RESUMEN

The Latarjet procedure is an established surgical treatment for recurrent glenohumeral joint instability with glenoid bone loss. Intraoperatively, the conjoint tendon and its attachement on the coracoid bone graft is routed through a split in subscapularis where the graft is fixed to and augments the anteroinferior glenoid. The objective of this in vitro study was to quantify the influence of glenohumeral joint position and conjoint tendon force on the lines of action and moment arms of subscapularis muscle sub-regions after Latarjet surgery. Eight fresh-frozen, entire upper extremities were mounted onto a testing apparatus, and a cable-pulley system was used to apply physiological muscle loading to the major shoulder muscles. The lines of action and moment arms of four subregions of subscapularis (superior, mid-superior, mid-inferior, and inferior) were quantified radiographically with the conjoint tendon unloaded and loaded while the shoulder was in (i) 0° abduction (ii) 90° abduction (iii) 90° abduction and full external rotation (ABER), and (iv) the apprehension position, defined as ABER with 30° horizontal extension. Conjoint tendon loading after Latarjet surgery significantly increased the inferior inclination of the lines of action of the mid-inferior and inferior subregions of subscapularis in the scapular plane in ABER and apprehension positions (p < 0.001), as well as decreased the horizontal flexion moment arm of the inferior subscapularis (p = 0.040). Increased subscapularis inferior inclination may ultimately increase inferior joint shear potential, while smaller horizontal flexion leverage may reduce joint flexion capacity. The findings have implications for Latarjet surgical planning and postoperative rehabilitation prescription.

Asunto(s)

Articulación del Hombro , Humanos , Articulación del Hombro/cirugía , Articulación del Hombro/fisiopatología , Articulación del Hombro/fisiología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Inestabilidad de la Articulación/cirugía , Inestabilidad de la Articulación/fisiopatología , Tendones/cirugía , Músculo Esquelético , Fenómenos Biomecánicos

RESUMEN

CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro- and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D µComputer tomography bioluminescence tomography (µCT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell (± IL-2) or control BDCA-2 CAR T cell (± IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 IU/mouse. CAR T cell distribution was measured in 2D BLI and 3D µCT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM. Results: 3D µCT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect. Conclusion: Overall, we demonstrated that 3D µCT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D- and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status.

Asunto(s)

Inmunoterapia Adoptiva , Neoplasias , Animales , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva/métodos , Interleucina-2 , Ratones , Imagen Multimodal , Neoplasias/diagnóstico por imagen , Neoplasias/terapia , Flujo de Trabajo

RESUMEN

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

Asunto(s)

Adenocarcinoma/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Inmunoterapia/métodos , Neoplasias Pancreáticas/metabolismo , Tetraspaninas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/terapia , Animales , Antígenos de Neoplasias/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/terapia , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Citocinas/metabolismo , Proteínas Ligadas a GPI/metabolismo , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Factores Inmunológicos , Activación de Linfocitos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Linfocitos T/inmunología , Tetraspaninas/genética , Neoplasias Pancreáticas

RESUMEN

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.

Asunto(s)

Antígenos CD20/inmunología , Carcinoma Ductal Pancreático/terapia , Inmunoterapia Adoptiva , Linfoma/terapia , Glicoproteína Asociada a Mielina/genética , Neoplasias Pancreáticas/terapia , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/trasplante , Animales , Antígenos CD20/genética , Antígenos CD20/metabolismo , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Linfoma/inmunología , Linfoma/metabolismo , Linfoma/patología , Ratones Endogámicos NOD , Ratones SCID , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Asociada a Mielina/metabolismo , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fenotipo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Pancreatic cancer has the worst prognosis and lowest survival rate among all types of cancers and thus, there exists a strong need for novel therapeutic strategies. Chimeric antigen receptor (CAR)-modified T cells present a new potential option after successful FDA-approval in hematologic malignancies, however, current CAR T cell clinical trials in pancreatic cancer failed to improve survival and were unable to demonstrate any significant response. The physical and environmental barriers created by the distinct tumor microenvironment (TME) as a result of the desmoplastic reaction in pancreatic cancer present major hurdles for CAR T cells as a viable therapeutic option in this tumor entity. Cancer cells and cancer-associated fibroblasts express extracellular matrix molecules, enzymes, and growth factors, which can attenuate CAR T cell infiltration and efficacy. Recent efforts demonstrate a niche shift where targeting the TME along CAR T cell therapy is believed or hoped to provide a substantial clinical added value to improve overall survival. This review summarizes therapeutic approaches targeting the TME and their effect on CAR T cells as well as their outcome in preclinical and clinical trials in pancreatic cancer.