An in situ depot for the sustained release of a TLR7/8 agonist in combination with a TGFβ inhibitor promotes anti-tumor immune responses

Abstract

Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming an intratumoral sustained drug release depot that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues, consequently generating systemic anticancer immunoreactivity. CarboCell can be injected through standard thin-needle technologies and has inherent imaging contrast which secure accurate intratumoral positioning. In particular, here we report the therapeutic performance for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor in preclinical tumor models in female mice.

Fig. 1. CarboCell allows for flexible administration and provides sustained intratumoral release of drug combinations with high tumor retention and minimal systemic spillover.

a CarboCell is compatible with clinical injection technologies, including image-guided endoscopy, enabling treatment of most lesions (left). Upon contact with tissue or water it self-assembles into a depot (right). b The main constituents of CarboCell are the esterified carbohydrate sucrose octabenzoate (SuBen), the triglyceride glycerol trioctanoate (GTO), the TLR7/8a Resiquimod (R848) and the TGFβi RepSox. c R848 and RepSox release profiles from two CarboCell compositions (CC and CC-ER, Supplementary Table 1) were evaluated by s.c. injection of 50 μL CarboCell in mice [n = 3 per time point] and is presented as percent cumulative release of the total amount injected. Drug retention was quantified by HPLC of excised CarboCell. d Blood concentration [n = 4 per time point] and (e) intratumoral activity of R848 administered intratumorally (i.t.), intravenously (i.v.), or intratumorally via CarboCell (CarboCell TLR) was determined by liquid scintillation counting (LSC) using ³H-R848 [n = 4 per time point]. ³H-R848 was formulated in PBS or CarboCell (CC-4 (Supplementary Table 1)). The results are presented as mean ± SEM. Source data are provided as a Source Data file. Illustration created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Fig. 2. Combinatorial CarboCell TLR:TGFb improves therapeutic efficacy and links the innate and adaptive immune response.

a Treatment schedule (CT26 tumors, CC (Supplementary Table 1)). b Individual tumor growth curves and c survival plots of mice bearing CT26 tumors [start size ~150 mm³, n = 9/group]. d–h Mice bearing established CT26 tumors were injected intratumorally with CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg, CC (Supplementary Table 1)). Untreated mice were included as controls. One day (1d), three days (3d) and seven days (7d) later, tumors and tdLNs were harvested for analysis. d Representative images of anti-MLKL immunohistochemistry (IHC) analysis of tumors. For remaining images see Supplementary Fig. 13. [bar = 200 µm, n = 3 (UT and d3), n = 4 (d1 and d7)]. e Gene expression analysis (one-way ANOVA with Tukey post-test) of tumors; Ccl4 (F(2,10) = 3.798, P = 0.0593), Cxcl10 (F(2,10) = 10.87, P = 0.0031), [n = 5 (UT), n = 5 (d1), n = 3 (d7)] and tdLNs; Ccl4 (F(2,12) = 15.10, P = 0.0005), Cxcl10 (F(2, 12) = 115.4, P < 0.0001) [n = 4 (UT), n = 5 (d1), n = 6 (d7)]. f Analysis of intratumoral levels (one-way ANOVA with Tukey post-test) of CXCL10 (F(3,11) = 3.661, P = 0.0475) and IFN-β (F(2,9) = 9.200, P = 0.0067) determined by ELISA [all groups n = 4, except CXCL10 d7 n = 3]. g, h Flow cytometry analysis (two-tailed unpaired t test). For gating strategies see Supplementary Fig. 14 and Supplementary Fig. 15. g %cDC1s, CD86 expression (MFI) on cDC1s, and %Inflammatory monocytes in tdLNs [n = 6]. h CD8⁺/Treg ratio and %AH1⁺ (of CD8⁺) T cells in tumors [n = 5]. i Treatment schedule. BALB/c [start size ~135 mm³, n = 8] and BALB/c-nu [start size ~ 121 mm³, n = 9] mice bearing established CT26 tumors received two weekly intratumoral injections with CarboCell TLR:TGFb. Untreated BALB/c [start size ~144 mm³, n = 8] and BALB/c-nu [start size ~133 mm³, n = 8] mice were included as controls. j Individual tumor growth curves. The results are presented as mean ± SEM. Source data and exact P-values are provided as a Source Data file.

