a hallmark of severity associated with the classic phenotype. The distinction of the clinical phenotypes should now be mandatory in studies dealing with Fabry disease and its current and future therapies.

α-Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α-galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α-galactosidase A is known as Fabry disease or Fabry-Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy-limiting, and eventually life-threatening complications of ERT. The present study focused on the epitope determination of human α-galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309-332) recognized by a human monoclonal anti-αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309-332), was synthesized by solid-phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K D =39×10 -9  m), which is nearly identical to that of the full-length enzyme (K D =16×10 -9  m). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full-length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Enzymes are attractive as immunotherapeutics because they can catalyze shifts in the local availability of immunostimulatory and immunosuppressive signals. Clinical success of enzyme immunotherapeutics frequently hinges upon achieving sustained biocatalysis over relevant time scales. The time scale and location of biocatalysis are often dictated by the location of the substrate. For example, therapeutic enzymes that convert substrates distributed systemically are typically designed to have a long half-life in circulation, whereas enzymes that convert substrates localized to a specific tissue or cell population can be more effective when designed to accumulate at the target site. This Topical Review surveys approaches to improve enzyme immunotherapeutic efficacy via chemical modification, encapsulation, and immobilization that increases enzyme accumulation at target sites or extends enzyme half-life in circulation. Examples provided illustrate "replacement therapies" to restore deficient enzyme function, as well as "enhancement therapies" that augment native enzyme function via supraphysiologic doses. Existing FDA-approved enzyme immunotherapies are highlighted, followed by discussion of emerging experimental strategies such as those designed to enhance antitumor immunity or resolve inflammation.

Summary: We report the case of a 38-year-old man who was bitten several times during his life by a tick. He didn't report any previous history of anaphylaxis after the ingestion of red meat. The serum specific IgE showed positivity to α-Gal. The proximity of the bits didn't increase the titer of IgE antibodies to alpha-gal. We could hypothesize that the frequency of the exposure to the tick Corresponding author bites and the amount of tick bites during his lifetime induced a sort of tolerance in this patient.

Objective: To investigate the prevalence of and factors associated with the presence of α-gal-specific IgE in a risk group of foresters and forest workers from La Rioja, Spain and in a control group., Methods: The study population comprised 169 workers and 100 individuals who did not recall having had tick bites. A questionnaire including demographic data and number of tick bites per year was completed by a physician. α-Gal sIgE was assessed using ImmunoCAP with serum samples that had been taken in 2010. In 2015, second serum specimens were taken from all but 1 of the workers, who had positive specific IgE to α-gal in 2010. These new samples were tested for IgE to the α-gal epitope and to mammalian meat., Results: The prevalence of positive sIgE to α-gal was 15% in the risk population and 4% in the control population. α-Gal sIgE positivity was associated with the number of tick bites per year and with seniority. Thirteen out of 21 patients sensitized to α-gal in 2010 showed positive specific IgE to α-gal in serum samples from 2015. Eleven had specific IgE to mammalian meat, but none reported symptoms of meat allergy., Conclusions: The prevalence of α-gal sIgE antibodies in this risk population was higher than in the control group and was associated with the number of tick bites per year and with seniority. None of the workers sensitized to mammalian meat developed meat allergy, possibly owing to the low levels of sIgE to α-gal.

