RESUMEN
Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.
RESUMEN
Most chemical techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Anticuerpos/química , Antineoplásicos/farmacología , Oligopéptidos/química , Transglutaminasas/química , Alquinos/química , Alquinos/metabolismo , Anticuerpos/metabolismo , Anticuerpos Monoclonales Humanizados/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Azidas/química , Azidas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Maleimidas/química , Maleimidas/metabolismo , Estructura Molecular , Oligopéptidos/metabolismo , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Transglutaminasas/metabolismo , TrastuzumabRESUMEN
Phosphoantigens enable the access to a new anti-tumoral and anti-infectious therapeutic pathway, based on innate immunity through the selective activation of Tγ9δ2 lymphocytes. The first proof of concept of this new immunotherapy approach was demonstrated with the synthetic phosphoantigen named bromohydrin pyrophosphate (BrHPP, IPH 1101) which was administrated in racemic form to about 200 patients in six clinical trials with good safety and promising early signals of efficacy in type C viral hepatitis and follicular non-Hodgkin's lymphoma. Enantiopure samples of BrHPP in gram scale are required for further studies on structure-bioactivity relationship. Thus we developed two complementary synthetic pathways, the first using transformation of a chiral compound and the second involving asymmetric synthesis starting from a prochiral building-block. The synthesis of a second-generation phosphoantigen, N-HDMAPP, which bears a phosphoramidate moiety, was also investigated.
Asunto(s)
Difosfatos/síntesis química , Compuestos Organofosforados/síntesis química , Difosfatos/administración & dosificación , Difosfatos/química , Humanos , Inmunoterapia , Estructura Molecular , Neoplasias/terapia , Compuestos Organofosforados/química , EstereoisomerismoRESUMEN
OBJECTIVE: To evaluate intraindividual variability of the BASDAI, BASFI, and HAQ in patients with spondyloarthropathies. METHODS: The three variables were collected prospectively in 24 patients, once a week for 30 weeks. They were also collected retrospectively in 31 patients on stable infliximab regimens with a mean of 11.5+/-4 injections, starting at the fourth infusion. RESULTS: The BASDAI and BASFI showed high intraindividual variability from week to week; SDs greater than 1 were found for the BASDAI in 14/24 patients and for the BASFI in 10/24 patients, and ranges greater than 4/10 occurred for the BASDAI in 13/24 patients and for the BASFI in 10/24 patients. Although the mean BASDAI was greater than 4 in only 6/24 patients, values greater than 4 occurred on one or more occasions in 19 (79%) patients. The retrospective study of infliximab-treated patients from the fourth infusion onward also showed high variability of BASDAI and BASFI values, with SDs greater than 1 in 18/31 patients and ranges greater than 4 in 14/31 patients and greater than 2 in 22/31 patients. CONCLUSION: Repeated determination of the BASDAI and BASFI by the patients (using paper forms or personal digital assessments) may help to identify candidates for treatment intensification, to optimize infliximab injection regimens in good responders, and to avoid unnecessary switching from one TNFalpha antagonist to another.