Fig. 3. Combinatorial CarboCell TLR:TGFb provides single agent therapeutic activity in MC38 and EMT-6 tumor bearing mice.

a Treatment schedule (MC38 tumors, CC (top) and CC-ER (bottom) (Supplementary Table 1)). b Individual tumor growth curves and (c) survival plots of mice bearing MC38 tumors [start size ~109 mm³, n = 9 (UT), n = 9 (Empty CarboCell), n = 8 (CarboCell TLR:TGFb), n = 8 (CarboCell TLR:TGFb extended release)]. d–f Mice bearing established MC38 tumors were injected intratumorally with CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg, (CC (Supplementary Table 1)). Untreated mice were included as controls. One day (1d) and seven days (7d) later, tumors and tdLNs were harvested for analysis. d Gene expression analysis (one-way ANOVA with Tukey post-test) of tumors; Ccl4 (F(2,12) = 10.87, P = 0.0020) and Cxcl9 (F(2,12) = 19.44, P = 0.0002) [n = 5 (UT), n = 5 (d1), n = 4 (d7)] and tdLNs; Ccl4 (F(2,12) = 10.87, P = 0.0020), Cxcl9 (F(2,12) = 19.44, P = 0.0002) and Irf7 (F(2,12) = 7.059, P = 0.0094) [all groups n = 5]. e Analysis of intratumoral levels (one-way ANOVA with Tukey post-test) of IFN-β determined by ELISA assay [n = 4]. f Representative images of anti-CD8 immunohistochemistry (IHC) analysis of tumors [bar=100 µm, n = 3]. g Treatment schedule EMT-6 tumors (CC (Supplementary Table 1)). h Individual tumor growth curves and (i) survival plots of mice bearing EMT-6 tumors [start size ~95 mm³, n = 8 (UT), n = 6 (CarboCell TLR), n = 5 (CarboCell TLR:TGFb]. j, k Flow cytometry analysis (two-tailed unpaired t test). For gating strategies see Supplementary Fig. 16 and Supplementary Fig. 17. Mice bearing established EMT-6 tumors were injected intratumorally with CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg, CC (Supplementary Table 1)) and untreated mice were included as controls [n = 6/group]. Analysis was performed seven days after treatment. j %cDC1s (of CD45⁺), CD86 expression (MFI) on cDC1s, and %inflammatory monocytes (of CD45⁺) in tdLNs. k %TAMs (of CD45⁺) in tumors. The results are presented as mean ± SEM. Source data and exact P values are provided as a Source Data file.

Fig. 4. Intratumoral CarboCell TLR:TGFb induces systemic anticancer immune activity and immunological memory.

