Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF-ß and PD-L1 enhances antitumor responses.

T cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-ß play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-ß inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-ß, termed AxF (scFv)2, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)2 antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3×EGFR TriTE with the AxF (scFv)2 delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-ß by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression.

Nanobody-Based EGFR-Targeting Immunotoxins for Colorectal Cancer Treatment.

Immunotoxins (ITXs) are chimeric molecules that combine the specificity of a targeting domain, usually derived from an antibody, and the cytotoxic potency of a toxin, leading to the selective death of tumor cells. However, several issues must be addressed and optimized in order to use ITXs as therapeutic tools, such as the selection of a suitable tumor-associated antigen (TAA), high tumor penetration and retention, low kidney elimination, or low immunogenicity of foreign proteins. To this end, we produced and characterized several ITX designs, using a nanobody against EGFR (VHH 7D12) as the targeting domain. First, we generated a nanoITX, combining VHH 7D12 and the fungal ribotoxin α-sarcin (αS) as the toxic moiety (VHHEGFRαS). Then, we incorporated a trimerization domain (TIEXVIII) into the construct, obtaining a trimeric nanoITX (TriVHHEGFRαS). Finally, we designed and characterized a bispecific ITX, combining the VHH 7D12 and the scFv against GPA33 as targeting domains, and a deimmunized (DI) variant of α-sarcin (BsITXαSDI). The results confirm the therapeutic potential of α-sarcin-based nanoITXs. The incorporation of nanobodies as target domains improves their therapeutic use due to their lower molecular size and binding features. The enhanced avidity and toxic load in the trimeric nanoITX and the combination of two different target domains in the bispecific nanoITX allow for increased antitumor effectiveness.

A New Optimized Version of a Colorectal Cancer-Targeted Immunotoxin Based on a Non-Immunogenic Variant of the Ribotoxin α-Sarcin.

Due to its incidence and mortality, cancer remains one of the main risks to human health and lifespans. In order to overcome this worldwide disease, immunotherapy and the therapeutic use of immunotoxins have arisen as promising approaches. However, the immunogenicity of foreign proteins limits the dose of immunotoxins administered, thereby leading to a decrease in its therapeutic benefit. In this study, we designed two different variants of non-immunogenic immunotoxins (IMTXA33αSDI and IMTXA33furαSDI) based on a deimmunized variant of the ribotoxin α-sarcin. The inclusion of a furin cleavage site in IMTXA33furαSDI would allow a more efficient release of the toxic domain to the cytosol. Both immunotoxins were produced and purified in the yeast Pichia pastoris and later functionally characterized (both in vitro and in vivo), and immunogenicity assays were carried out. The results showed that both immunotoxins were functionally active and less immunogenic than the wild-type immunotoxin. In addition, IMTXA33furαSDI showed a more efficient antitumor effect (both in vitro and in vivo) due to the inclusion of the furin linker. These results constituted a step forward in the optimization of immunotoxins with low immunogenicity and enhanced antitumor activity, which can lead to potential better outcomes in cancer treatment.

Effect of an intervention New Medicine Service to improve adherence in the Spanish community pharmacies: a protocol of a pragmatic randomised trial / Eficacia del Servicio de Asistencia en Nuevos Medicamentos para mejorar su adherencia en pacientes con enfermedades crónicas en farmacias comunitarias españolas: protocolo de un ensayo pragmático aleatorizado

Introduction: Non-adherence to medications is one of the challenges health systems faces. Patients with poor adherence to treatment fail to benefit from effective medication, and this is associated with reductions in quality of life, poorer outcomes, increased hospitalisations, deaths, and, consequently, higher healthcare costs. Community pharmacies are shown to be key elements in improving adherence to prescribed medications, optimising patient outcomes and increasing the efficiency of care. Objectives: (1) assess the effectiveness of the New Medicine Service (NMS) intervention delivered by community pharmacists to improve adherence to treatment in patients who have been prescribed a new medicine for a specific chronic condition; and (2) to conduct an economic evaluation of this intervention. Methods: A pragmatic randomized clinical trial at community pharmacy-level (clusters) will be performed. Patients identified in the collaborating community pharmacy as starting treatment for the following conditions, will be invited to join the study: chronic obstructive pulmonary disease, hypertension, diabetes mellitus or on an anticoagulant/antiplatelet agent. The intervention is based on the pharmacist-patient communication, aiming to assess the patient's relationship with his/her new prescription, and identify potential issues, concerns and false beliefs or expectations. Ethics and dissemination: The study protocol has been reviewed and ethics approval obtained from the regional ethics committee. The results from this study will be actively disseminated through manuscript publications and conference presentations. (AU)

Introducción: La falta de adherencia a los medicamentos es uno de los desafíos a los que se enfrentan los sistemas de salud. Los pacientes con mala adherencia al tratamiento no se benefician de la eficacia de la medicación, lo que se asocia con peor calidad de vida, aumento en hospitalizaciones y muertes y, en consecuencia, mayores costes sanitarios. Se ha demostrado que las farmacias comunitarias son elementos clave para mejorar la adherencia a los medicamentos prescritos, optimizar los resultados en pacientes con enfermedades crónicas y aumentar la eficiencia de la atención sanitaria. Objetivos: (1) evaluar la efectividad de la intervención Asistencia a Nuevos Medicamentos (ANM) administrada por farmacéuticos comunitarios para mejorar la adherencia al tratamiento en pacientes a los que se les ha recetado un nuevo medicamento para una enfermedad crónica específica; y (2) realizar una evaluación económica de esta intervención. Métodos: Se realizará un ensayo clínico pragmático aleatorizado a nivel de farmacia comunitaria (clúster). Se invitará a unirse al estudio a los pacientes identificados en la farmacia comunitaria que inicien tratamiento para: enfermedad pulmonar obstructiva crónica, hipertensión arterial, diabetes mellitus o en tratamiento con un anticoagulante/ antiagregante plaquetario. La intervención se basa en la comunicación farmacéutico-paciente, con el objetivo de evaluar la relación del paciente con su nuevo medicamento, e identificar posibles problemas, preocupaciones y falsas creencias-expectativas. Ética y difusión: Se ha obtenido el dictamen favorable del Comité de Ética de la Investigación Biomédica de Andalucía. Los resultados de este estudio se difundirán activamente a través de publicaciones y presentaciones en congresos. (AU)

Trispecific T-cell engagers for dual tumor-targeting of colorectal cancer.

Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR-EpCAM-) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity.