CD70‐targeting CAR‐T cells have potential activity against CD19‐negative B‐cell Lymphoma
Authors: Wenhai Deng, Panpan Chen, Wen Lei, Yang Xu, Nengwen Xu, Jeffrey J. Pu, Aibin Liang, Wenbin Qian
Journal: Cancer Communications (2021)

To the Editor: CD19-targeting chimeric antigen receptor T (CAR-T) cell therapy is a revolutionary immunotherapy in treating relapsed or refractory B lineage malignancies. However, relapse with CD19-negative tumor after treatment with anti-CD19 CAR-T cells has been reported in different types of B-cell lymphoid malignancies, with a percentage exceeding 10% in patients with acute lymphoblastic leukemia [1] and up to 38% in patients with non-Hodgkin lymphoma (NHL) [2]. Thus, antigen escape constitutes a significant obstacle for anti-CD19 CAR-T therapy, underscoring the need to develop CAR-T cell therapies directed to alternative antigens. CD70, the membrane-binding ligand of the CD27 (a tumor necrosis factor receptor superfamily), has been reported to be expressed on the malignant cells of diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL) and follicular lymphomas (FL), as well as Hodgkin's lymphoma, Waldenström macroglobulinemia, and multiple myeloma, but rarely on normal B cells or T cells [2-4]. In addition, CD70 is also expressed in various solid tumor types, such as renal cell carcinoma and mesothelioma [3]. Recently, targeting CD70 has emerged as potential novel immunotherapeutic strategy [4-6]. Here, we report on the development of novel, second-generation CAR-T cells against CD70, a target for CAR-T cell therapy of B-cell lymphoma that has not been fully realized. CAR-T cell-based cancer immunotherapy relies on not only the specificity but also association constant of single-chain variable fragment (scFv). The scFv used in the CAR is synthesized from parent antibody by fusing their light- (VL) and heavy (VH)-chain variable domains into a single chain using a peptide flexible linker. A previous study demonstrated that the CAR targeting CD79b, in which the scFv was synthesized in a light-heavy (L/H) orientation of variable domain, had superior antigen-specific effector functions than that of the CAR with heavy-light (H/L) orientation of the variable domain [7]. Using this strategy, we generated two second-generation anti-CD70 CAR constructs, with either a L/H or a H/L scFv configuration derived from a reported anti-CD70 monoclonal antibody, denoted as anti-CD70 (L/H) or anti-CD70 (H/L), respectively (Figure 1A). We also generated a CAR consisting of truncated CD27 (without its intracellular signaling domain) (Figure 1A) coupled with the co-stimulatory domain of 4-1BB and then CD3ζ signaling domain (denoted as trCD27), which had previously been shown to have CD70-specific tumor recognition [8]. In contrast to solid tumor cell lines, CD70 mRNA was highly expressed in NHL cell lines such as 60 B-cell lymphoma cell lines (Supplementary Figure S1A). Flow cytometry analysis confirmed the high expression of CD70 protein in B-cell lymphoma cell lines Raji, JeKo-1, Mino, and WSU-DLCL2 compared with K562 cells serving as a CD70-negative control (Supplementary Figure S1B). We next characterized three types of anti-CD70 CAR-T cells lentivirally transduced to express anti-CD70 (L/H), anti-CD70 (H/L) or trCD27 CAR constructs, and assessed their effector functions. The anti-CD19 CAR, as the control lentiviral vector, was also generated. The representative transduction efficiency of anti-CD70 (L/H), anti-CD70 (H/L), trCD27, and anti-CD19 CAR was 55.1%, 44.5%, 74.5%, and 74.5%, respectively (Supplementary Figure S2). Proliferation of CAR-T cells following stimulation with anti-CD3/CD28 beads was measured. The anti-CD70 (L/H) CAR-T cells showed a lower rate of ex vivo expansion, while anti-CD70 (H/L), trCD27, and anti-CD19 CAR-T cells showed significantly greater proliferative capacity (Figure 1B). It has been well known that some scFvs are associated with ligand-independent tonic signaling, which could contribute to the reduction of efficacy of CAR-T therapy by leading to terminal effector T-cell differentiation and exhaustion [1]. We therefore compared the subsets of anti-CD70 (L/H), anti-CD70 (H/L), trCD27, or anti-CD19 CAR-T cells at 12 days after culture with anti-CD3/CD28 beads. The similar subset distribution of T cells was observed in anti-CD70 (L/H), anti-CD70 (H/L), and trCD27 CAR-T cells. However, anti-CD70 CAR-T cells showed a higher frequency of stem cell memory T cells when compared to anti-CD19 CAR-T cells (Figure 1C and Supplementary Figure S3). Inclusion of costimulatory 4-1BB in anti-CD19 CAR construct had been reported to cause tonic signaling via continuous TRAF2-dependent activation of the NF-κB pathway [9]. We verified significantly increased NF-κB