Tick-induced allergy to red meat is associated with anti-α-Gal IgE antibody levels. We propose that tick salivary prostaglandin E2 triggers antibody class switching in mature B cells, increasing the levels of anti-α-Gal IgE antibodies. Immune tolerance to α-Gal in blood type B individuals might reduce the risk to this allergy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Antivenoms are mammalian immunoglobulins with the ability to neutralize snake venom components and to mitigate the progression of toxic effects. Immediate hypersensitivity to antivenoms often occurs during the first administration of these heterologous antibodies. A comparable clinical situation occurred after introduction of cetuximab, a chimeric mouse-human antibody, for cancer treatment. The carbohydrate epitope galactose-alpha-1,3-galactose, located on the Fab region of cetuximab, was identified as the target responsible for IgE reactivity., Objective: To investigate whether serum IgE antibodies directed to the α-gal epitope are associated with hypersensitivity to equine antivenoms., Methods: Antivenoms were screened for α-gal epitopes via immunoblot and in comparison with cetuximab and pork kidney by IgE reactivity assays. Basophil activation tests were used to investigate reactivity to antivenoms in samples from 20 patients with specific IgE antibodies to α-gal and 10 controls. Additional IgE detection, IgE inhibition, ImmunoCAP inhibition, and skin prick tests were performed using samples from selected patients., Results: Both antivenoms and cetuximab induced positive skin prick test results in patients with sIgE to α-gal. Alpha-gal epitopes were detected by immunoblotting on antivenoms. Measurements of IgE reactivity and ImmunoCAP inhibition indicated that the antivenoms contained lower α-gal contents than cetuximab. Deglycosylation assays and IgE inhibition tests confirmed that IgE-mediated reactivity to antivenom is associated with α-gal. Antivenoms, pork kidney, and cetuximab activated basophils from patients with IgE to α-gal., Conclusion: Alpha-gal is a potential target of IgE-mediated reactivity to equine antivenom and a possible cause of the high incidence of hypersensitivity reactions during the first application of equine antivenom., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Formation of antibodies against a therapeutic enzyme is an important complication during enzyme replacement therapy (ERT) for lysosomal storage diseases. Fabry disease (FD) is caused by a deficiency of alpha-galactosidase (GLA), which results in the accumulation of globotriaosylceramide (GL-3). We have shown immune tolerance induction (ITI) during ERT in FD model mice by using an anti-B lymphocyte stimulator (anti-BlyS) antibody (belimumab). A single dose of the anti-BlyS antibody temporarily lowered the percentage of B cells and IgG antibody titer against recombinant human GLA. Administration of a low maintenance dose of the anti-BlyS antibody suppressed the B cell population and immunotolerance was induced in 20% of mice, but antibody formation could not be prevented. We then increased the maintenance dose of the anti-BlyS antibody and immunotolerance was induced in 50% of mice. Therapeutic enzyme distribution and clearance of GL-3 were also enhanced by a high maintenance dose of the anti-BlyS antibody., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Glutaraldehyde-fixed porcine heart valves (ga-pV) are one of the most frequently used substitutes for insufficient aortic and pulmonary heart valves which, however, degenerate after 10-15 years. Yet, xeno-immunogenicity of ga-pV in humans including identification of immunogens still needs to be investigated. We here determined the immunogenicity of ga-pV in patients with respect to antibody formation, identity of immunogens and potential options to reduce antibody levels., Methods: Levels of tissue-specific and anti-αGal antibodies were determined retrospectively in patients who received ga-pV for 51 months (n=4), 25 months (n=6) or 5 months (n=4) and compared to age-matched untreated subjects (n=10) or younger subjects with or without vegetarian diet (n=12/15). Immunogenic proteins were investigated by Western blot approaches., Results: Tissue-specific antibodies in patients were elevated after 5 (1.73-fold) and 25 (1.46-fold, both P<.0001) months but not after 51 months, whereas anti-Gal antibodies were induced 4.75-fold and 3.66-fold after 5 and 25 months (both P<.0001) and still were significantly elevated after 51 months (2.85-fold, P<.05). Western blots of porcine valve extracts with and without enzymatic deglycosylation revealed strong specific staining at ≈65 and ≈140 kDa by patient sera in either group which were identified by 2D Western blots and mass spectrometry as serum albumin and collagen 6A1. Vegetarian diet reduced significantly (0.63-fold, P<.01) the