Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction

Abstract

Background: Intratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events.

Methods: To address these limitations, TransCon™ TLR7/8 Agonist-an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod-was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice.

Results: Following a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions.

Conclusions: Our findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

Keywords: Adaptive immunity; Innate immunity; Intratumoral injection; Intratumoral treatment; Toll-like receptor; Tumor microenvironment.

Conflict of interest statement

All authors are or were employees of Ascendis Pharma and declare competing financial interests.

© 2022. The Author(s).

Figures

Fig. 1

In vivo pharmacokinetic characteristics of TransCon TLR7/8 Agonist. A TransCon TLR7/8 Agonist was designed as a sustained-release prodrug of resiquimod consisting of resiquimod bound, at multiple conjugation sites, to a suspension of polymeric PEG hydrogel microspheres via a covalent and cleavable linker (R = resiquimod; L = linker). B Male Wistar rats (n = 3/group) received a single subcutaneous injection of either soluble resiquimod (25 μg) or TransCon TLR7/8 Agonist [25 μg equivalent (eq.) of resiquimod]. Blood was withdrawn and used for plasma generation over 28 days. The resiquimod concentration in the plasma samples was quantified by LC–MS/MS. Values are represented as mean resiquimod concentration (pg/ml) per group ± SEM. C Female BALB/c mice were SC implanted with 3 × 105 CT26 tumor cells in their flank. When tumors were grown to a mean tumor volume of ~ 115 mm3, mice were randomized into treatment cohorts (Day 0). The day following randomization, animals received 5 or 20 μg (eq. of resiquimod) of TransCon TLR7/8 Agonist as a single intratumoral dose. Blood was withdrawn (n = 3/time point) and used for plasma generation at 6 h, 3 days and 7 days. The resiquimod concentration in the plasma samples was quantified by LC–MS/MS. Values are represented as mean resiquimod concentration (pg/ml) per dose group ± SEM

Fig. 2

TransCon TLR7/8 Agonist promoted TGI, minimal systemic cytokine release, and sustained intratumoral cytokine expression. A Female BALB/c mice were SC implanted (flank) with 3 × 105 CT26 tumor cells. At a mean tumor volume of ~ 90 mm3, mice were randomized (Day 0; n = 10–15/group). The following day, animals received either TransCon Vehicle, 20 μg (eq. of resiquimod) of TransCon TLR7/8 Agonist, or 20 μg of soluble resiquimod (single IT dose, arrow). Values are represented as mean tumor volume ± SEM. On Day 14, *p < 0.005 vs all other groups, n = 6–11/group. B Female BALB/c mice were SC implanted (flank) with 3 × 105 CT26 tumor cells. At a mean tumor volume of ~ 120 mm3, mice were randomized (Day 0). The following day, animals received either TransCon Vehicle, 20 μg of TransCon TLR7/8 Agonist, or 20 μg of soluble resiquimod as a single IT dose. Plasma samples were (n = 3 independent mice/group/timepoint) assessed for cytokines by ProcartaPlex Multiplex Immunoassay. Values are represented as median plasma analyte concentration (pg/ml) with whiskers representing minimum and maximum replicate values. Points are connected for visualization purposes. *p < 0.05 Soluble Resiquimod vs TransCon Vehicle (same day), †p < 0.05 TransCon TLR7/8 Agonist vs TransCon Vehicle (same day), ‡p < 0.05 TransCon TLR7/8 Agonist vs Soluble Resiquimod (same day). C Female BALB/c mice were SC implanted (flank) with 3 × 105 CT26 tumor cells. At a mean tumor volume of ~ 115 mm3, mice were randomized (Day 0). The following day, animals received either TransCon Vehicle or 20 or 80 μg of TransCon TLR7/8 Agonist. Tumors were harvested and assessed for cytokine levels. Values are represented as the minimum, maximum, and median (black line) analyte concentrations normalized to protein input for each treatment group (n = 3/treatment/timepoint, except for the TransCon TLR7/8 Agonist treated group at the 168-h timepoint, where n = 2). *p < 0.05 vs TransCon Vehicle (same day)

