https://doi.org/10.5061/dryad.wwpzgmsw5

Zhou, Y., Li, C., Chen, X. et al. Development of folate receptor targeting chimeras for cancer selective degradation of extracellular proteins. Nat Commun 15, 8695 (2024). https://doi.org/10.1038/s41467-024-52685-9

Description of the data and file structure

Figure 1: FRTACs mediate the uptake and lysosomal degradation of soluble proteins. d. Uptake of anti-FITC-594 (50 nM) in HepG2 cells treated with Ab-FA (25 nM) and FA-FITC (200 nM) for 3 h (n = 3). e. In-gel fluorescence analysis of anti-biotin-647 (50 nM) internalization and degradation in HepG2 cells by Ab-FA (25 nM) in the presence or absence of Chloroquine (CQ, 20 μM), Bafilomycin A1 (BAF, 200 nM), and MG132 (1 μM) for 24 h (n = 3). f. Cellular uptake and lysosome colocalization of anti-Rabbit-647 (50 nM) in the presence of Ab (25 nM), Ab (25 nM) + free FA (125 nM), and Ab-FA (25 nM) in Hela cells for 24 h by immunofluorescent staining. Scale bar: 50 μm. The colocalization of internalized anti-Rabbit-647 with lysosomes was analyzed by Pearson's correlation coefficients. The intracellular fluorescence intensity is presented as mean fluorescence intensity (MFI) (n = 15 images from three biologically independent experiments). Box plot: minima (lower whisker), maxima (upper whisker), center (median), bounds of the box (25th and 75th percentiles), whiskers (range from minima to maxima). g. Uptake of anti-Rabbit-647 (50 nM) mediated by Ab-FA (25 nM) in Hela cells transfected with scramble siRNA or Rab7 siRNA for 3 h (n = 3).  N indicates biologically independent experiments except for figure 2f.

Figure 2: FRTACs recruit FR to induce lysosomal degradation of soluble proteins. a. Inhibition of anti-biotin-647 (50 nM) internalization in the presence of Ab-FA (25 nM) by free FA (300 μM) in HepG2 cells for 3 h (n = 3). b. Uptake of anti-rabbit-647 (50 nM) mediated by Ab-FA (50 nM) in Hela cells transfected with scramble or FR1 siRNA for 6 h (n = 3). c. Uptake of anti-rabbit-647 (50 nM) mediated by Ab-FA (50 nM) in Hela cells transfected with plasmid expressing FLAG-FR1 for 6 h. Non-transfected (NT) cells and cells transfected with empty vector (EV) were used as negative controls (n = 3). d. Uptake of anti-Rabbit-647 (50 nM) in FR2 overexpression cells. Non-transfected (NT) cells and cells transfected with empty vector (EV) were used as negative controls (n = 3). e. Inhibition of anti-FITC-594 (50 nM) internalization in the presence of Ab-FA (25 nM) by methyl-β-cyclodextrin (MβCD, 20 mM), chlorpromazine (CHP, 5 μg/ml) or cytochalasin D (cyto-D, 5 μM) in Hela cells for 3 h (n = 3). f. Uptake of anti-biotin-594 (50 nM) in different cancer cell lines (n = 3). N indicates biologically independent experiments.

