Invention and characterization of a systemically administered, attenuated and killed bacteria-based multiple immune receptor agonist for anti-tumor immunotherapy

Abstract

Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is critical for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley's Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced uncontrollable toxicity. The discovery of TLRs and their agonists, particularly the potent TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that LPS may be both a critical active ingredient and responsible for dose-limiting i.v. toxicity of G-NB. This communication reports the production of killed, stabilized, intact bacteria products from non-pathogenic G-NB with ~96% reduction of LPS-endotoxin activity. One resulting product candidate, Decoy10, was resistant to standard methods of cell disruption and contained TLR2,4,8,9, NOD2 and STING agonist activity. Decoy10 also exhibited reduced i.v. toxicity in mice and rabbits, and a largely uncompromised ability to induce cytokine and chemokine secretion by human immune cells in vitro, all relative to unprocessed, parental bacterial cells. Decoy10 and a closely related product, Decoy20, produced single agent anti-tumor activity or combination-mediated durable regression of established subcutaneous, metastatic or orthotopic colorectal, hepatocellular (HCC), pancreatic, and non-Hodgkin's lymphoma (NHL) tumors in mice, with induction of both innate and adaptive immunological memory (syngeneic and human tumor xenograft models). Decoy bacteria combination-mediated regressions were observed with a low-dose, oral non-steroidal anti-inflammatory drug (NSAID), anti-PD-1 checkpoint therapy, low-dose cyclophosphamide (LDC), and/or a targeted antibody (rituximab). Efficient tumor eradication was associated with plasma expression of 15-23 cytokines and chemokines, broad induction of cytokine, chemokine, innate and adaptive immune pathway genes in tumors, cold to hot tumor inflammation signature transition, and required NK, CD4+ and CD8+ T cells, collectively demonstrating a role for both innate and adaptive immune activation in the anti-tumor immune response.

Keywords: TLR agonist; adaptive; anti-cancer; anti-tumor; bacteria; immunotherapy; innate; toll-like receptor.

Figure 1

Light and electron microscope images of untreated ATCC 13070 bacteria and Decoy10. The bacteria were grown, treated to produce Decoy10, and processing were carried out as described under Materials and Methods. Grey-scale light microscope images of heat-fixed and crystal violet-stained, untreated ATCC 13070 bacteria (A) and Decoy10 (B) at ~1,000 magnification. Transmission electron microscope images of 2% uranyl acetate negatively-stained, untreated ATCC 13070 bacteria (C) and Decoy10 (D) at 23,000 magnification.

Figure 2

Decoy10 inhibits the growth of s.c. CT-26 murine colorectal carcinoma, and extends survival and inhibits metastasis of an orthotopic CT-26 model. The experiments were carried out as described under Materials and Methods. (A) Tumor cells (2x10⁵ in PBS) were implanted s.c. on Day 0 and i.v. treatment with Decoy10 (QDx2 per week for 3 weeks)) with 8 mice per group was initiated one day after randomization on Day 11 when the average tumor volume was 202 mm³. Arrows denote dosing days. Body weight was measured 4 times per week. The highest Decoy10 dose group produced statistically significant tumor growth inhibition relative to the control (untreated) group (*Log-rank p=0.023). (B) Mean body weights recorded during the study are presented. There were no treatment-related deaths or requirements for dosing holidays. (C) CT-26-GFP tumor fragments were surgically implanted on the cecum. Randomization and treatments were initiated 5 days after tumor fragment implant (7 mice per group), with 5-FU starting the same day and Decoy10 starting one day after randomization. Body weights were measured twice per week. Average group body weight loss with Decoy10 treatment relative to randomization was only observed once on Day 15 (4.0%).

Figure 3

Decoy20 extends survival in a metastatic, murine pancreatic carcinoma model. The experiment was carried out as described under Materials and Methods. Randomization and treatments were initiated 5 days after tumor cell implant (7 mice per group), with i.p. Gemcitabine at 50 mg/kg QDx4 per week for 3 weeks starting the same day and i.v. Decoy20 at 5x10⁷ or 2x10⁸ per mouse QDx2 per week starting one day after randomization. Median survival was 27 Days (No Treatment), 41 Days (Gemcitabine), 35 Days (5x10⁷ Decoy20), and 75 Days (2x10⁸ Decoy20). All median survival increases were statistically significant relative to No Treatment by Log-rank analysis (p<0.001). Body weights were measured before and at least daily for three days after Decoy20 treatment. Maximum transient average group weight loss was 8.2% and 9.8% for the low and high Decoy20 doses, respectively, during the first week of treatment, and 2.1% and 2.6% in the second week of treatment, with no weight loss in the third week of treatment, demonstrating toxicity tolerance with repeat dosing.

