Camptothesome-based combination nanotherapeutic regimen for improved colorectal cancer immunochemotherapy

Abstract

Camptothesome is a sphingomyelin-conjugated camptothecin (SM-CSS-CPT) nanovesicle that fortified the therapeutic delivery of CPT in diverse cancer types. To mitigate the Camptothesome-induced IDO1 negative feedback mechanism, we had co-encapsulated, indoximod (IND, IDO1 inhibitor) into Camptothesome using doxorubicin-derived IND (DOX-IND). To maximize the therapeutic potential of DOX-IND/Camptothesome, herein, we first dissected the synergistic drug ratio (DOX-IND/SM-CSS-CPT) via systematical in vitro screening. DOX-IND/Camptothesome with optimal drug ratio synchronized in vivo drug delivery with significantly higher tumor uptake compared to free drugs. This optimum DOX-IND/Camptothesome outperformed the combination of Camptothesome, Doxil and IND or other IDO1 inhibitors (BMS-986205 or epacadostat) in treating mice bearing late-stage MC38 tumors, and combination with immune checkpoint blockade (ICB) enabled it to eradicate 60 % of large tumors. Further, this optimized co-delivery Camptothesome beat Folfox and Folfiri, two first-line combination chemotherapies for colorectal cancer in antitumor efficacy and exhibited no side effects as compared to the severe systemic toxicities associated with Folfox and Folfiri. Finally, we demonstrated that the synergistic DOX-IND/Camptothesome was superior to the combined use of Onivyde + Doxil + IND in curbing the advanced orthotopic CT26-Luc tumors and eliminated 40 % tumors with complete metastasis remission when cooperated with ICB, eliciting stronger anti-CRC immune responses and greater reversal of immunosuppression. These results corroborated that with precise optimal synergistic drug ratio, the therapeutic potential of DOX-IND/Camptothesome can be fully unleased, which warrants further clinical investigation to benefit the cancer patients.

Keywords: Camptothesome; Colorectal cancer; Immunochemotherapy; Indoleamine 2,3-dioxygenase; Indoximod.

Fig. 1.. Determination of synergistic drug molar ratio and development of the optimal co-delivery DOX-IND/Camptothesome.

a-f, cytotoxicity study of drug combination with varied molar ratios in MC38 (a-c) and CT26 (d-f) cells for 48 h by MTT assay. c,f, the IC₅₀ values and combination index at different drug ratios. g-j, Schematic depicting the synthesis and preparation (g), representative size distribution by dynamic light scattering (DLS, h), cryoEM (i), and formulation stability monitoring (j) for DOX-IND/Camptothesome with optimal drug ratio (2/1). k, Physicochemical characterizations. l, differential scanning calorimetry (DSC) thermograms of DOX-IND/Camptothesome. Thermotropic phase transition behavior was determined by MicroCal VP-capillary DSC. m, Leakage profile of DOX-IND in DOX-IND/Camptothesome under 4 °C. n, The release kinetics of DOX-IND from DOX-IND/Camptothesome under different conditions at 37 °C. Data in a,b,d,e,j,k,m,n are expressed as mean ± s.d. (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 2.. DOX-IND/Camptothesome synchronized drug delivery to orthotopic tumors with synergistic drug ratio.

a,b, maximum tolerated dose (MTD) investigation. a, mouse weight change after intravenously injecting DOX-IND/Camptothesome (DOX-IND/SM-CSS-CPT, 2/1 molar ratio) once to mouse at various doses. b, a table depicting the mouse death and weight loss. c-g, blood kinetics (c-e) and tumor delivery (f-j) in orthotopic CRC CT26 tumor model (tumor: ~400 mg, n = 3) following single i.v. injection of DOX-IND/Camptothesome (DOX-IND/SM-CSS-CPT, 21.5/15.1 mg/kg), free DOX (12.5 mg/kg), free IND (5 mg/kg), free DOX-IND (21.5 mg/kg), Doxil (12.5 mg/kg), SM-CSS-CPT (15.1 mg/kg) and free CPT (4 mg/kg). Blood kinetics and drug tumor uptake were analyzed by HPLC according to the established method[1]. In addition, CPT (g,h) and DOX (i,j) biodistribution was also assessed by drug’s fluorescence intensity through Lago optical imager and HPLC, respectively. Data in a-e, g-j are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 3.. DOX-IND/Camptothesome is superior to the combination of Camptothesome, IND, and Doxil in large subcutaneous MC38 tumor mouse model.

a-c, therapeutic efficacy of DOX-IND/Camptothesome in comparison to the combination of Camptothesome + IND + Doxil in MC38 tumor model (tumors: ~300 mm³, n = 5). On day 16 post tumor cells inoculation, mice were intravenously injected once at 21.5/15.1 (DOX-IND/SM-CSS-CPT) mg/kg, Doxil 12.5 DOX mg/kg, 5 mg IND/kg. a, tumor growth curves. b, Kaplan-Meier survival curves. c, the mice tumor images taken on day 22. d-f, combination with PD-L1/PD-1 ICB in treating large MC38 tumor model (tumors ~300 mm³, n = 5). ICB was intrapearinatally injected at 100 μg per mouse per 3 days for a total of 3 times. d, tumor growth curves. e, Kaplan-Meier survival curves. f, the mice tumor images taken on day 22. Red circle: tumor-free mouse. g-j, therapeutic efficacy in large MC38 tumor model (~300 mm³, n = 5). The MTD dose of Doxil (25 mg/kg) was adopted according to previous report[29]. g, tumor growth curves. The ratio of Trp (nM)/kyn (nM) in plasma (h) and tumors (i) on day 22. j, the mice tumor images taken on day 22. Red circle: tumor-free mouse. Data in a,d,g-i are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test; survival curves were compared using the log-rank Mantel–Cox test.

