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Abstract(s)
A doença de Fabry (DF) é uma doença de sobrecarga lisossomal (DAL) ligada ao cromossoma X
causada por mutações no gene GLA que codifica a α-galactosidase A. Isto, leva a um acúmulo
progressivo de globotriaosilceramida (GL-3, Gb3) em todos os tecidos do corpo. As manifestações
cardíacas, renais e neurológicas são comuns e a esperança de vida é significativamente reduzida em
relação à população em geral.
O controle da DF envolve a administração de terapia de reposição enzimática intravenosa (TRE).
Duas formas, agalsidase alfa e agalsidase beta, foram aprovadas em vários países e são geralmente
bem toleradas. Embora a TRE tenha demonstrado melhorar a evolução do doente e o curso da doença,
especialmente quando iniciada no início da doença, a infusão intravenosa das proteínas recombinantes
pode desencadear uma resposta humoral imune, resultando em reações associadas à infusão (RAIs),
e/ou o desenvolvimento de anticorpos antifármaco neutralizantes (ADAs), que parecem atenuar a
eficácia da terapia em doentes, mediando a progressão da doença apesar da TRE 1-3.
Anticorpos imunoglobulina (Ig) G têm sido frequentemente descritos em doentes com DF
recebendo TRE. As respostas de IgG são relatadas numa proporção maior de doentes que recebem
agalsidase beta do que em doentes que recebem agalsidase alfa. Os anticorpos IgE são menos comuns
do que os anticorpos IgG e não foram observados em doentes recebendo agalsidase alfa. O impacto
clínico do desenvolvimento de anticorpos IgG para TRE em doentes com DF permanece obscuro,
devido à falta de dados e à acentuada heterogeneidade dos pacientes em termos de manifestações da
doença e resposta à terapia 4.
Outros estudos que examinam o desenvolvimento de anticorpos em doentes com DF e o potencial
impacto de tais anticorpos na eficácia da TRE são necessários.
O objetivo principal deste projeto é estudar a imunogenicidade de um conjunto de doentes com DF
e verificar o impacto da imunogenicidade na eficácia e no perfil de segurança da TRE. Para isso,
fizemos o screning de anticorpos IgG1, IgG4 e IgE contra as terapias agalsidase alfa e beta, pré e pós
infusão da TRE, através ELISA; e dissociamos os IC associados da TRE e fizemos o screning de IgG
libertados dos IC, através ELISA, para identificarmos os falsos negativos.
Relacionamos os resultados obtidos com os dados de segurança, dados clínicos e funcionais
cardíacos e renais do doentes, para verificarmos o impacto da presença de ADA contra agalsidase
alfa e beta, na eficácia e segurança da TRE, o que nos permitirá, ainda, fazem uma correlação direta
entre as duas terapias TRE, agalsidase alfa e beta.
No nosso estudo verificamos que os anticorpos IgG1 foram os mais frequentemente observados
contra ambas as terapias, agalsidase alfa e beta, seguidos pelos anticorpos IgG4 e por último os
anticorpos IgE menos frequentes, mas também presentes em doentes tratados com terapia com
agalsidase alfa e em mulheres, ao contrário de o que alguns estudos demonstram.
À semelhança de outros estudos, um maior desenvolvimento de ADA foi observado em doentes
tratados com terapia com agalsidase beta versus terapia com agalsidase alfa. E também maior
desenvolvimento, e em maiores quantidades de ADA em homens versus mulheres.
Ao comparar os níveis de anticorpos IgG e IgE contra ambas as terapias, observamos apenas
diferenças estatisticamente significativas para a terapia com agalsidase beta.
Em relação aos IC, foi observada a presença de IC no pré e pós-infusão, porém não houve diferenças
estatisticamente significativas, possivelmente porque os 30 minutos após a infusão da TRE não foram
suficientes para que ocorresse a produção de IC.
