Tolerogenic nanoparticles restore the antitumor activity of recombinant immunotoxins by mitigating immunogenicity

Abstract

Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.

Keywords: antidrug antibodies; cancer; mesothelin; nanoparticle; rapamycin.

Fig. 1.

The combination of LMB-100 + SVP-R prevents the ADA response against LMB-100. (A) A ribbon diagram of LMB-100 and an illustration of SVP-R. (B) Mice were injected seven times every other week with LMB-100 or a combination of LMB-100 and SVP-R one, three, or seven times (indicated by arrows). Anti–LMB-100 antibodies were evaluated by ELISA (n = 8). (C) Mice were injected with LMB-100 and SVP-R as indicated by arrows (n = 7). (D) Mice were injected with LMB-100 and SVP-R as indicated by arrows. Final mean titer on week 10 is shown (n = 7). (E) Neutralization assay using plasma from the mice treated as shown in C (n = 7). KLM-1 cells were seeded and treated with plasma-LMB-100 mixture. Cell viability was assessed after 72 h. Curves represent mean of seven viability curves (n = 7, six replicas per samples). (F) Mice were injected with LMB-100 and SVP-R as indicated by arrows (n = 8). ELISA plates were coated with LMB-100, Fab, or anti–TAC-PE24. Plasma samples from week 6 were evaluated. The dilution factor for 50% of binding is shown. Lines indicate mean; error bars, SEM. For statistical analysis in B and C, the AUC for each curve was calculated, and AUCs were compared using one-way ANOVA.

Fig. 2.

Combination of LMB-100 with SVP-R induces a specific, transferable, and regulatory T-cell–mediated immune tolerance. (A) Mice were injected three times weekly with LMB-100 (2.5 mg/kg i.v.) or a combination of LMB-100 with SVP-R (2.5 mg/kg i.v.). During weeks 4–8, mice were challenged with weekly doses of LMB-100 (i.v.) and ovalbumin (s.c.). Plasma was collected and analyzed for anti–LMB-100 and anti-ovalbumin antibodies by ELISA. For statistical analysis, AUC for each curve was calculated and analyzed using the Mann–Whitney U test. Error bars SEM, n = 13. (B) Mice were injected six times with vehicle, LMB-100, SVP-R, or LMB-100 + SVP-R. During week 4, splenocytes from donor mice were isolated and adoptively transferred to recipient naïve mice. Recipient mice were injected with LMB-100 six times. Plasma was collected and analyzed for anti–LMB-100 antibodies by ELISA. Results from two separate experiments with identical schedules were combined. n = 5–10. Error bars represent SEM. (C) Mice were injected with LMB-100 on days 1, 3, 5, 29, 31, 33, 43, 45, and 47. SVP-R was given on days 1, 3, and 5. Anti-mouse CD-25–depleting antibody (PC61) or isotype control were injected i.p. on days 15 and 16. Titers on day 55 are shown. (D) Plasma from mice that were injected seven times every other week with LMB-100 or a combination of LMB-100 + SVP-R. Anti–LMB-100 isotypes were analyzed using sandwich ELISA with subclasses IgG1, IgG2a, IgG2b, IgG3, and IgM specific to LMB-100 (n = 8).

Fig. 3.

LMB-100 and SVP-R colocalize preferentially on DCs and macrophages. (A) Dye-conjugated SVP-Cy5 and LMB-100-Alexa Fluor 488 were injected i.v. alone or in combination (n = 3–4 mice per group). Spleen cells were analyzed by FACS at 2 h after injection for dye-conjugate uptake. (B and C) Representative FACS plots show gating for macrophages (F4/80⁺CD11b⁺) and DCs (CD11c⁺MHC-II⁺), and in vivo uptake by the gated populations. Red quadrants indicate the percent of positive cells analyzed for each experimental condition. (D) Summary of SVP-R and LMB-100 in vivo uptake by macrophages, DCs, monocytes, CD4⁺ T cells, B cells, neutrophils and CD8⁺ T cells. The gating strategy for all cells is shown in Fig. S5.

