Ex vivo tumor-fragment cultures and in vivo PBMC-humanized mouse models as preclinical tools for cancer immunotherapy development
- Autores: Iñaki Eguren Santamaría
- Directores de la Tesis: Ignacio Melero Bermejo (dir. tes.), Miguel Fernández de Sanmamed Gutierrez (dir. tes.)
- Lectura: En la Universidad de Navarra ( España ) en 2024
- Idioma: inglés
- Tribunal Calificador de la Tesis: Jose Luis Monserrat Pérez (presid.), Fernando Pastor Rodríguez (secret.), María Paloma Sánchez Mateos Rubio (voc.), Jon Zugazagoitia (voc.), Alberto Villanueva Garatachea (voc.)
- Programa de doctorado: Programa de Doctorado en Medicina Aplicada y Biomedicina por la Universidad de Navarra
- Calificación: Sobresaliente Cum Laude
- Materias:
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- Resumen
Almost every successful anticancer treatment is preceded by preclinical breakthroughs that encouraged clinical trials. However, therapeutic strategies showing promising preclinical results often fail to confirm activity in clinical trials, particularly for immunotherapy drugs. Better preclinical methods are required to bridge the gap between preclinical research and clinical practice.
PART 1: D. Thommen et al. recently reported on a methodology based on short-term cultures of patient-derived tumor fragments whose cytokine secretion and activation markers on infiltrating T cells were associated with clinical response to PD-1 blockade. We set up similar cultures with tumor fragments using mouse tumors to test an agonist anti-CD137 mAb and its combinations with anti-PD-1 and/or anti-TGF-β. Increases in IFNγ in the culture supernatant were detected upon activation with anti-CD137 and anti-PD-1 mAb combinations. Interestingly, increases in Ki67 and other activation markers were observed in tumor-infiltrating lymphocytes. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137 + anti-PD-1 treatment, no association was found between in-culture IFNγ production and the in vivo therapeutic outcome. Using a series of patient-derived tumor fragments from excised solid malignancies, we showed IFNγ production in a fraction of studied cases.
PART 2: Immunodeficient mice engrafted with peripheral blood mononuclear cells (PBMCs) are models to study cancer immunotherapy agents. However, this approach is limited by xenograft-versus-host disease (xGVHD). Here, we explore different approaches to overcome xGVHD.
Immunodeficient NOD-scid IL2Rgnull (NSG) mice were intravenously transferred with human PBMCs and subcutaneously co-engrafted with HT29 human colon carcinoma cells. Diverse strategies to reduce xGVHD while preserving the antitumor activity of human immune cells were tested: (1) ex vivo depleting CD4+ T cells, (2) in vivo cyclophosphamide administration to eliminate proliferating xenoreactive T-cell clones and (3) using major histocompatibility complex (MHC) class I and II-deficient NSG mice: (Kb Db)null (IA)null (MHC-dKO NSG). Body weight and plasma murine alanine aminotransferase levels were measured as indicators of xGVHD and tumor size monitored. The antitumor effects of nivolumab plus ipilimumab and an anti-epithelial cell adhesion molecule (EpCAM)/CD3 bispecific antibody (αEpCAM/CD3 BsAb) were evaluated.
CD4+ T-cell depletion and early cyclophosphamide attenuate xGVHD but also abrogate the antitumor activity. In contrast, xGVHD was significantly attenuated in MHC-dKO NSG recipients, while the antitumor effect of human PBMCs was preserved. Furthermore, the administration of nivolumab plus ipilimumab caused exacerbated xGVHD in conventional NSG mice, precluding the observation of their antitumor effects. Severe xGVHD did not occur in MHC-dKO NSG mice, enabling the study of complete and durable tumor rejections. Similarly, NSG mice treated with an αEpCAM/CD3 BsAb showed complete tumor regressions, but died due to xGVHD. In contrast, MHC-dKO NSG mice upon treatment with the αEpCAM/CD3 BsAb achieved complete tumor responses without severe xGVHD. A significant proportion of mice rendered tumor-free showed tumor rejection on rechallenge with HT29 cells without further treatment. Finally, tumor-infiltrating CD8+ T-cell number increase, activation and CD137 upregulation were observed upon αEpCAM/CD3 BsAb treatment.
Humanized MHC-dKO immunodeficient mice allow and refine the preclinical testing of immunotherapy agents for which experimentation is precluded in conventional immunodeficient mice due to severe xGVHD.
