An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses

Abstract

Targeted immunomodulation for reactivating innate cells, especially macrophages, holds great promise to complement current adaptive immunotherapy. Nevertheless, there is still a lack of high-performance therapeutics for blocking macrophage phagocytosis checkpoint inhibitors in solid tumors. Herein, a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) is described, which undergoes biomimetic surface propagation on cancer cell membranes through ligand-receptor binding and enzyme-triggered reactions. By simultaneously blocking CD47 and CD24 signaling, PAC-SABI enhances the phagocytic ability of macrophages in vitro and in vivo, promoting anti-tumor responses in breast and pancreatic cancer mouse models. Moreover, building on the foundation of PAC-SABI-induced macrophage repolarization and increased CD8⁺ T cell tumor infiltration, sequential anti-PD-1 therapy further suppresses 4T1 tumor progression, prolonging survival rate. The in vivo construction of PAC-SABI-based nano-architectonics provides an efficient platform for bridging innate and adaptive immunity to maximize therapeutic potency.

Fig. 1. Design and proposed mechanism of PAC-SABIs.

a Peptide molecular structure design of Pep-PEG. b Schematic illustration of PAC-SABIs formation process including peptide self-assembling, antibody modification, and ALP catalysis. c Schematic illustration of immune quiescent microenvironment and in vivo construction of PAC-SABIs. Figure was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 international license. d Proposed mechanism of PAC-SABIs-mediated activation of macrophage phagocytosis against cancer cell.

Fig. 2. Characterization of PAC-SABIs.

a CACs of Pep and Pep-PEG. Three independent experiments were performed. b Fluorescence spectrum of NBD-labeled Pep. Insert: fluorescence emission image of NBD-labeled Pep and Pep plus ALP (1 U/ml). Three independent experiments were performed. c Fluorescence spectrum of NBD-labeled Pep-PEG. Insert: fluorescence emission image of NBD-labeled Pep-PEG and Pep-PEG plus ALP (1 U/ml). Three independent experiments were performed. d Zeta potentials of Pep, Pep-PEG and PAC-SABIs detected by dynamic light scattering. The error bars represent the mean ± SD (n = 3 independent experiments). e CD spectrum of Pep and PAC-SABIs in the presence or absence of ALP (1 U/ml). Three independent experiments were performed. f Secondary structure calculation of Pep and PAC-SABIs in the presence or absence of ALP (1 U/ml). Three independent experiments were performed. g FTIR spectra of Pep and PAC-SABIs in the presence or absence of ALP (1 U/ml). The green area refers to absorption band around 1629 cm⁻¹. Three independent experiments were performed. h Conversion rates of Pep, Pep-PEG and PAC-SABIs incubated with ALP (1 U/ml) over time. The black dashed line refers to conversion rates of 90%. Three independent experiments were performed. i Time-dependent TEM images of Pep and PAC-SABIs. The red arrows point to the formation of nanofibers. Scale bar: 200 nm. Three independent experiments were performed. Source data are provided as a Source Data file.

Fig. 3. In situ self-assembly of PAC-SABIs on BC and PC cell membranes.

a Schematic illustration of tumor immune quiescent microenvironment, in situ assembly of PAC-SABIs on the cancer cell surface, and blockage of CD47 and CD24 phagocytic checkpoints by PAC-SABIs. Figure was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 international license. b Time-dependent CLSM images of 4T1 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 μm. Three independent experiments were performed. c Time-dependent CLSM images of PAN02 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 μm. Three independent experiments were performed. d Merged bright field and fluorescence images of 4T1 cell treated with NBD-labeled PAC-SABIs for 120 min. Scale bar: 20 μm. Three independent experiments were performed. e CLSM images of 4T1 cell treated with Dil dye (red) and NBD-labeled PAC-SABIs for 120 min. Scale bar: 10 μm. Three independent experiments were performed. f, g Fluorescence distribution of NBD-labeled PAC-SABIs on 4T1 cell. Three independent experiments were performed. h Time-dependent SEM images of 4T1 cells treated with PAC-SABIs. The red arrows point to PAC-SABIs on cell membrane. Scale bar: 2 μm. Three independent experiments were performed. i Time-dependent SEM images of PAN02 cells treated with PAC-SABIs. The red arrows point to the PAC-SABIs on cell membrane. Scale bar: 2 μm. Three independent experiments were performed. j CLSM images of 3D 4T1 spheroids treated with ^Cy5.5PAC-SABIs, Calcein-AM, and Hoechst 33342. Scale bar: 50 μm. Three independent experiments were performed. k CLSM images of 3D 4T1 spheroids along the z-axis position. Scale bar: 50 μm. Three independent experiments were performed.