No entanto, ao analisar as diferenças de sinal entre as amostras tratadas e não tratadas com ácido,
verificamos que o tratamento com ácido permite a dissociação de AAA dos IC. Isso é de suma
importância para a medição e caracterização da imunogenicidade e mostra a presença de IC estáveis
entre a droga e os anticorpos com diferentes perfis de afinidade circulando no sangue.
Devido ao fato de não nos ter sido possível aceder os dados de segurança e dados clínicos e
funcionais cardíacos e renais dos doentes, não fomos capazes de aferir e correlacionar a presença de
ADA contra agalsidase alfa e beta, na eficácia e segurança da TRE e com o aparecimento de RAIs.
Fabry disease (FD) is a X-linked lysosomal storage disorder (LSD) caused by mutations on the GLA gene encoding for α -galactosidase A. This leads to a progressive accumulation of globotriaosylceramide (GL-3, Gb3) in tissues throughout the body. Cardiac, renal and neurological manifestations are common and life expectancy is significantly reduced relative to the general population. Management of FD involves the administration of intravenous enzyme replacement therapy (ERT). Two forms, agalsidase alfa and agalsidase beta, have been licensed in certain jurisdictions and are generally well tolerated. Although ERT has been demonstrated to improve patient outcomes and disease course, especially when initiated early in the course of the disease, the intravenous infusion of the recombinant proteins can trigger a humoral immune response, resulting in infusion-associated reactions (IARs), and/or the development of neutralizing antidrug antibodies (ADAs), which seem to attenuate therapy efficacy in patients, mediating disease progression despite ERT 1-3. Immunoglobulin (Ig) G antibodies have frequently been reported in patients with FD receiving ERT. Immunoglobulin G (IgG) responses are reported in a greater proportion of patients receiving agalsidase beta than in patients receiving agalsidase alfa. Immunoglobulin E (IgE) antibodies are less common than IgG antibodies and have not been observed in patients receiving agalsidase alfa. The clinical impact of the development of IgG antibodies to ERT in patients with FD remains unclear, due to lack of data and to the marked heterogeneity of patients both in terms of disease manifestations and response to therapy 4. Further studies that examine the development of antibodies in patients with FD and the potential impact of such antibodies on the outcome of ERT are necessary. The general purpose of this project is to study the immunogenicity of a court of Fabry patients and see the impact of immunogenicity on the effectiveness and safety profile of ERT. For this, we will screnn for antibodies IgG1, IgG4 and IgE against agalsidase alfa and beta therapy, pre and pos ERT infusion, by ELISA; and dissociation of Immuno-Complexes (IC) associated to ERT and screening of IgG released from IC through ELISA, in order to identify false negatives. We will relate the results obtained with the safety data, and clinical and functional cardiac and renal data of patients, to verify the impact of the presence of ADA against agalsidase alfa and beta, on the effectiveness and safety of ERT, which will allow us to still make a direct correlation between the two ERT therapies, agalsidase alpha and beta. In our study we verified than, IgG1 antibodies were the most frequently observed against both therapies, agalsidase alfa and beta, followed by IgG4 antibodies and lastly the least frequent IgE antibodies, but also present in patients treated with agalsidase alfa therapy and in women, contrary to what some studies demonstrate. Similar to other studies, a greater development of ADA was observed in patients treated with agalsidase beta therapy versus agalsidase alfa therapy. Also, there is a greater development and in greater amounts of ADA in men versus women. When comparing the levels of IgG and IgE antibodies against both therapies, we only observed statistically significant differences for agalsidase beta therapy. In relation to IC, the presence of IC was observed in the pre and post infusion, however there were no statistically significant differences, possibly because the 30 minutes after the infusion of ERT, were not enough for the production of IC to occur. However, when analyzing the signal differences between samples treated and untreated with acid, we found that the acid treatment allows the dissociation of Anti-Agalsidase antibodies (AAA) from IC. This is of paramount importance for immunogenicity measurement and characterization and shows the presence of stable IC between drug and antibodies with different affinity profiles circulating in blood´s patients. Due to the fact that we were unable to access the safety data, and clinical and functional cardiac and renal data of patients, we were unable to measure and correlate of the presence of ADA against agalsidase alfa and beta, on the effectiveness and safety of ERT and with the appearance of IARs.
