RESUMEN
Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti-programmed cell death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1-targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti-PD-L1 Pb-Tx CX-072, which is currently in clinical trials.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígeno B7-H1/uso terapéutico , Inmunoterapia/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Microambiente TumoralRESUMEN
We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.
Asunto(s)
Sustitución de Aminoácidos , Anticuerpos Monoclonales Humanizados , Antineoplásicos , Regiones Determinantes de Complementariedad , Receptores ErbB/inmunología , Mutación Missense , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/química , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Cetuximab , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Receptores ErbB/genética , Células HEK293 , HumanosRESUMEN
Monoclonal antibodies represent an attractive therapeutic tool as they are highly specific for their targets, convey effector functions and enjoy robust manufacturing procedures. Humanization of murine monoclonal antibodies has vastly improved their in vivo tolerability. Humanization, the replacement of mouse constant regions and V framework regions for human sequences, results in a significantly less immunogenic product. However, some humanized and even fully human sequence-derived antibody molecules still carry immunological risk. To more fully understand the immunologic potential of humanized and human antibodies, we analyzed CD4(+) helper T cell epitopes in a set of eight humanized antibodies. The antibodies studied represented a number of different VH and VL family members carrying unique CDR regions. In spite of these differences, CD4(+) T cell epitopes were found only in CDR-sequence containing regions. We were able to incorporate up to two amino acid modifications in a single epitope that reduced the immunogenic potential while retaining full biologic function. We propose that immunogenicity will always be present in some antibody molecules due to the nature of the antigen-specific combining sites. A consequence of this result is modifications to reduce immunogenicity will be centered on the affinity-determining regions. Modifications to CDR regions can be designed that reduce the immunogenic potential while maintaining the bioactivity of the antibody molecule.