activation in anti-CD70 (L/H) CAR-T cells (Figure 1D). Due to the dysfunction of anti-CD70 (L/H) CAR-T cells characterized by the aberrant proliferation and abnormal NF-κB activation, we chose anti-CD70 (H/L) CAR-T cells to define the antitumor activity of the CAR-T cells targeting CD70 antigen. We performed cytotoxicity assays against CD19- and CD70-positive Raji cells and JeKo-1 cells at an effector-to-target ratio of 20:1 for 4 h, and CD107a degranulation and cytokine expression by CAR-T cells were also determined. The T cells transduced with anti-CD70 (H/L) and anti-CD19 CAR constructs demonstrated comparable cytolytic activity and CD107a degranulation against B lymphoma cell lines (Supplementary Figure S4A and S4B). In consistent with these results, the pattern of cytokine production in response to Raji and JeKo-1 cells, especially with upregulation of Th1 cytokines IFN-γ and TNFα, was similar in anti-CD70 (H/L) and anti-CD19 CAR-T cells (Supplementary Figure S4C). We also detected that anti-CD70 (H/L) CAR-T cells, but not BFP-transduced T cells, effectively killed CD19-negative Raji cells (Supplementary Figure S4D). Next, anti-CD70 (H/L) CAR-T cells showed comparable killing efficacy when co-cultured with CD19-positive and CD19-negative Raji cells, respectively (Supplementary Figure S4E). We finally used anti-CD70 (H/L) CAR-T cells to test therapeutic efficacy against both CD19-positive and CD19-knockout Raji xenografts in NSG mice because our in vitro assays demonstrated superior ex vivo growth functions of anti-CD70 (H/L) CAR-T cells compared to anti-CD70 (L/H) CAR-T cells (Figure 1B). Anti-CD70 (H/L) CAR-T cells and anti-CD19 CAR-T cells were injected into NSG mice 3 days after intravenous injection of CD19-positive Raji cells. Tumor burden was assessed by bioluminescence imaging, which showed complete elimination of lymphoma by day 12 for anti-CD19 CAR-T cells and day 25 for anti-CD70 (H/L) CAR-T cells (Figure 1E). To evaluate the ability of anti-CD70 (H/L) CAR-T cells to prevent antigen escape, NSG mice were engrafted with CD19-knockout Raji cells. Tumor-bearing mice were administrated with one intravenous infusion of anti-CD19 or anti-CD70 (H/L) CAR-T cells. The bioluminescence imaging results revealed that tumors were significantly suppressed in mice treated with anti-CD70 (H/L) CAR-T cells, yielding near-complete tumor clearance (Figures 1F and 1G), which highlights the potential effect of anti-CD70 (H/L) CAR-T cells against CD19-negitive B-cell malignancies. Conversely, there was no significant difference of tumor burden between mice treated with anti-CD19 CAR-T cells and T cells without CAR transduction. Moreover, anti-CD70 (H/L) CAR-T cell-treated mice also demonstrated significantly prolonged survival compared with the other two groups (Figure 1H). Herein, we report on the development of a novel CAR product targeting CD70 using an anti-CD70 scFv and demonstrated potential antitumor effects of anti-CD70 CAR-T cells in CD19-positive and -negative B-cell lymphoma models. However, CD70 expression levels in tumor cells were different amongst the B-cell tumor types. For example, CD70 positivity has been reported in 33% of FL cases, 25% of MCL, and 71% of DLBCL [3, 4]. One potential way to combat the problem of antigen escape following CAR-T cell therapy is to target alternative or more than one antigen receptor, and CARs targeting both CD19 and CD20 in B-cell lymphoma are in clinical development [10]. Together, our findings suggest that anti-CD70 CAR-T cells represent a new therapeutic option for the treatment of patients who have CD19-negative recurrence of lymphoma. Future strategies combining dual targeting of CD19 and CD70 are warranted. The animal experiments were approved by the Institutional Animal Care and Use Committee, Zhejiang University. The studies involving human participants were reviewed and approved by an independent Ethics Committee of The Second Affiliated Hospital, College of Medicine, Zhejiang University. The participants provided their written informed consent to participate in this study. Not applicable. The raw data and analyses supporting the conclusions of this article are openly provided upon request, to any qualified researcher. This study was supported by funds from Translational Research Grant of NCRCH(2020ZKZC01), and the National Natural Science Foundation of China (No. 81830006, 81800315). Conception, design and manuscript writing: W. Qian, A. Liang, J. J. Pu, and W. Deng. Collection, analysis, and interpretation: W. Deng, P. Chen, W. Lei, Y. Xu and N. Xu. All authors were involved at each stage of manuscript preparation and approved the final version. All authors declare no competing interests. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.