level of pre-formed αGal but not of tissue-specific antibodies., Conclusion: Immune response in patients towards ga-pV is induced by the porcine proteins albumin and collagen 6A1 as well as αGal epitopes, which seemed to be more sustained. In contrast, in healthy young subjects pre-formed anti-Gal antibodies were reduced by a meat-free nutrition., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: Immune reaction against the α-Gal(Galα1,3-Galβ1-4GlcNAc-R) epitope is known to be one of the important factors for calcification of bioprostheses. The genetically manipulated α-Gal knock-out (KO) mice lacking the α-Gal epitope and containing anti-α-Gal antibodies may simulate an immune response against xenoantigens in primates including humans. The aims of this study were (i) to compare calcification and immune reactions of glutaraldehyde (GA)-fixed bovine with primate pericardium and (ii) to assess the differences in bovine pericardium treated with decellularization and α-galactosidase, using an α-Gal KO mouse implantation model., Methods: Four types of GA-fixed xenogeneic tissues were implanted into α-Gal KO mice for 16 weeks (Group 1: primate pericardium, n = 14; Group 2: bovine pericardium, n = 19; Group 3: decellularized bovine pericardium, n = 20; Group 4: decellularized and α-galactosidase-treated bovine pericardium, n = 21). Serum enzyme-linked immunosorbent assays for immune response were performed before implantation and 2, 4, 8, 12 and 16 weeks after implantation. Implanted tissues were harvested and studied for histopathology and quantification of calcification., Results: Anti-α-Gal IgG antibody titres of Groups 2-4 increased after implantation although the titres did not change in Group 1. When compared with preimplantation titres, the increased IgG titres were highest at 8 weeks after implantation in Groups 2-4 (0.4358 ± 0.0524 vs 0.5462 ± 0.0519 in Group 2, 0.4712 ± 0.0500 vs 0.6424 ± 0.0613 in Group 3 and 0.4892 ± 0.0801 vs 0.6112 ± 0.0339 in Group 4; P = 0.028 in each group). Median calcium levels were higher in Group 2 than in Group 1 (9.21 vs 3.86 μg/mg, P < 0.001), but were not significantly higher in Groups 3 and 4 than in Group 1 (4.09 μg/mg in Group 3, P = 0.904; 5.47 μg/mg in Group 4, P = 0.210)., Conclusions: GA-fixed bovine pericardium showed higher calcium levels than primate pericardium. Increased IgG antibody titres in the bovine but not in the primate group suggested that an immune response was involved in implanted tissue. Differences in calcium levels of bovine pericardium treated with decellularization and α-galactosidase were insignificant compared with primate pericardium, suggesting those treatments to be effective methods in preventing calcification of the bioprostheses., (© The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Background: The study aim was to evaluate the immune reaction, difference of degenerative calcification, and anti-calcification effect of decellularization with or without α-galactosidase in bovine pericardium and porcine heart valves, using an α1,3-galactosyltransferase (α-Gal) knockout (KO) mouse model., Methods: In order to elucidate the anti-calcification effect of decellularization with or without α-galactosidase, bovine pericardium and porcine heart valve tissues were assigned to four groups according to the tissue preparation method: (i) glutaraldehyde (GA) fixation only; (ii) decellularization + GA fixation (Decell); (iii) α-galactosidase + GA fixation (α-galactosidase); and (iv) decellularization +α-galactosidase + GA fixation (Decell + α-galactosidase). Each prepared tissue was implanted subcutaneously into α-Gal KO mice. Anti-α-Gal immunoglobulin (Ig) G and IgM antibody titers were monitored prior to implantation and at four, eight and 12 weeks after implantation using an enzyme-linked immunosorbent assay. Calcium contents of explanted tissues were measured at 12 weeks after implantation., Results: There were no significant differences in the anti-α-Gal IgG antibody titers according to the type of bioprosthetic material or tissue preparation method (p >0.05). The calcium content was significantly lower in porcine heart valves than in bovine pericardium when implanted in α-Gal-KO mice (p <0.001). Calcium contents in bovine pericardium and porcine heart valves were significantly lower in the Decell, α-galactosidase and Decell + α-galactosidase groups than in the GA group (all p <0.05)., Conclusions: The porcine heart valve induced lower levels of calcium deposition than did the bovine pericardium, but the anti-α-Gal IgG antibody titers did not differ significantly between the bioprosthetic tissues. Decellularization had significant anticalcification effects in both the bovine pericardium and porcine heart valves, though there was no significant difference in the anti-α-Gal IgG antibody titers among tissue preparation methods.