Fabry disease (FD) is a X-linked lysosomal storage disorder (LSD) caused by mutations on the GLA gene encoding for α -galactosidase A. This leads to a progressive accumulation of globotriaosylceramide (GL-3, Gb3) in tissues throughout the body. Cardiac, renal and neurological manifestations are common and life expectancy is significantly reduced relative to the general population. Management of FD involves the administration of intravenous enzyme replacement therapy (ERT). Two forms, agalsidase alfa and agalsidase beta, have been licensed in certain jurisdictions and are generally well tolerated. Although ERT has been demonstrated to improve patient outcomes and disease course, especially when initiated early in the course of the disease, the intravenous infusion of the recombinant proteins can trigger a humoral immune response, resulting in infusion-associated reactions (IARs), and/or the development of neutralizing antidrug antibodies (ADAs), which seem to attenuate therapy efficacy in patients, mediating disease progression despite ERT 1-3. Immunoglobulin (Ig) G antibodies have frequently been reported in patients with FD receiving ERT. Immunoglobulin G (IgG) responses are reported in a greater proportion of patients receiving agalsidase beta than in patients receiving agalsidase alfa. Immunoglobulin E (IgE) antibodies are less common than IgG antibodies and have not been observed in patients receiving agalsidase alfa. The clinical impact of the development of IgG antibodies to ERT in patients with FD remains unclear, due to lack of data and to the marked heterogeneity of patients both in terms of disease manifestations and response to therapy 4. Further studies that examine the development of antibodies in patients with FD and the potential impact of such antibodies on the outcome of ERT are necessary. The general purpose of this project is to study the immunogenicity of a court of Fabry patients and see the impact of immunogenicity on the effectiveness and safety profile of ERT. For this, we will screnn for antibodies IgG1, IgG4 and IgE against agalsidase alfa and beta therapy, pre and pos ERT infusion, by ELISA; and dissociation of Immuno-Complexes (IC) associated to ERT and screening of IgG released from IC through ELISA, in order to identify false negatives. We will relate the results obtained with the safety data, and clinical and functional cardiac and renal data of patients, to verify the impact of the presence of ADA against agalsidase alfa and beta, on the effectiveness and safety of ERT, which will allow us to still make a direct correlation between the two ERT therapies, agalsidase alpha and beta. In our study we verified than, IgG1 antibodies were the most frequently observed against both therapies, agalsidase alfa and beta, followed by IgG4 antibodies and lastly the least frequent IgE antibodies, but also present in patients treated with agalsidase alfa therapy and in women, contrary to what some studies demonstrate. Similar to other studies, a greater development of ADA was observed in patients treated with agalsidase beta therapy versus agalsidase alfa therapy. Also, there is a greater development and in greater amounts of ADA in men versus women. When comparing the levels of IgG and IgE antibodies against both therapies, we only observed statistically significant differences for agalsidase beta therapy. In relation to IC, the presence of IC was observed in the pre and post infusion, however there were no statistically significant differences, possibly because the 30 minutes after the infusion of ERT, were not enough for the production of IC to occur. However, when analyzing the signal differences between samples treated and untreated with acid, we found that the acid treatment allows the dissociation of Anti-Agalsidase antibodies (AAA) from IC. This is of paramount importance for immunogenicity measurement and characterization and shows the presence of stable IC between drug and antibodies with different affinity profiles circulating in blood´s patients. Due to the fact that we were unable to access the safety data, and clinical and functional cardiac and renal data of patients, we were unable to measure and correlate of the presence of ADA against agalsidase alfa and beta, on the effectiveness and safety of ERT and with the appearance of IARs.
Description
Tese de mestrado, Ciências Biofarmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Lysosomal storage disorder Fabry disease α galactosidase A Enzyme replacement therapy Infusion-associated reactions Neutralizing antidrug antibodies Teses de mestrado -2021