Fig 2: Effects of treatment with urelumab variants on human monocytes and NK cells in the peripheral blood of humanized mice. Humanized mice were injected with 3µg/g (hIgG4: n=3-4; αCD137: n=6; αCD137 PNGaseF: n=4) (A, C, E) or 6µg/g (hIgG4: n=7; αCD137: n=5; αCD137 PNGaseF: n=4) (B, D, F) of the human IgG4 isotype control, urelumab (αCD137), or deglycosylated urelumab (αCD137 PNGaseF) and studied for twenty days after antibody injection. (A, B) Shown are the relative changes in CD33+ monocyte numbers (left panel) as well as in CD137 expression (right panel, ΔMFI) on CD33+ monocytes at the indicated time-points after injection of 3 (A) or 6 (B) µg/g of the respective antibody preparations. (C-F) Shown are the relative changes in CD56+CD16- (C, D) and CD56+CD16+ (E, F) NK cell subset abundance (left panel) as well as in CD137 expression (right panel) on the respective NK cell subsets at the indicated time-points after injection of 3 (C, E) or 6 (D, F) µg/g of the respective antibody preparations. Results are presented as mean +/-SEM. For statistical testing a 2-Way Anova with Tukey’s multiple comparison test was used. *p<0.05; **p<0.01.

Fig 3: Differences in peripheral blood mononuclear cell (PBMC) counts between lung cancer patients and healthy controls. (a) Flow cytometry was performed to detect myeloid cell subsets in PBMCs and analyze the proportions of monocytes (CD11b+) and myeloid‐derived suppressor cells (MDSCs; HLA‐DR−CD11b+CD33+) among CD45+ cells. The results are presented using scatter plots. (b) Differences in the proportions of monocytes, eosinophils, and MDSCs in PBMCs between lung cancer patients and healthy controls. (c) Correlations between monocyte and eosinophil counts in PBMCs in lung cancer patients. (d) Correlations between histories of smoking and drinking and monocyte counts in PBMCs in lung cancer patients. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 4: In vivo evaluation of CD33 targeted SdAb-based CAR-T cells on mouse xenograft AML model(A) Schematic representation of the optimized procedure on NGS mice created with BioRender.com. (B) Kaplan-Meier survival curves of low-dose (0.5 × 106 CAR-T cells/mice), intermediate-dose (1.5 × 106 CAR-T cells/mice), and high-dose (3 × 106 CAR-T cells/mice) treatments of AML (MOLM13) xenografted mice (n = 8). For statistical analysis, survival curves were compared using the Log rank (Mantel-Cox) test, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) In vivo tumoral progression in AML (MOLM13 luciferase+) xenografted mice treated with SdAb/ScFv-based CAR-T cells, and UTD T cells. Top panel: luminescence images of a sample of the treated mice (two males on the left and two females on the right), injected with luciferin at different time points. Bottom panel: progression of MOLM13 cells on each mouse, measured as luciferase activity (Total Flux), and corresponding survival curves, analyzed using the Log rank (Mantel-Cox) test, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 5: Identification of CD33-specific SdAbs(A) CD33 indirect ELISA curves of llama plasma at different stages during immunization (percentage of signal vs. plasma dilution factor [DF]). (B) Output/input (O/I) phage ratio for each panning round. (C) Phage ELISA of the original library and the final output AO3. (D) Preliminary ELISA screening of isolated E. coli clones infected with bacteriophages. As positive control, one positive clone from a previous round output (C+) was offered. (E) Final ELISA screening of isolated E. coli clones expressing the SdAbs from the expression vector pETMod. The positive control (C+) was C4 from the preliminary screening, and the negative control was an irrelevant SdAb-expressing clone culture supernatant from a panning round against another target. (F) Sequence alignment of the five candidate SdAbs selected for further characterization. (G) Phylogenetic tree of the identified SdAb sequences against CD33, clustered into five families based on half distance. (H) Coomassie blue-stained SDS-PAGE and anti-HA Western blot of the IMAC and IEC purified SdAbs. (I) Cross reactivity test (ELISA) of the five candidate SdAbs against antigens from the same llama library.