Fig. 4.

Combination of LMB-100 with SVP-R induces immune tolerance in mice with preexisting antibodies. (A) Female BALB/c mice were injected six times with LMB-100 (2.5 mg/kg i.v.) on weeks 1 and 3 to induce a titer of ADA against LMB-100. On week 10, mice were challenged with three doses of LMB-100, vehicle (PBS), or LMB-100 + SVP-R. The mice treated with LMB-100 + SVP-R were challenged with three additional doses of LMB-100 on week 12. Plasma was collected and analyzed for LMB-100 ADAs by ELISA. n = 7 or 12. Error bars represent SEM. (B) Female BALB/c mice were injected 12 times with LMB-100 over the course of 14 wk to induce a high titer of ADA against LMB-100. In week 15, mice were immunized with LMB-100 or LMB-100 + SVP-R. ADA titers prechallenge and postchallenge are shown. (C and D) BM and spleen were isolated from mice with preexisting ADA and were challenged with PBS, LMB-100, SVP-R, or LMB-100 + SVP-R. BM cells and splenocytes (100,000 cells/well) were seeded in ELISpot plates precoated with LMB-100 (n = 8).

Fig. 5.

Combinations of SVP-R and LMB-100 restores neutralized antitumor activity. (A) AB1-L9 cells were inoculated into mice and treated with PBS, LMB-100, or SVP-R as indicated by arrows (n = 7). (B) Mice were immunized with LMB-100 four times to induce a baseline titer and then inoculated with AB1-L9. Mice were treated with vehicle, LMB-100, or SVP-R as indicated by arrows. Tumor size was measured using a caliper (n = 7). (C) Plasma from days 5 and 19 was analyzed for anti–LMB-100 antibodies by ELISA. Titer was interpolated at 10% of the signal. (D) Mice were treated as described in C. The experiment was terminated on day 31. The Kaplan–Meier plot shows the time to the experimental endpoint (once tumor volume was >400 mm³ or if a mouse lost >30% of its body weight). n = 7. (E) Mice were inoculated with CT26 cells on day 1 and treated with SVP-R or vehicle on days 10 and 16. Values indicate average tumor size (n = 7). Error bars represent SEM. (F) Mice were inoculated with 66C14 cells on day 1 and treated with SVP-R or vehicle on days 10, 12, and 14. Values indicate average tumor size (n = 5). For statistical analysis, AUC for each curve was calculated, and AUCs were compared using one-way ANOVA. Error bars represent SEM.

Fig. 6.

SVP-R enhances the cytotoxic activity of LMB-100 in human cell lines. KLM-1 and HAY cells were seeded in 96-well plates and treated with various concentrations of SVP-R, LMB-100, or LMB-100 + SVP-R. After 72 h, cell viability was assessed using WST-8 or crystal violet. Viability curves were fitted to each sample and IC₅₀ was calculated. (A) Cytotoxic activity of SVP-R in both cell lines. (B) Activity of LMB-100 in KLM-1 cells with or without 5 μg/mL of rapamycin encapsulated in SVP. (C) Activity of LMB-100 in HAY cells with or without 1 μg/mL of rapamycin encapsulated in SVP. Curves show a mean of six replicates. Error bars represent SEM. (D) Representative well images taken after HAY cells were fixed and stained with crystal violet.

Fig. 7.

SVP-R activity is not diminished by checkpoint inhibitor antibodies. BALB/c mice were immunized weekly with LMB-100 or LMB-100 + SVP-R five times (2.5 mg/kg i.v.), and at 5 d after each injection were immunized with anti-mouse CTLA-4 antagonist (A) or anti–OX-40 antagonist (B) or vehicle (i.p.). Plasma samples were collected on day 6 of each week, and LMB-100 ADA titer was evaluated using direct ELISA. n = 8. Error bars represent SEM. The experiments were repeated with n = 3 and 5, with similar results.