Figure 3: FRTACs mediate the lysosomal degradation of membrane proteins (EGFR, PD-L1, and CD47) via their interaction with FR. b. Dose response of EGFR degradation (24 h) in Fadu cells (n = 3). c. Time course of EGFR degradation mediated by Ctx-FA (10 nM) in Fadu cells (n = 3). d. Immunofluorescent staining of EGFR degradation and lysosome colocalization after treatment of Ctx (10 nM), FA (50 nM), Ctx (10 nM) + free FA (50 nM), IgG-FA (10 nM) and Ctx-FA (10 nM) for 24 h in Fadu cells. Scale bar: 25 μm. The colocalization was analyzed by Pearson's correlation coefficients (n = 15). The intracellular fluorescence intensity is presented as mean fluorescence intensity (MFI)  (n = 10). Box plot: minima (lower whisker), maxima (upper whisker), center (median), bounds of the box (25th and 75th percentiles), whiskers (range from minima to maxima). e. Inhibition of EGFR degradation in the presence of Ctx-FA (10 nM) by free FA (3 mM) and Bafilomycin A1 (BAF, 200 nM) in Fadu cells for 6 h (n = 3). f. Inhibition of EGFR degradation in the presence of Ctx-FA (10 nM) by MG132 (3 μM) in Fadu cells for 6 h (n = 3). g. EGFR degradation mediated by Ctx-FA (10 nM) in Hela cells transfected with scramble siRNA or Rab7 siRNA for 6 h (n = 3). h. Downregulation of EGFR and MAPK phosphorylation in Fadu cells. Representative blots from three biologically independent experiments. **i. **Schematic of Atz-FA targeting PD-L1. j,k. Degradation of PD-L1 in MDA-MB-231 and A549 cells treated with Atz-FA (10 nM) for 24 h (n = 3). m,n. Degradation of CD47 in MDA-MB-231 and A549 cells treated with Ab2-FA (100 nM) for 24 h (n = 3).

Figure 4: Evaluation of the pharmacokinetics (PK) for PD-L1 degraders in vivo. a. Blot intensity of rat IgG in the plasma of C57BL/6 mice treated with Ab3, Ab3-FA-12x, Ab3-FA-25x at different time points (2.5 mg/kg via IP injection). Data are presented as mean ± SD, n = 4. b. Concentration of Ab3-FA-25x in C57BL/6 mice bearing B16F10 tumor at different time points (2.5 mg/kg via IP injection) Data are presented as mean ± SD, n = 3. (12x and 25x: 12 or 25 molar equivalents of DBCO-NHS ester in the first step; 25 equivalents of folate-azide were used in the second step). N indicates mice.

Figure 5: FRTAC targeting PD-L1 inhibits tumor growth in B16F10, CT26 and MOC1 syngeneic mouse models. d. Tumor growth curves after different treatments as indicated by a (n = 8). e. Tumor growth curves after different treatments as indicated by b (n = 10). f. Tumor growth curves after different treatments as indicated by c (n = 9). g. Body weight curves after different treatments as indicated by  a (n = 8). h. Body weight curves after different treatments as indicated by b (n = 10).  i. Body weight curves after different treatments as indicated by c (n = 9). j. weight of excised tumors on day 24 after different treatments as indicated by b (n = 10). N indicates mice.

Figure 6: FRTACs show cancer selectivity for the degradation of EGFR and PD-L1. a. Comparison of EGFR degradation efficiency mediated by Ctx-FA (10 nM) in normal cell line HACAT, and cancer cell line Huh7 (n = 3). b. Comparison of PD-L1 degradation efficiency of Atz-FA in normal cell line, HACAT, and cancer cell line Huh7 and TU138 (n = 3). c. Quantification of FR expression levels on HACAT, Huh7, and TU138 by western blot (n = 3).

Figure S1: Uptake of soluble protein targets mediated by FRTACs is dose- and cell line-dependent. a. Dose response of anti-FITC-594 (50 nM) uptake induced by FA-FITC and comparison with 50 nM Ab-FA in HepG2 cells for 3 h (n = 3). B. Dose response of anti-FITC-594 (50 nM) uptake induced by Ab-FA in HepG2 cells for 3 h (n = 3). c. Uptake of anti-biotin-594 (50 nM) in HepG2 cells treated with Ab-FA (25 nM) for 3 h. d. Comparison of anti-biotin-594 (50 nM) uptake in different cancer cell lines treated with Ab-FA (25 nM) for 24 h. N indicates biologically independent experiments.