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.

Substitution of the defective lysosomal enzyme in lysosomal storage disorders (LSDs) often elicits antibody formation towards the infused protein. Aside from Gaucher disease, antibodies often lead to infusion associated reactions and a reduced biochemical response. In Pompe disease, antibody titer is predictive of clinical outcome, but this is less apparent in other LSDs and warrants further study. Few laboratories are capable of enzyme-antibody determination: often physicians need to rely on the enzyme manufacturer for analysis. Currently, laboratories employ different antibody assays which hamper comparisons between cohorts or treatment regimens. Assay standardisation, including measurement of antibody-related enzyme inhibition, is therefore urgently needed. Successful immunomodulation has been reported in Pompe and in Gaucher disease, with variable success. Immunomodulation regimens that contain temporary depletion of B-cells (anti-CD20) are most used. Bone marrow transplantation in MPS-I results in disappearance of antibodies. No other clinical studies have been conducted in humans with immunomodulation in other LSDs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Pancreatic cancer is a lethal disease that remains one of the most resistant to traditional therapies. Immunotherapy in pancreatic cancer induces the recruitment and activation of T cells that recognize tumor-associated antigens (TAAs); thus, the mechanism differs from that of chemotherapy and radiotherapy. The goal of cancer immunotherapy is to elicit immune responses against autologous tumors, and especially to induce multiple T cell clones against a variety of TAAs. In the present study, we prepared a polyvalent tumor lysate vaccine engineered to express the α-gal epitopes, Galα1-3Galβ1-4 GlcNAc-R (i.e., α-gal tumor lysate), from primary tumors. The vaccine elicited strong antibody production against multiple TAAs in pancreatic cancer cells and induced activation of multiple tumor-specific T cells in α1,3-galactosyltransferase (α1,3GT) knockout (KO) mice. The tumor lysate vaccine exhibited a similar effect on pancreatic cancer stem cells (CSCs) with the CD44+CD24+ phenotype. Furthermore, in vivo experiments using NOD/SCID mice, inoculated with splenocytes from KO mice vaccinated with the α-gal tumor lysate and injected with pancreatic cancer cells, showed successful induction of a marked immune response that resulted in suppression of tumorigenesis and significant improvement in overall survival. In contrast, inoculation of lymphocytes from KO mice vaccinated with control tumor lysate vaccine had no effects on tumor growth and survival. The results of both in vitro and in vivo experiments emphasize the efficiency of tumor lysate vaccines expressing α-gal epitopes in targeting all pancreatic cancer cells, including differentiated cancer cells and pancreatic CSCs. The α-gal tumor lysate vaccine could be the basis for a novel therapeutic approach in human clinical trials.

Hyperacute rejection (HAR) depends on the response of xenoreactive antibodies principally against porcine α-Gal epitope. Methods eliminating HAR include GGTA1 inactivation, regulation of the complement system and modification of the oligosaccharide structure of surface proteins in donor's cells. Transgenic animals designed for the purpose of xenotransplantation with single modification do not display full reduction of the α-Gal epitope level, which means that a accumulation of several modifications in one transgenic individual is needed. The aim of the study was to create a molecular and cytogenetic profile of a double transgenic animal with α1,2-fucosyltransferase and α-galactosidase expression. As a result of interbreeding of an individual with α1,2-fucosyltransferase expression with an individual with α-galactosidase expression 12 living piglets were obtained. PCR revealed the pCMVFUT gene construct was present in four individuals and pGAL-GFPBsd in three, including one with a confirmed integration of both the gene constructs. Fluorescence in situ hybridization confirmed the site of transgene integration, which corresponded to the mapping site of the transgenes which occurred in the parental generations. Karyotype analysis did not show any changes in the structure or the number of chromosomes (2n = 38, XX). As for the results pertaining to the single transgenic individuals, expression analysis demonstrated a high extent of α-Gal epitope level reduction on the surface of cells, whereas human serum cytotoxicity tests revealed the smallest decrease in longevity of cells in the obtained double transgenic individual (4.35 %). The tests suggest that the co-expression of both the transgenes leads to a considerable reduction of the α-Gal antigen level on the surface of cells and a decrease of xenotransplant immunogenicity.