HLA-G gene editing: a novel therapeutic alternative in cancer immunotherapy

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLAG was reached to different degrees, including complete silencing. Most importantly, HLA-G – cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.Fil: Palma, María Belén. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citología, Histología y Embriología; Argentina. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Tronik-Le Roux, Diana. Saint-Louis Hospital; Francia. Université de Paris; Francia.Fil: Amín, Guadalupe. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Castañeda, Sheila. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Möbbs, Alan M. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Scarafia, María Agustina. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: La Greca, Alejandro. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.Fil: Daouya, Marina. Saint-Louis Hospital; Francia. Université de Paris; Francia.Fil: Poras, Isabelle. Saint-Louis Hospital; Francia. Université de Paris; Francia.Fil: Inda, Ana María. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citología, Histología y Embriología; Argentina. Comisión de Investigaciones Científicas; Argentina.Fil: Moro, Lucía Natalia. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Carosella, Edgardo D. Saint-Louis Hospital; Francia. Université de Paris; Francia.Fil: García, Marcela N. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citología, Histología y Embriología; Argentina.Fil: Miriuka, Santiago G. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citología, Histología y Embriología; Argentina. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The immune-checkpoint HLA-G/ILT4 is involved in the regulation of VEGF expression in clear cell renal cell carcinoma

Background: Clear cell renal cell carcinoma (ccRCC), the most aggressive renal cancer, is characterized by early lymph node metastases and bad prognosis. Most therapies targeting advanced or metastatic ccRCC are based, as first-line treatment, on the administration of the vascular endothelial growth factor (VEGF) neutralizing antibody termed Bevacizumab. Despite proven benefits, the expected results were not obtained for the majority of patients. The possibility that an intricate interplay between angiogenesis and immune-checkpoints might exist lead us to evaluate tumor angiogenesis, by means of VEGF expression together with the immune checkpoint HLA-G/ILT4. Methods: Tumor specimens were obtained from patients from two separate cohorts: One from “Evita Pueblo” Hospital from Berazategui, (Buenos Aires, Argentina) and the second includes patients surgically operated at the Urology Department of Saint-Louis Hospital (Paris, France) with a confirmed ccRCC diagnosis. Immunohistochemistry was performed with specific antibodies directed against HLA-G, VEGF-A, VEGF-C, D240, CD34, ILT4 and Ca-IX. In addition, gene expression levels were measured in a cell line derived from a ccRCC patient by semi-quantitative RT-PCR. Results: Our results show that the highly vascularized tumors of ccRCC patients express high levels of VEGF and the immune-checkpoint HLA-G. In addition, ILT4, one of the HLA-G receptors, was detected on macrophages surrounding tumor cells, suggesting the generation of an immune-tolerant microenvironment that might favor tumorigenesis. Notably, RT-qPCR analysis provided the first evidence on the transcriptional relationship between HLA-G/ILT4 and the VEGF family. Namely, in the presence of HLA-G or ILT4, the levels of VEGF-A are diminished whereas those of VEGF-C are increased. Conclusions: In an effort to find new therapeutic molecules and fight against metastasis dissemination associated with the poor survival rates of ccRCC patients, these findings provide the rationale for co-targeting angiogenesis and the immune checkpoint HLA-G.Facultad de Ciencias Médica

Two isoforms of the kidney androgen-regulated protein are encoded by two alleles of a single gene in OFl mice

SummaryTwo cDNA clones coding for two forms of the mouse kidney androgen-regulated protein (KAP) distinguished by their electrophoretic mobilities on SDS gel electrophoresis have been isolated from libraries prepared from strains of mice having one (BALB/c) or two (OFl) forms of the KAP protein. The corresponding mRNAs have identical sizes, as well as identical sequences in their 5' non-translated regions. The size difference observed between the two proteins is due to two point mutations in the coding region of the KAP mRNA, leading to two amino-acid changes one of which resulted in the substitution of a glycine for a glutamic acid. As shown byin vitrotranscription/translation experiments, these two amino-acid differences are responsible for the shift in the apparent molecular weight of the protein on SDS gels. Both forms of the protein are more abundant in males than in females.In vitrotranslation of kidney RNAs isolated from six different strains and species of mice revealed the presence of other forms of the KAP protein, characterized by small variations of their molecular weights. Southern blot analysis data are consistent with the presence of only onekapgene in the mouse genome. A restriction fragment length polymorphism has been observed, which does not correlate with the protein polymorphism, indicating the presence of another allele in the OF1 mouse genome.</jats:p