Figure S2: FRTACs generated by two-step labeling method with more FA labeling and longer linker length have higher protein degradation efficiency. a. Generation of FRTAC by one-step labeling method.b. EGFR degradation mediated by FRTACs prepared by one-step labeling method using different amounts of folate-NHS ester (3x, 12x, and 25x) (n = 3). c. Comparison of EGFR degradation induced by FRTACs prepared by one- or two-step labeling method with various amounts of reagents (n = 3). 3x: 3 molar equivalents, 12x: 12 molar equivalents, 25x: 25 molar equivalents, 1k: PEG1k linker, 2k: PEG2k linker. N3: two-step labeling (12x and 25x indicate the equivalence of the DBCO-NHS ester in the first step; 25 equivalents of folate-azide was used in the second step). NHS: one-step labelling. N indicates biologically independent experiments.

Figure S3: EGFR degradation is related to both FR and EGFR expression levels. a. Dose response of EGFR degradation mediated by Ctx-FA (24 h) in Hela cell (n = 3). b. Time course of EGFR degradation mediated by Ctx-FA (10 nM) in Hela cell (n = 3). c. EGFR degradation mediated by Ctx-FA and negative controls (n = 3).** ** **d. **FR1 and FR2 level before and after degrader treatment (n = 2). **e. **Endogenous EGFR expression level in different cancer cell lines. f. Quantification of e (n = 3). g. EGFR degradation in different cancer cell lines. h. Quantification of g (n = 3). i. Quantification of FR expression levels on different cancer cell lines by flow cytometry (n = 3). j. Correlation of EGFR degradation efficiency with FR expression level alone or the ratio of FR and endogenous EGFR on different cancer cell lines. N indicates biologically independent experiments.

Figure S4: FRTACs mediate the lysosomal degradation of mPD-L1 via their interaction with FR in three mouse cell lines (CT26: c, e, g; B16F10: d, f, h; MOC1: i). a. Schematic of Ab3-FA targeting mouse PD-L1 (mPD-L1).b. Representative plot of the binding affinity of Ab3 and Ab3-FA to mPD-L1 characterized by MicroScale Thermophoresis (MST) (n = 2).c, d. Dose response of mPD-L1 degradation (24 h) mediated by Ab3 and Ab3-FA (n = 3). e, f. Time course of mPD-L1 degradation mediated by Ab3-FA (10 nM) (n = 3). g, h. Inhibition of Ab3-FA (10 nM) mediated mPD-L1 degradation by free FA (3 mM for CT26, 1 mM for B16F10) and Bafilomycin A1 (BAF, 50 nM) for 24 h (n = 3). i. Cellular mPD-L1 degradation in MOC1 cells mediated by Ab3-FA (10 nM, 24 h) (n = 3). N indicates biologically independent experiments.

Figure S5: Evaluation of the pharmacokinetics (PK) for PD-L1 degraders in-vivo. a. blot of rat IgG in the plasma of C57BL/6 mice treated with Ab3, Ab3-FA-12x, Ab3-FA-25x at different time points (2.5 mg/kg via IP injection) from 4 mice. b. Representative blot of rat IgG in the plasma of C57BL/6 mice bearing B16F10 tumor treated with Ab3-FA-25x at different time points (2.5 mg/kg via IP injection) from 3 mice. Ab3-FA-25x at different concentrations were used as standard. (12x and 25x: 12 or 25 molar equivalents of DBCO-NHS ester in the first step; 25 equivalents of folate-azide were used in the second step).

Figure S6:  Non-targeting FA conjugates and free FA have no effects on tumor growth in CT26 syngeneic mouse model. Tumor growth curves after different treatments.

Figure S7: FRTAC reveals anti-tumor effect by degrading PD-L1 and recruiting cytotoxic T cell into the tumor. A. Western blot analysis of PD-L1 and CD8a level in tumor tissues isolated from CT26 tumor-bearing mice (7.5 mg/kg daily for 3 days, 3 mice/cohort).

Figure S8: FRTACs exhibit cancer selectivity both in-vitro and in-vivo. a. Basal levels of EGFR and PD-L1 in normal HACAT and cancer cell line Huh7 and TU138 (n = 3 biologically independent experiments). b. Mouse PD-L1 level in spleen, n = 3 mice. c. Mouse PD-L1 level in lung, n = 3 mice.

Code/software

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