Fig. 4. Promotion of phagocytic clearance of cancer cells via PAC-SABIs treatment in vitro.

a Phagocytosis images of pHrodo-red⁺ over time. Scale bar: 50 μm. Three independent experiments were performed. b Phagocytosis of 4T1 cells, in the presence of IgG control or PAC-SABIs. The error bars represent the mean ± SD (n = 3 independent experiments). c Representative 3D CLSM image reconstruction of in vitro phagocytosis of 4T1 cells (pHrodo-red⁺, red) by BMDMs and RAW264.7 cells (Calcein-AM, green). Three independent experiments were performed. d Representative flow cytometry plots depicting BMDM phagocytosis of 4T1 cells treated with IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs. e Quantitative analysis of BMDM flow cytometry results. The error bars represent the mean ± SD (n = 3 independent experiments; **p < 0.01, ***p < 0.001; the p value was analyzed by a two-tailed unpaired Student’s t test). f Representative flow cytometry plots depicting RAW264.7 cell phagocytosis of 4T1 cells treated with IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs. g Quantitative analysis of RAW264.7 cell flow cytometry results. The error bars represent the mean ± SD (n = 3 independent experiments; **p < 0.01; the p value was analyzed by a two-tailed unpaired Student’s t test). Source data are provided as a Source Data file.

Fig. 5. Biodistribution and tumor targeting of ^Cy5.5PAC-SABIs in vivo.

a Representative NIR fluorescence images of free Cy5.5, ^Cy5.5SAMIs and ^Cy5.5PAC-SABIs on 4T1 subcutaneous tumor-bearing mice after intravenous injection. Images were acquired at 0, 2, 6, 12, 24, 48, 72, 96 and 120 h post injection. The white dashed circles refer to the tumor site. b Time-dependent quantitative calculation of the average fluorescence intensity in 4T1 tumor area and AUCs of ^Cy5.5SAMIs and ^Cy5.5PAC-SABIs. The error bars represent the mean ± SD (n = 3 mice). c Representative NIR fluorescence images of free Cy5.5, ^Cy5.5SAMIs and ^Cy5.5PAC-SABIs on PAN02 subcutaneous tumor-bearing mice after intravenous injection. Images were acquired at 0, 2, 6, 12, 24, 48, 72, 96 and 120 h post injection. The black dashed circles refer to the tumor site. d Time-dependent quantitative calculation of the average fluorescence intensity in PAN02 tumor area and AUCs of ^Cy5.5SAMIs and ^Cy5.5PAC-SABIs. The error bars represent the mean ± SD (n = 3 mice). e Ex vivo NIR fluorescence images of 4T1 tumor and major organs (H heart, Li liver, S spleen, Lu lung, K kidney) collected post 48 h injection. Three independent experiments were performed. f Ex vivo NIR fluorescence images of PAN02 tumor and major organs (H heart, Li liver, S spleen, Lu lung, K kidney) collected post 48 h injection. Three independent experiments were performed. g Fluorescence images of 4T1 and PAN02 subcutaneous tumor sections at 48 h post-injection of ^Cy5.5PAC-SABIs. A refers to the area of fibroids; B refers to the area of cancer cells; C refers to the paracancerous area. Scale bar: 100 μm. Three independent experiments were performed. Source data are provided as a Source Data file.

Fig. 6. Antitumor efficacy of PAC-SABIs in vivo.