Figure 4

Decoy10 synergizes with indomethacin and anti-PD-1 to regress established tumors in the s.c. murine H22 hepatocellular carcinoma model. The experiment was carried out as described under Materials and Methods. Tumor cell implantation was carried out s.c. with 2x10⁶ H22 cells in PBS. Mice were randomized and indomethacin (Indo) treatments were initiated on Day 7 when the average tumor volume was 187 mm3. (A) Indo was administered QD for 6 weeks at 10 µg/mL in the drinking water (no regressions, Log-rank p=0.007 vs no treatment). Decoy10 (D10) was not tested alone in this experiment but produced slight statistically significant tumor growth delay without producing any regressions at QDx2 per week ( Figure 5 ). D10 was administered starting one day after Indo. Indo + QDx2 D10 per week produced optimal combination synergy (4/6 CR, Log-rank p=0.018 vs indomethacin). All regressions in this part of the study were durable until termination on Day 91. Transient, average group body weight loss during each week of treatment, relative to randomization day, for the Indo + QDx2 per week D10 group was 9.5%, 7.7%, 4.1%, 1.2%, 0.9%, and 0, respectively. (B) Anti-PD-1 was tested alone (Q3-4 days per week for 2 weeks) (Log-rank p=0.002 vs no treatment), with Indo administered QD for 2 weeks, and with Indo (6 weeks) + the three different D10 schedules in part A (only the optimal triple combination is shown). Indo + anti-PD-1 produced 1 durable CR, Indo + once per week D10 produced 2 durable CRs. With the triple combination, once per week D10 produced the best result (6/6 CRs, with 5/6 durable to study termination on day 91) (Log-rank p=0.018 vs Indo + D10, and p=0.004 vs Indo + Anti-PD-1. Transient, average weekly group weight loss with this triple combination (8.8%, 6.1%, 3.7%, 2.2%, 5.2%, and 0.8%) was similar to the weight loss observed with the optimal double combination of Indo + Decoy10 in part (A) The QDx2 per week D10 triple combination schedule produced 3/6 CRs (1 durable) and the Q3-4 day per week D10 triple combination schedule produced 4/6 CRs (3 durable) (not shown).

Figure 5

Decoy10 synergizes with anti-PD1 to regress established tumors in the s.c. H22 hepatocellular carcinoma model and the triple combination induces immunological memory. The experiment was carried out as described under Materials and Methods. Implantation was carried out with 2x10⁶ H22 cells in PBS. Treatments were initiated on Day 7 when tumors averaged 194 mm³ with 6 mice per group. Indomethacin (Indo) was administered p.o. at 10 µg/mL in drinking water QD for 6 weeks starting on Day 7. Decoy10 was administered i.v. at 2x10⁸ QDx2 per week as single agent or once per week in combinations, both for 6 weeks starting on Day 8. Anti-PD-1 (aPD-1) was administered i.p. at 10 mg/kg Q3-4 days per week for 2 weeks starting on Day 7. (A) All treatments (single and combination) produced a statistically significant enhancement of survival relative to no treatment (Log-rank p ≤ 0.006). There were no regressions with single agent treatments. The 2-way combinations each produced 2/6 full, durable regressions (to termination at Day 101) and the 3-way combinations (two different Decoy10 doses) produced 10/12 full regressions with 9 durable to termination at Day 140. The maximum transient, weekly average group body weight loss was 6.9%, 5.3%, 5.5%, 3.3%, 1%, and 0 respectively after each of the six QDx2 weekly doses of single agent Decoy10, and was 8.30%, 5.0%, 5.4%, 2.9%, 3.7%, and 0 for the triple combination with the higher dose of once per week Decoy10. Decoy10 + indomethacin was not tested in this experiment. (B) The nine triple combination mice with durable regressions were re-challenged with H22 cells on the opposite flank from the first tumor challenge on Day 91 (no further treatment). All rechallenge tumors started to grow and then were fully rejected demonstrating 100% immunological memory. Full tumor take was recorded in naïve mice that received the same tumor cells on the same day as the re-challenge.

Figure 6

Decoy20, anti-PD-1 and indomethacin induce durable regression of established H22 HCC tumors with a Decoy20 therapeutic index of >33 and induction of immunological memory. The experiment was carried out as described under Materials and Methods. Implantation was carried out with 2x10⁶ H22 cells in PBS. Treatments were initiated on Day 7 when tumors averaged 205 mm³ in volume with 6 mice per group. (A) Indomethacin (6 weeks) and anti-PD-1 (two weeks) were administered starting on Day 7 as described in Figures 4 and 5 . Decoy20 was administered i.v. once per week for 6 weeks starting on Day 8 at 3x10⁷, 1x10⁸, 3x10⁸, or 1x10⁹ per mouse. All doses produced 5/6 or 6/6 durable regressions (*maximum average group weight loss). (B) The 11 tumor-regressed mice from the two middle dose groups in part A were rechallenged on Day 91 with fresh HCC tumor cells on the opposite flank relative to the first tumor challenge. Naïve mice were challenged with the same cells on the same day. There was no further treatment.

Figure 7

Tumor-eradicating combination therapy increases tumor inflammation signature (TIS) score. The experiment was carried out as described under Materials and Methods and in the text (extension of the experiment described for Table 5 ). Indomethacin (NSAID or N) was administered p.o. in drinking water QD x 7 at 10 µg/mL starting on Day 1. Anti-PD-1 (P) was administered i.p. on days 1 and 4 at 10 mg/kg. Decoy10 (D) was administered i.v. once at 2x10⁸ on Day 2. RNA was isolated from tumors harvested on Day 8 and Tumor Inflammation Signature Score (TIS) analysis was carried out using nanoString technology. The TIS p values for each treatment compared to no treatment were 0.037 NSAID, 0.053 Decoy, 0.001 anti-PD-1, 0.002 NSAID + Decoy, <0.001 NSAID + anti-PD-1, <0.001 Decoy + anti-PD-1, <0.001 NSAID + Decoy + anti-PD-1.

Figure 8

Tumor-eradicating combination therapy is associated with increased innate and adaptive immune pathway gene and cell signature expression in HCC tumors. The experiment was carried out as described under Materials and Methods, in the text, and Figure 7 . Tumors were harvested after one week of treatment, RNA was isolated and analyzed using nanoString technology. (A) Analysis of general immune pathways, cells and genes in individual tumors (6 mice per group). (B) Results in 8A averaged within each group. (C–F). Expression of cytokine, chemokine, innate and adaptive pathway-associated genes, respectively, in individual tumors.