To the Editor:
Despite new drug development and improved understanding of molecular pathways leading to acute myeloid leukemia (AML), the cure rate of the disease is still poor. For high-risk patients or in relapse, the only long-term curative therapy is still allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is not feasible in a considerable number of patients and poses significant risks in morbidity and mortality. New strategies are urgently needed to improve the cure rate in AML.
The success of chimeric antigen receptor (CAR)-modified T-cells directed to the CD19 antigen for the treatment of lymphoid malignancies of B-cell lineage boosted the research on CAR T-cells targeting myeloid antigens, such as CD33, CD123, and Fsm-like tyrosine kinase 3 (FLT3) [1, 2]. FLT3, a tyrosine kinase receptor thought to be specific to hematopoietic lineage, is an attractive target especially due to overexpression in AML blasts. In the past few years, several independent groups developed CAR T-cells targeting FLT3 with promising in vitro results [3,4,5]. The expression of FLT3 in HSCs might indeed cause concern [5]. Recent publications have shown CD34+-HSC are less susceptible to CAR T-cell treatment compared to AML cells [3, 4]. This is likely due to different affinities of the antibodies used for CAR generation, suggesting different CARs might have different toxicities in vivo.
T-cell receptor (TCR)-modified T-cell therapy is an alternative form of adoptive T-cell therapy. Unlike CARs, which recognize cell surface antigens, TCRs recognize peptides derived from any cellular proteins presented by major histocompatibility complex (MHC) molecules. Therefore, TCRs should not be affected by down modulation of surface antigen expression when intracellular expression is retained, which is an escape mechanism in CAR therapies. We have generated an HLA-A2-restricted, FLT3-specific TCR to target AML cells with the aim of using it in the context of HLA-A2-mismatched allo-HSCT. In the proposed set-up, FLT3-directed CD8+ T-cells would kill any remaining HLA-A2+/FLT3+ cells possibly eradicating all leukemic cells while allowing the engraftment and establishment of an HLA-A2-, leukemia free hematopoiesis.
It is not possible to generate optimal-affinity TCRs against self-antigens in humans because of thymic selection. To circumvent tolerance and develop optimal-affinity TCR against FLT3, we immunized mice expressing a diverse human TCR repertoire and a mouse CD8-binding HLA-A2 molecule (ABabDII) with an in silico-predicted FLT3 epitope [6]. The FLT3982-991 peptide GLLSPQAQV (GLL), with an affinity of 56 nM to HLA-A2 predicted by NetMHC 3.4, was selected for immunizations (Fig. 1a). ABabDII mice were immunized and boosted with GLL peptide and peripheral blood was analyzed for peptide-specific T-cells 7 days after immunization. Reactive CD8+ T-cells were collected from inguinal lymph nodes and spleen of two mice (#6780 and #6782) and expanded with peptide in vitro for 10 days. By an IFN-γ capture assay, 2,500 and 65,300 IFN-γ+-CD8+ T-cells were sorted, respectively (Supp. Figure 1). TCRs were isolated by RACE-PCR and Vα and Vβ pairs were identified based on their abundance (Supp. Table 1). We constructed codon-optimized TCR cassettes with mouse constant region for better expression in human T-cells (named TCR-6780 and TCR-6782) in the γ-retroviral vector pMP71 as described [7].
Following retroviral transduction, GLL-specific TCRs were expressed in human CD8+ T-cells, as confirmed by staining against the mouse constant TCRβ region (mTRBC) and FLT3982-991-HLA-A2 tetramers (Fig. 1b). Transduced T-cells were co-cultured with T2 cells loaded with decreasing concentration of GLL peptide at 1:1 ratio overnight, IFN-γ was detected in the supernatant by ELISA. Both TCR-6780 and TCR-6782 recognized GLL peptide at a concentration as low as 10 nM (Fig. 1c). Half-maximum IFN-γ release at 17 and 100 nM peptide concentrations, respectively, indicates TCR-6780 has a higher functional avidity, although the affinity still appears to be suboptimal.
We generated K562 cell lines expressing HLA-A2, FLT3, or both by γ-retroviral transduction for co-cultivation assays (Supp. Figure 2). TCR-transduced T-cells recognized K562/HLA-A2 cells loaded with the GLL peptide (Fig. 1d). TCR-6780-transduced T-cells secreted IFN-γ to K562 cells co-expressing HLA-A2 and FLT3, proving the epitope was naturally processed and presented while TCR-6782-transduced T-cells did not secrete IFN-γ, indicating a lower functional TCR avidity (Fig. 1d). Next, TCR-6780 and TCR-6782 transduced T-cells were co-cultured with cells expressing FLT3 endogenously (THP1 is derived from AML patient; MV;4-11 is a mixed-lineage myelomonocytic leukemia line carrying the 4;11 chromosomal translocation; SEM is a B-cell precursor leukemia cell line). SEM and MV;4-11 cells were retrovirally transduced to stably express HLA-A2. The target cells were labeled with 1 µM CFSE prior to overnight co-culture with TCR-transduced CD8+ T-cells and CD137 upregulation specifically on TCR-transduced CD8+ T-cells was analyzed (Supp. Fig 3 for gating strategy). TCR-6780-transduced T-cells recognized cell lines co-expressing FLT3 and HLA-A2 to a different extent. 1.8% of TCR-6780-transduced CD8+ T-cells upregulated CD137 in response to THP1 cells in contrast to 25.33% in response to THP-1 overexpressing FLT3. SEM and MV;4-11 were recognized when they expressed HLA-A2 only (14.9% and 14.3%, respectively). TCR-6782-transduced CD8+ T-cells did not upregulate CD137, likely because of low avidity (Fig. 1e). We could not detect IFN-γ from effector cells upon co-culture with target cells indicating low avidity of both TCRs (data not shown).
