Fig 1: B cell aptamer interaction with CD22 protein(A) B-ALL aptamer (B-ALL1–B-ALL5 and B-ALL31) and control aptamer (Cont.) specificity for CD22 was evaluated using an aptamer qRT-PCR internalization assay with CHO cells expressing CD22 (CHO 22+) and wild-type CHO cells not expressing CD22 (CHO WT). Data are plotted as mean ± SEM; n = 3 biological replicates; mixed effect analysis p > 0.005 with post hoc Bonferroni’s multiple-comparisons test; ∗∗∗p < 0.0001. (B) Dose dependence of B cell aptamer B-ALL1 and Cont. aptamer for CD22. Data are plotted as mean ± SEM; n = 3 biological replicates; 2-way ANOVA, p < 0.0001 with Sidak’s multiple-comparisons post hoc test; ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.0001. (C) Knockdown of CD22 expression by treatment with anti-CD22 siRNAs. CD22 was detected by anti-human CD22 (green), the cytoplasm was stained with AF568-phalloidin (red), and the nucleus was visualized by TOPRO-3 (blue). Scale bar, 20 μm. (D) qRT-PCR aptamer internalization assay of aptamers B-ALL1, B-ALL2, and Cont. aptamer with CHO 22+ cells and CHO 22+ (siRNA-treated) cells. Data are plotted as mean ± SEM; n = 3 biological replicates; 2-way ANOVA, p < 0.0001 with Bonferroni’s multiple-comparisons post hoc test; ∗∗∗p < 0.0001. (E) B-ALL1 aptamer and Cont. aptamer specificity for recombinant histidine-tagged CD22 or control histidine-tagged protein (glyoxalase) in the presence of epratuzumab or a control IgG1 (bevacizumab). Data are plotted as mean ± SEM; n = 4 biological replicates; one-way ANOVA with multiple-comparisons post hoc test; ∗∗p < 0.001, ∗p < 0.05.
Fig 2: In silico docking between CD22 and candidate aptamers(A) The number of H-bonds after 30 ns of molecular dynamics simulation between candidate B cell aptamers and the epratuzumab region of the CD22 protein. The top five selected candidates (aptamers B-ALL1–B-ALL5), and the most abundant aptamer observed during B-ALL cell internalization SELEX (aptamer B-ALL31) were identified (red) for experimental validation. (B) Predicted secondary structure of aptamers B-ALL1–B-ALL5 and B-ALL31 using the RNA structure.109
Fig 3: CAR T analysis by flow cytometry(A) Flow cytometric analysis of CAR expression on transduced primary human T cells from 3 donors by CD19-Fc and CD22-His showed no CAR expression on UTD cells (upper right quadrant) and 40%–60% of engineered T cells co-expressing both CAR molecules.(B) All donors maintained high viability, with mean ± standard deviation (SD) expression plotted for each marker, as well as the combined signal (x axis). Data are represented as mean ± SD.
Fig 4: RNAsky-based transcript detection(A) Padlock probes are synthetic oligonucleotides that contain two anchor regions that hybridize to target sequence of interest and a backbone region (black curve) that contains a pre-defined barcode sequence. Sixteen padlock probes were designed to target specific regions of the CAR construct.(B) The CD19.20.22 CAR construct comprises 11 regions, including a tandem CD19.20 binder (binding 1) and a CD22 binder (binding 2), separated by the ribosomal skip element 2A to facilitate equal expression of the two CARs. Each CAR comprises a leader peptide (LP), target-binding region, hinge region, transmembrane region, and intracellular signaling domain region (ICD).(C) RNAsky preparation requires seven steps of hybridization of padlock probes, ligation of the probe, RCA (rolling circle amplification) reaction, rolonies detection, rolonies localization, secondary antibody staining, and data quantification and analysis using MACS iQ View software (Miltenyi Biotec).RNAsky assay detects CAR RNA transcripts in a sequence-specific manner using fluorescence microscopy.
Supplier Page from Thermo Fisher Scientific for Recombinant Human Siglec-2 (CD22) His-tag Protein