RESUMEN
Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.
Asunto(s)
Epítopos de Linfocito T/inmunología , Inmunotoxinas/inmunología , Neoplasias/inmunología , Proteínas Recombinantes de Fusión/inmunología , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/inmunología , Aminoácidos/genética , Aminoácidos/inmunología , Animales , Formación de Anticuerpos/inmunología , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Exotoxinas/genética , Exotoxinas/inmunología , Femenino , Humanos , Inmunoterapia/métodos , Inmunotoxinas/genética , Inmunotoxinas/uso terapéutico , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones SCID , Modelos Moleculares , Neoplasias/patología , Neoplasias/terapia , Péptidos/genética , Péptidos/inmunología , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Recombinant immunotoxins (RITs) are chimeric proteins that are being developed for cancer treatment. We have produced RITs that contain PE38, a portion of the bacterial protein Pseudomonas exotoxin A. Because the toxin is bacterial, it often induces neutralizing antibodies, which limit the number of treatment cycles and the effectiveness of the therapy. Because T cells are essential for antibody responses to proteins, we adopted an assay to map the CD4(+) T-cell epitopes in PE38. We incubated peripheral blood mononuclear cells with an immunotoxin to stimulate T-cell expansion, followed by exposure to overlapping peptide fragments of PE38 and an IL-2 ELISpot assay to measure responses. Our observation of T-cell responses in 50 of 50 individuals correlates with the frequency of antibody formation in patients with normal immune systems. We found a single, highly immunodominant epitope in 46% (23/50) of the donors. The immunodominant epitope is DRB1-restricted and was observed in subjects with different HLA alleles, indicating promiscuity. We identified two amino acids that, when deleted or mutated to alanine, eliminated the immunodominant epitope, and we used this information to construct mutant RITs that are highly cytotoxic and do not stimulate T-cell responses in many donors.