Several FLT3 inhibitors are being used for the treatment of AMLs without life-threatening side effects or toxicity [8]. Anti-FLT3 monoclonal antibodies have entered a clinical trial in AML. TCR-engineered T-cells are more potent in terms of killing target cells. Theoretically, a single molecule of the antigen presented on the MHC can elicit T-cell toxicity [9]. Hence, TCR-mediated on-target recognition of vital tissues may induce severe toxicity [10,11,12]. So far, there was no evidence in the literature that non-hematopoietic human cells express FLT3 and Flt3 knock-out mice develop normally but have an impaired lymphoid progenitor cell compartment [13], suggesting FLT3 may indeed be specific to the hematopoietic compartment.
To assess the safety of FLT3 as a target antigen, we used a cDNA library representing 48 healthy human tissues. We first verified the sensitivity of the PCR reaction on a titration of FLT3+ THP1 cell line (Supp. Fig 4a). In RT-PCR, a 236 bp FLT3 amplicon was detected in several tissues including the brain (Supp. Fig 4b). Testing an additional cDNA library spanning 24 tissues of the central nervous system (CNS) by RT-PCR revealed that FLT3 was highly expressed in the cerebellar hemispheres and vermis (Fig. 2a). Detected PCR products were confirmed to be FLT3 by Sanger sequencing (Supp. Fig 4c), thus, unveiling those sites as potential sites of on-target off-tumor toxicity.
Next, we analyzed postmortem human brain tissue. Total protein and total RNA from three different individuals were isolated from the cerebellar vermis (CV) and cerebellar hemispheres (CH). FLT3 gene expression was verified by RT-PCR (Fig. 2b). FLT3 is synthesized and glycosylated in the endoplasmic reticulum (ER), then transported to the cell membrane. The membrane-bound, glycosylated FLT3 is detected as 160 kDa band while ER-bound immature FLT3 is 135 kDa. We detected only 135 kDa immature FLT3 in the cerebellar lysate by western blot analysis suggesting FLT3 is found primarily intracellular, most likely bound to the ER membrane in cerebellum tissue (Fig. 2b). In mice, Flt3 has been detected in the mouse cerebellum, specifically in Purkinje cells (PC) [14]. Immunohistochemical (IHC) and immunofluorescent (IF) staining were used to further elucidate which cerebellar cell type expresses FLT3. Immunofluorescent co-staining of CD56 (neural cell adhesion molecule (NCAM)) and FLT3 revealed a cytoplasmic and intracellular expression of FLT3 in Purkinje cells, confirming the western blot results (Fig. 2c).
In conclusion, we generated two TCRs recognizing the self-epitope FLT3982-991 presented in the context of HLA-A2. We showed for the first time that the FLT3982-991 epitope is naturally processed and presented and therefore a potential TCR target. The first TCRs we generated did not show optimal-affinity, hence, could not be used for kill assays on AML cell lines as well as on-target toxicity assessment. However, the unexpected intracellular expression of FLT3 in Purkinje cells appears to bear a risk that is too high to move forward with TCR into the clinic because of the anticipated on-target off-tumor recognition. Purkinje cells integrate afferent signals mainly from the brainstem and send their axons down to the deep cerebellar nuclei. PC degeneration causes coordination deficits, such as limb ataxia, dysarthria, and oculomotor disturbances, as seen in paraneoplastic syndromes. Therefore, we abandoned the effort to generate a TCR with improved affinity by novel immunizations and the whole strategy of targeting FLT3 with TCR-transduced T-cells. Nevertheless, our findings do not necessarily infer that FLT3 is not a suitable target for CAR therapy. First, antibody-based therapeutics may not be efficient enough to kill a cell if the surface expression is weak, while few pMHC complexes might be enough for recognition and killing by TCRs [9, 15]. Second and most important, according to our data, FLT3 is likely to be present intracellularly but not on the surface of human Purkinje cells and is therefore unlikely to be recognized by a CAR. The urgent need for novel therapies in AML might well justify pursuing this strategy in high-risk patients.
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Acknowledgements
This study is funded by SFB-TR36, Berlin Institute of Health and the German Cancer Consortium (DKTK). The authors thank Margarete Gries, Nicole Hellmig, Kiara Freitag, Petra Matylewski, Randi Koll, Fabienne Pritsch, and Francisca Egelhofer for excellent technical assistance. NCG is supported by a grant of the Berlin Institute of Health (BIH) CRG-TP7. JR is a participant of the BIH-Charité Clinical Scientist Program funded by the Charité-Universitätsmedizin Berlin and the Berlin Institute of Health (BIH).
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Çakmak-Görür, N., Radke, J., Rhein, S. et al. Intracellular expression of FLT3 in Purkinje cells: implications for adoptive T-cell therapies. Leukemia 33, 1039–1043 (2019). https://doi.org/10.1038/s41375-018-0330-7
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DOI: https://doi.org/10.1038/s41375-018-0330-7