Fig. 3

TransCon TLR7/8 Agonist associated with low systemic cytokines and sustained activation of peripheral immune cells. In two independent experiments, Female BALB/c mice were SC implanted (flank) with 2 × 106 CT26 tumor cells. At a mean tumor volume of ~ 185–200 mm3, mice were randomized into treatment cohorts (Day 0; n = 18–20/group). On the same day of randomization, animals received either buffer control or TransCon TLR7/8 Agonist as a single IT dose (arrow). For each experiment, samples from 5 to 6 animals were collected for processing and analysis at each timepoint. Data from the two experiments was concatenated prior to analysis. A Mean tumor volume (mm3) ± SEM over time is shown (n = 13–38/group/timepoint). On Day 14, *p < 0.05 vs Buffer, n = 13–16/group. B Mean body weight (g) ± SEM is shown (n = 13–38/group/timepoint). C Plasma cytokine/chemokine concentrations over time are shown. D Frequency of indicated immune cell sub-type within the CD45+ gate assessed in PBMCs one day after treatment is shown. E PBMCs were isolated at various times following treatment and assessed for markers of adaptive immune-cell subsets and activation via flow cytometry. For C–F *p < 0.05 vs Buffer on the same day, n = 5–11/ group/timepoint

Fig. 4

IT TransCon TLR7/8 Agonist potentiated activation of APCs and T cells in tDLNs. Mice were treated as described in Fig. 3. Lymphocytes from tDLNs were isolated at various times following treatment and assessed for markers of immune-cell subsets and activation via flow cytometry. A Antigen presenting cell immunophenotyping. B Adaptive and cytolytic immune cell immunophenotyping. For A and B *p < 0.05 vs Buffer on the same day, n = 9–11/group/timepoint

Fig. 5

IT TransCon TLR7/8 Agonist potentiated activation of APCs and antigen-specific CD8 T cells in tumors. Mice were treated as described in Fig. 3. Dissociated tumor cells were isolated at various times following treatment and assessed for markers of immune-cell subsets and activation via flow cytometry. A Antigen presenting cell immunophenotyping following treatment initiation. *p < 0.05 vs Buffer on the same day, n = 5–11 per group per timepoint. B CD8 T cell granzyme B and PD-1 immunophenotyping on day 14 following treatment initiation. *p < 0.05 vs Buffer, n = 9–11/group. C %AH1-tetramer+ CD8 T cells on Day 14 following treatment initiation. n = 9–11/group

Fig. 6

TransCon TLR7/8 Agonist with immunotherapy promoted TGI in injected and non-injected tumors and immunological memory. A Female BALB/c mice were SC implanted (flank) with 2 × 106 CT26 tumor cells. At a mean tumor volume of ~ 80 mm3, mice were randomized into treatment cohorts (Day 0; n = 10/group). On the same day of randomization, animals received either anti-PD1, 100 μg (eq. of resiquimod) TransCon TLR7/8 Agonist as a single IT dose (arrow) or were left untreated (Control). On Day 18, *p < 0.05 vs Control, and **p < 0.05 vs control, n = 9–10/group. B Female BALB/c mice were SC implanted with 5 × 105 CT26 tumor cells into the left and right flanks. At a mean tumor volume of ~ 100 mm3, mice were randomized into treatment cohorts (Day 0). On the day of randomization, animals received either TransCon Vehicle or 216 μg (eq. of resiquimod) of TransCon TLR7/8 Agonist as a single intratumoral dose (arrow). Some cohorts were further treated with 20 μg human IL-2 dosed twice daily on Days 0–4 and once daily on Days 8–12. In this experiment, 3 out of 7 mice treated with TransCon TLR7/8 Agonist + IL-2 experienced complete regressions in injected and non-injected tumors. On Day 16, *p < 0.05 vs TransCon Vehicle and **p < 0.05 vs all other groups, n = 7/group. For non-injected tumors on Day 18, †p < 0.05 vs Control and ‡p < 0.05 vs Control and TransCon TLR7/8 Agonist, n = 7/group. C Mice from B that were treated with TransCon TLR7/8 Agonist and IL-2, and that experienced complete regressions in both treated and untreated tumors for at least 10 days (n = 3), were SC rechallenged with CT26 tumor cells in their front right dorsal region and observed for tumor growth. Naïve mice (n = 10) were used as controls for tumor growth. Values are represented as mean tumor volume ± SEM