Fig. 4.. DOX-IND/Camptothesome outperforms the combination of Camptothesome and Doxil with other IDO1 inhibitors in large subcutaneous MC38 tumor model.

a-c, anticancer activity of co-delivery Camptothesome in comparison to the co-injection of Camptothesome + Doxil + other IDO1 inhibitors (BMS-986205 (BMS), epacadostat (EPA)) in large MC38 tumor model (tumors: 200 mm³, n = 5). Mice received a single intravenous injection of 21.5/15.1 mg DOX-IND/SM-CSS-CPT/kg, Doxil 12.5 DOX mg/kg. Free IDO1 inhibitors were daily oral administration at 5 mg IND/kg, 5 mg BMS/kg, 5 mg EPA/kg from day 14 post cancer cells inoculation. a, tumor growth curves. b, Kaplan-Meier survival curves. c, the mice tumor images taken on day 20. d-f, combination with various ICB in treating large MC38 tumor model (tumors: 400 mm³, n = 5), drug dose were given as same a-c, ICB was intraperitoneally injected at 100 μg per mouse per 3 days for a total of 3 times. d, tumor growth curves. e, Kaplan-Meier survival curve. f, the mice tumor images taken on day 20. Red circle: tumor-free mouse. Data in a,d are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test; survival curves were compared using the log-rank Mantel–Cox test.

Fig. 5.. DOX-IND/Camptothesome performed better than FOLFOX and FOLFIRI, two standard of care combination chemotherapies for CRC with diminished adverse effects.

a-c, anti-CRC activity compared to FOLFOX and FOLFIRI in a late-stage subcutaneous MC38 tumor mouse model (tumors: ~400 mm³, n = 5). In FOLFOX: 5-FU (100 mg/kg), oxaliplatin (2.5 mg/kg), and leucovorin (90 mg/kg); and in FOLFIRI: 5-FU (100 mg/kg), irinotecan (24 mg/kg), and leucovorin (90 mg/kg) were intravenously injected to mice, these doses were inferred from the reported literatures[–33]. DOX-IND/Camptothsome was injected at 21.5 mg/kg for DOX-IND, and 15.1 mg/kg for SM-CSS-CPT. ICB was used as Fig. 3. a, Monitoring of the tumor development. b, Kaplan-Meier survival curves. c, mice tumor images on day 20. Red circle: tumor-free mouse. d-g, the serum chemistry, and hematological cell studies on day 20 from an independent study after healthy C57BL/6 mice received the same treatment as Fig. 5a. Data in a,d-g are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test; survival curves were compared using the log-rank Mantel–Cox test.

Fig. 6.. DOX-IND/Camptothesome defeated the Onivyde-based drug combination in advanced metastatic orthotopic CT26-Luc CRC tumor models.

Late-stage orthotopic CRC model was established by inoculating the CT26-Luc cells into the cecum subserosa of mouse[1, 36]. On day 9, when primary tumors reached ~ 400 mg, various drug combinations were given to mice at equivalent doses (DOX-IND/SM-CSS-CPT 21.5/15.1 mg/kg, Doxil 12.5 DOX mg/kg, IND 5 mg/kg, 6.7 mg/kg for irinotecan in Onivyde). ICB was injected as described in Fig. 3. a, the whole mouse body and ex vivo images showing the primary tumor and metastasis. b, Primary tumor growth curve based on bioluminescence imaging (BLI). c, BLI to show the whole body tumor development. Red circle: tumor-free mouse. d, ex vivo tumor metastasis imaging (upper panel: photograph; lower panel: BLI). e, Normalized organ BLI radiance (vs liver in vehicle control). f, Heat map summarizing the metastasis frequency in diverse organs and tissues. Data in b,e are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons.

Fig. 7.. Potent anti-CRC immune responses with reversal of immunosuppression.

On day 19, the tumors (from Fig. 6b) were excised and processed for comprehensive immunophenotyping analysis using flow cytometry. Representative flow cytometric plots of tumor-infiltrating IFN-γ⁺/CD8⁺, Granzyme B⁺/CD8⁺, and CD25⁺/CD8⁺ T cells (a); CD103⁺/CD11c⁺ (b) and CD86⁺/CD80⁺ (c) dendric cells and Foxp3⁺/CD4⁺ Tregs (d) cells and their respective quantification (right panel). Data (right panel) are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons.