CD70 Deregulation in Follicular Lymphoma at Diagnosis Is Associated with Relapse and Opens New Avenues for Dual CD19-CD70 CAR-T Therapy
Authors: Araujo-Ayala Ferran , Ros Maria , Bachiller Mireia , Alexandru Raluca , Mozas Pablo , Colell Salut , Mateos-Jaimez Judith , López Cristina , Villalba-Esparza María , Nadeu Ferran , Rivero Andrea , Dobaño-López Cèlia , Valero Juan Garcia , Serrat Neus , Gimenez-Alejandre Marta , Giró Ariadna , Hodson Daniel J , Lopez-Guillermo Armando , Campo Elias , Colomer Dolors , Maiques-Diaz Alba , Martin-Subero Jose Ignacio , Bea Silvia , Magnano Laura , Guedan Sonia , De Andrea Carlos Eduardo , Perez Galan Patricia 
Published: December 2024

Follicular lymphoma (FL) is the most common indolent B-cell lymphoma. Although achieving a complete response with first-line treatment, most of the patients eventually relapse, which worsens the prognosis and increases the risk of histological transformation to aggressive lymphoma. Therefore, there is the need to better stratify patients at diagnosis and develop tailored therapies for high-risk patients.
Here we assessed the immune profile (Nanostring® nCounter) in formalin-fixed paraffin-embedded (FFPE)-derived biopsies in a series of FL patients at diagnosis, homogenously treated with immunochemotherapy (mostly R-CHOP), and followed at Hospital Clínic de Barcelona. By comparing patients who experienced relapse (n=20) with those who did not relapse after a median follow-up of 14.1 years (n=12), we found 31 differentially expressed genes, 25 of them upregulated in the relapsed group. Among them, we focused on the role of CD70 in FL pathogenesis. CD70 mRNA was higher in patients who eventually relapsed and was associated with inferior progression-free survival (PFS). Moreover, when analyzing the presence of recurrent somatic mutations CD70low cases more frequently bear mutations in CARD11, CIITA, and TNFRSF14 genes.
We next sought to determine the specific subpopulations expressing CD70 by multiplex immunofluorescence (Vectra Polaris). Interestingly, we demonstrated that CD70 is mainly expressed in tumor cells, and it is associated with inferior PFS of the patients. Moreover, a fraction of T cells from the tumor microenvironment also expressed CD70. Intriguingly, higher levels of CD70 were detected in both CD4+ and CD8+ T cells in the relapsed group, including T regulatory and T follicular helper cells. CD70 interacts with CD27, its only known ligand, and can be mutually activated. While CD27 RNA levels were comparable between relapsed and non-relapse groups, when analyzing the CD27 protein, those patients who will eventually relapse, display high CD27 protein levels in B and TFH cells, while low CD27 levels in CD8+ and CD4+ T helper non-follicular cells. Furthermore, we found a correlation between the percentage of CD70+ cells within the B cell population and CD70+ in CD4+ and CD8+ cells (R=0.51, p &lt; 0.05).
To investigate the role of CD70 in FL pathogenesis, we generated CD70 knock-out (CD70-KO) cell lines (n=2) using CRISPR/Cas9. Then T cells from healthy donors were co-cultured with the original FL cell line (CD70+) or the generated CD70-KO FL cells, and we observed that CD70+ tumor cells promoted CD70 expression in T cells. Moreover, CD70 CRISPR/Cas9 in primary samples (n=3), showed that CD70-KO tumor cells exhibit reduced response to proliferative stimuli (CD40L, IL-4 and IL-21). Finally, to advance towards personalized therapies for these high-risk patients and taking advantage of the CAR-T program at our institution, we generated a dual CD19-CD70 CAR-T combining an approved academic product, ARI-0001 (Martinez-Cibrian N, Br J Haematol 2024), and a truncated CD27 protein. We demonstrated that this novel CAR-T is effective in both cell lines and ex vivo primary FL samples, improving tumor depletion in CD19dim samples. Nevertheless, we observed that CAR-T activity is limited by fratricide, which may be overcome by knocking-out CD70 expression in CAR-T cells. Ongoing work includes the optimization of the CRISPR protocol during the CAR-T generation and in vivo studies.
In summary, this multipronged study has allowed us to uncover the prognosis potential of CD70, its biological role in FL patients and the generation of a novel dual CD19-CD70 CAR-T with therapeutic potential in FL as well as other B-NHL.