RESUMEN
PURPOSE: Although programmed death-ligand 1 (PD-L1) antibody-based therapy has improved the outcome of patients with cancer, acquired resistance to these treatments limits their clinical efficacy. FS118 is a novel bispecific, tetravalent antibody (mAb2) against human lymphocyte activation gene-3 (LAG-3) and PD-L1 with the potential to reinvigorate exhausted immune cells and overcome resistance mechanisms to PD-L1 blockade. Here, using FS118 and a murine surrogate, we characterized the activity and report a novel mechanism of action of this bispecific antibody. EXPERIMENTAL DESIGN: This study characterizes the binding activity and immune function of FS118 in cell lines and human peripheral blood mononuclear cells and further investigates its antitumor activity and mechanism of action using a surrogate murine bispecific antibody (mLAG-3/PD-L1 mAb2). RESULTS: FS118 demonstrated simultaneous binding to LAG-3 and PD-L1 with high affinity and comparable or better activity than the combination of the single component parts of the mAb2 in blocking LAG-3- and PD-L1-mediated immune suppression and enhancing T-cell activity. In syngeneic tumor mouse models, mLAG-3/PD-L1 mAb2 significantly suppressed tumor growth. Mechanistic studies revealed decreased LAG-3 expression on T cells following treatment with the mouse surrogate mLAG-3/PD-L1 mAb2, whereas LAG-3 expression increased upon treatment with the combination of mAbs targeting LAG-3 and PD-L1. Moreover, following binding of mLAG-3/PD-L1 mAb2 to target-expressing cells, mouse LAG-3 is rapidly shed into the blood. CONCLUSIONS: This study demonstrates a novel benefit of the bispecific approach over a combination of mAbs and supports the further development of FS118 for the treatment of patients with cancer.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígenos CD/metabolismo , Antineoplásicos Inmunológicos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Animales , Afinidad de Anticuerpos , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Inmunofenotipificación , Activación de Linfocitos/inmunología , Ratones , Unión Proteica , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
The immunoglobulin superfamily protein lymphocyte-activation gene 3 (LAG-3) participates in immune suppression and has been identified as a suitable target for cancer therapies. In order to generate bispecific antibodies targeting LAG-3, Fcabs (Fc-region with antigen binding) targeting human and murine LAG-3 were generated from phage libraries. These Fcabs bind to LAG-3, inhibiting its interaction with MHC class II, and induce IL-2 production in a T cell assay. Bispecific antibodies, known as mAb2, were produced by replacing the Fc region of a monoclonal antibody with Fcab sequences in the CH3 domain. mAb2 containing anti-LAG-3 Fcabs have mAb-like biophysical characteristics and retain LAG-3 binding and functional activity. mAb2 can thus be generated using multiple Fabs to investigate bispecific parings and develop novel therapeutics.
Asunto(s)
Anticuerpos Biespecíficos , Antígenos CD/inmunología , Fragmentos Fc de Inmunoglobulinas , Animales , Humanos , Macaca fascicularis/metabolismo , Ratones , Ingeniería de Proteínas , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Representational difference analysis (RDA) has great potential for preferential amplification of unique but uncharacterised DNA sequences present in one source such as a whole genome, but absent from a related genome or other complex population of sequences. While a few examples of its successful exploitation have been published, the method has not been well dissected and robust, detailed published protocols are lacking. Here we examine the method in detail, suggest improvements and provide a protocol that has yielded key unique sequences from a pathogenic bacterial genome.