a Treatment schedule (CT26 tumors, CC Supplementary Table 1). Mice bearing two established CT26 tumors were injected intratumorally in one tumor with CarboCell. b Individual tumor growth curves and (c) survival plots of mice bearing two CT26 tumors [start size ~100 mm³ (injected tumor), start size ~87 mm³ (uninjected tumor), n = 9 (UT) and n = 15 (CarboCell TLR:TGFb)]. d Flow cytometry analysis (one-way ANOVA with Tukey post-test) of %CD36 (of Tregs) (F(2,19) = 20.99, P < 0.0001) and %Tregs (of CD45 + ) (F(2,19) = 0.6498, P = 0.5334) in tumors 3 days [n = 6 (UT)] or 14 days [n = 5 (CarboCell TLR:TGFb)] after injection of CarboCell. Mice bearing two established CT26 tumors were injected intratumorally in one tumor with CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg, CC (Supplementary Table 1)). Untreated mice were included as controls. For gating strategy see Supplementary Fig. 18. e TCRβ CDR3 nucleotide sequence overlap (two-tailed unpaired t-test) between intratumoral TCRβ CarboCell injected tumors and uninjected tumors from mouse bilateral tumor model [n = 6 (UT), n = 4 (CarboCell TLR:TGFb)]. f Morisita overlap index of intratumor TCRβ in CarboCell injected (R) and contralateral uninjected tumor (L) from CarboCell CarboCell TLR:TGFb treated mice [n = 4 (M01-M04)]. g, h Lung metastases were evaluated in mice bearing s.c. 4T1 flank tumors after three weekly intratumoral injections of CarboCell TLR (7.5 mg/kg), CarboCell TGFb (20 mg/kg), or CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg) starting at day 7 post inoculation (CC (Supplementary Table 1)), or no treatment (UT) [n = 8 (CarboCell groups), n = 7 (UT)]. 24 days post inoculation, lung metastases were quantified by 6-thioguanine clonogenic assay (Supplementary Fig. 19). g Percent of mice displaying complete clearance of pulmonary metastases. h Analysis of lung colonies (one-way ANOVA with Tukey post-test) (F(3,27) = 5.765, P = 0.0035) i Flow cytometry evaluation of tumor arginase-1 expression (MFI) in 4T1 tumors injected with CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg, CC (Supplementary Table 1)) analyzed (one-way ANOVA with Tukey post-test) one (1d), three (3d), and seven days (7d) after treatment [n = 6 (UT), n = 5 (d1) n = 5 (d3) n = 6 (d7)] on Mo-MDSCs (F(3,18) = 8.852, P = 0.0008), PMos (F(3,18) = 22.24, P < 0.0001), and TAMs (F(3,18) = 7.347, P = 0.0020). For gating strategy see Supplementary Fig. 20. j Treatment schedule (CT26 tumors, splenocyte transfer). Mice bearing established CT26 tumors were treated with intraperitoneal injection (i.p.) of cyclophosphamide (CTX) one day prior to splenocyte transfer. The mice received either no splenocytes, splenocytes from naïve mice, splenocytes from mice bearing established CT26 tumors, or splenocytes from mice rejecting two CT26 tumors (data shown in a–c). k Mean tumor growth curves and (l) survival plots of mice bearing CT26 tumors [palpable tumors, n = 8/group]. The results are presented as mean ± SEM. Source data and exact P values are provided as a Source Data file. Illustrations created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Fig. 5. Intratumoral CarboCell TLR:TGFb decreases viable tumor tissue volume and augments conventional aPD-L1 immunotherapy.

a–f Mice bearing established CT26 tumors were injected intratumorally with empty CarboCell, CarboCell TLR (7.5 mg/kg), CarboCell TGFb (20 mg/kg), or CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg) (CC (Supplementary Table 1)). Untreated mice were included as reference. Five days after treatment, tumors were harvested for analysis. a 3D rendering of the representative preprocessed images of AF555 lectin-labelled tumor vasculature (red) and AF647-labelled anti-PD-L1 (turquoise) extravasation in optically cleared tumors. b 3D isosurface rendering of representative masks of total tumor tissue (semi-transparent grey) and viable tumor tissue (cyan). Scale bar, 1 mm. c–e Results of image analysis describing effects of the treatments (one-way ANOVA with Tukey post-test) [n = 4/group] on (c). the volume of viable tumor tissue (F(3, 12) = 4.769, P = 0.0206), d targeting of viable extravascular space (EVS) (F(3,12) = 2.658, P = 0.0958), e fraction of anti-PD-L1 extravasation volume ending up in viable EVS from total extravasation volume (F(3,12) = 11.07, P = 0.0009), and (f). total extravasation volume per total vessel surface (vessel permeability) (Kruskal-Wallis test (F) P = 0.0248, Dunn’s post-test). g Treatment schedule (MC38 tumors, CC-ER (Supplementary Table 1)). h Individual tumor growth curves and (i) survival plots of mice bearing MC38 tumors [start size ~129 mm³, n = 8 (UT), n = 7 (aPD1), n = 8 (CarboCell TLR:TGFb), n = 6 (CarboCell TLR:TGFb + aPD1)]. The results are presented as mean ± SEM. Source data and exact P values are provided as a Source Data file.