Efficacy Proof of Concept for Allogeneic CD123 Targeting CAR T-Cells Against Primary Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Efficient Control of Tumor Progression in PDX Model and Potential Loss of CD123 Expression in Relapsed Disease

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy with historically poor outcomes and no established standard of care. Nearly 100% of patients with BPDCN overexpress CD123, and targeting CD123 has therefore emerged as an attractive therapeutic target.

UCART123v1 is an allogeneic "off the shelf" product composed of genetically modified T-cells expressing an anti-CD123 CAR and a RQR8 depletion ligand, which confers susceptibility to rituximab. The expression of the T-cell receptor αβ (TCRαβ) is abrogated through the inactivation of the TRAC gene, using Cellectis' TALEN^® gene-editing technology.

We have previously reported the selective in vitro anti-tumor activity of UCART123v1 cells against primary BPDCN samples using cytotoxicity and T-cell degranulation assays, as well as the secretion of IFNγ and other cytokines (IL2, IL5, IL6, IL-13 and TNF-α) by UCART123v1 cells when cultured in the presence of BPDCN cells (Cai et al, 2017 ASH).

To evaluate anti-tumor activity of UCART123v1 cells in vivo, we established two relapsed BPDCN patient-derived xenografts (PDX1 and 2) in NSG-SGM3 mice. In PDX-1 model, mice were randomized upon tumour engraftment (D21 after primary BPDCN injection) into 4 groups and received an IV injection of either vehicle, 10×10⁶ TCRαβ KO control T-cells, or UCART123v1 cells (3×10⁶ or 10×10⁶ cells). Mice from vehicle group died by D53 after BPDCN injection with high tumor burden in PB, spleen and BM. 3 out of 9 (33%) mice treated with 3×10⁶ and 6 out of 9 (67%) mice treated with 10×10⁶ UCART123v1 were alive and disease-free at the end of the study (D299 after primary BPDCN injection).

In PDX-2 model, which received the same treatment as PDX-1 (at D19 after primary BPDCN cell injection), all vehicle-treated mice died by D49. UCART123v1 therapy extended survival of treated mice to 104-241 days, but tumors relapsed at 90-155 days (Fig. 1A). The relapses in UCART123v1 treated mice were associated with the emergence of CD123-, CD56+CD45+ BPDCN cells infiltrating spleens and BMs (Fig. 1B).

To understand the molecular basis for CD123 loss, we isolated RNA from CD123+ cells from two of the vehicle-treated mice and CD123- cells from four of the UCART123v1-treated mice and performed RT-PCR and RNA-sequencing. The cells from all samples were hCD45+ and hCD56+, indicating leukemic origin. These analyses detected the presence of full-length transcripts (exons 2-12) in both CD123+ control samples (Sample 1 and 2in Fig. 1C). In 2 of the 4 CD123- samples, CD123 transcripts were absent, as were transcripts of neighbouring genes (samples 3 and 9 in Fig. 1C). RNA-sequencing reads aligned to Genome Browser tracks for CD123 and housekeeping gene GPI showed no reads present for CD123 but reads present for GPI in the two samples with CD123 loss. The aCGH (Array‐Based Comparative Genomic Hybridization) results showed that samples 3 and 9 (CD123-) had large regional deletions on chromosome X, which includes the CD123 gene. In another sample (sample 5), the splicing analysis algorithm MAJIQ detected CD123 transcripts containing only exons 2-9, indicating premature transcription termination. If translated, this truncated transcript would produce a protein isoform lacking the transmembrane domain in exon 10. Finally, MAJIQ also revealed canonical splicing of exon 2 to exon 3 in all CD123⁺ samples but a sharp increase in skipping from exon 2 to exon 5 in sample 16 (Fig. 1D). This exon-skipping event preserves the open-reading frame and yields the previously reported transcript variant 2. Per UniProt, the resultant protein will retain the ligand-binding domain but lack several glycosylation sites and two beta sheets in the extracellular domain, potentially compromising recognition by UCART123v1 cells. The aCGH and FISH results further showed that this patient sample harbored TP53 deletion, which could have contributed to DNA instability observed in different mice engrafted with these tumor cells.

In summary, allogeneic anti-CD123 CAR T therapy resulted in eradication of BPDCN in vitro and in increased disease-free survival in primary BPDCN PDX models. However, CD123 loss was observed in one PDX model harboring a TP53 deletion. These results provide preclinical proof-of-principle that UCART123v1 cells have potent anti-BPDCN activity, and indicate potential mechanisms leading to antigen loss and disease relapse.

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