a Scheme of the PAC-SABIs therapeutic strategy for 4T1 orthotopic tumor. b BLIs of 4T1 tumor-bearing mice on days 7, 14, 21, and 28 (n = 5 mice). c Quantification analysis of 4T1 tumor BLI signals in each treatment group. The error bars represent the mean ± SD (n = 5 mice; **p < 0.01, ***p < 0.001, ****p < 0.0001; the p value was analyzed by a two-tailed unpaired Student’s t test). d Kaplan–Meier survival curves of 4T1 tumor-bearing mice (n = 5 mice; **p < 0.01; the p value was analyzed by log-rank test). e Scheme of the PAC-SABIs therapeutic strategy for PAN02 orthotopic tumor. f BLIs of PAN02 tumor-bearing mice on days 7, 14, 21, and 28 (n = 5 mice). g Quantification analysis of PAN02 tumor BLI signals in each treatment group. The error bars represent the mean ± SD (n = 5 mice; *p < 0.05, ***p < 0.001; the p value was analyzed by a two-tailed unpaired Student’s t test). h Kaplan–Meier survival curves of PAN02 tumor-bearing mice (n = 5 mice; *p < 0.05; the p value was analyzed by log-rank test). i Scheme of macrophage depletion and therapeutic strategy for 4T1 subcutaneous tumor. j Representative flow cytometry plots of tissue-resident macrophages. k Quantification analysis of tissue-resident macrophages. Boxplots represent the median and interquartile range, and the whiskers denote minimum and maximum values (n = 4 mice; ****p < 0.0001; the p value was analyzed by a two-tailed unpaired Student’s t test). l BLIs of 4T1 tumor-bearing mice on days 7, 14, 21, and 28 (n = 5 mice). m Quantification analysis of BLI signals of 4T1 tumor in each treatment group. The error bars represent the mean ± SD (n = 5 mice; *p < 0.05, **p < 0.01; the p value was analyzed by a two-tailed unpaired Student’s t test). n Kaplan–Meier survival curves of 4T1 tumor-bearing mice (n = 5 mice; **p < 0.01; the p value was analyzed by log-rank test). a, e, i were created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 international license. Source data are provided as a Source Data file.

Fig. 7. Immune response in vivo.

a Representative H&E and IF staining of cytokeratin 19 (CK19), F4/80⁺ macrophages and CD8⁺ T cells for the corresponding 4T1 tumor tissues after different treatments. Scale bars are marked in the figures. b Representative flow cytometry plots depicting CD45⁺CD11b⁺F4/80⁺ macrophages phagocytosis, CD11b⁺F4/80⁺CD206^hi macrophages, CD11b⁺F4/80⁺CD86^hi macrophages, CD45⁺CD3⁺CD4⁺/CD8⁺ T cells, and CD45⁺CD3⁺CD4⁺Foxp3⁺ T cells in 4T1 tumors after different treatments. c–g Quantification analysis of CD45⁺CD11b⁺F4/80⁺ macrophages phagocytosis, CD11b⁺F4/80⁺CD206^hi macrophages, CD11b⁺F4/80⁺CD86^hi macrophages, CD45⁺CD3⁺CD4⁺/CD8⁺ T cells, and CD45⁺CD3⁺CD4⁺Foxp3⁺ T cells in 4T1 tumors after different treatments. The error bars represent the mean ± SD (n = 4 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; the p value was analyzed by a two-tailed unpaired Student’s t test). h Identification of differentially expressed genes in 4T1 tumors after treating with IgG Control or PAC-SABIs (n = 5 mice). i Number of differential genes enriched in GO term of 4T1 tumors treated with PAC-SABIs versus IgG Control (n = 5 mice). j GO enrichment analysis of the differential pathways in 4T1 tumors treated with PAC-SABIs versus IgG Control (n = 5 mice). k Volcano plot for the transcriptome sequencing of 4T1 tumors treated with PAC-SABIs versus IgG Control. Differentially expressed genes were calculated using a two-sided limma moderated t-test with Benjamini–Hochberg correction for multiple testing (n = 5 mice). Source data are provided as a Source Data file.

Fig. 8. Enhanced anti-PD-1 therapy after phagocytosis modulation.

a Scheme of the combination therapeutic strategy. Figure was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 international license. b Spider plots of individual tumor growth curves for 4T1-tumor-bearing BALB/c mice in each treatment group. c Average tumor growth curve of 4T1-tumor-bearing BALB/c mice in each treatment group. The error bars represent the mean ± SD (n = 7 mice; ****p < 0.0001; the p value was analyzed by a two-tailed unpaired Student’s t test). d Kaplan–Meier survival curves of 4T1 tumor-bearing BALB/c mice treated with the indicated formulation. (n = 7 mice; **p < 0.01, ***p < 0.001; the p value was analyzed by log-rank test). e Representative H&E staining of lungs harvested from the indicated treatment group. Scale bar: 100 μm. Three independent experiments were performed. f Quantitative analysis of phagocytosis in CD45⁺CD11b⁺F4/80⁺ macrophages flow cytometry results. The error bars represent the mean ± SD (n = 4 independent experiments; ***p < 0.001, ****p < 0.0001; the p value was analyzed by a two-tailed unpaired Student’s t test). g Representative IF images of tumor-infiltrated CD8⁺ T cells. Scale bar: 100 μm. Three independent experiments were performed. Source data are provided as a Source Data file.