Immunotherapeutic activity of a conjugate of a Toll-like receptor 7 ligand

Abstract

The immunotherapeutic activity of Toll-like receptor (TLR) activators has been difficult to exploit because of side effects related to the release and systemic dispersion of proinflammatory cytokines. To overcome this barrier, we have synthesized a versatile TLR7 agonist, 4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde (UC-1V150), bearing a free aldehyde that could be coupled to many different auxiliary chemical entities through a linker molecule with a hydrazine or amino group without any loss of activity. UC-1V150 was covalently coupled to mouse serum albumin (MSA) at a 5:1 molar ratio to yield a stable molecule with a characteristically altered UV spectrum. Compared with the unconjugated TLR7 agonist, the UC-1V150/MSA was a 10- to 100-fold more potent inducer of cytokine production in vitro by mouse bone marrow-derived macrophage and human peripheral blood mononuclear cells. When administrated to the lung, the conjugate induced a prolonged local release of cytokines at levels 10-fold or more higher than those found in serum. Under the same conditions, the untethered TLR7 ligand induced quick systemic cytokine release with resultant toxicity. In addition, two pulmonary infectious disease models were investigated wherein mice were pretreated with the conjugate and then challenged with either Bacillus anthracis spores or H1N1 influenza A virus. Significant delay in mortality was observed in both disease models with UC-1V150/MSA-pretreated mice, indicating the potential usefulness of the conjugate as a localized and targeted immunotherapeutic agent.

Fig. 1.

Synthesis of UC-1V150. Reagents and conditions were as follows: α-bromo-p-tolunitrile, K₂CO₃/dimethylformamide, 25°C (step a); NH₃-MeOH, 60°C (step b); CH₃OCH₂CH₂CH₂OH, 100°C (step c); Br₂/CH₂Cl₂, 25°C (step d); CH₃ONa/CH₃OH, reflux (step e); lithium N,N′-diemthylenediamino aluminum hydride/THF, 0°C (step f); and HCl, 25°C (step g).

Fig. 2.

Conjugation of UC-1V150 to MSA. Covalent linkage is formed via a linker, SANH, between the UC-1V150 and MSA containing an amino group.

Fig. 3.

Quantification of UC-1V150 molecules conjugated to MSA. (A) Different concentrations of UC-1V150-SANH were subjected to spectrophotometric analysis. (B) The readout of UC-1V150-SANH was used to derive a standard curve. (C) Conjugation of UC-1V150 to MSA was indicated by a UV absorption peak at 342 nm due to hydrazone formation. The concentration of UC-1V150/MSA used in this analysis at 0.25 μg was based on MSA concentration, which is equivalent to 3.63 μM MSA. The concentration of UC-1V150 in conjugate was calculated as (0.6901 − 0.0056)/0.0366 = 18.7 μM; thus, the UC-1V150:MSA ratio is 18.7/3.63 = 5.2.

Fig. 4.

In vitro cytokine release in response to UC-1V150/MSA conjugates. Murine BMDM were treated with UC-1V150 or UC-1V50/MSA conjugates at various concentrations as indicated. Culture supernatants were harvested 24 h later, and cytokine levels were measured by immunoassay. The results are a representative of at least two separate experiments in triplicate per treatment.

Fig. 5.

In vivo efficacy of UC-1V150/MSA conjugates. C57BL/6 mice were administered various amounts of UC-1V150 or UC-1V150/MSA via the tail vein as indicated. Sera were collected 2 h later, and cytokine levels were determined by multiplex immunoassay. Each group had four mice. The error bars indicate the SEM.

Fig. 6.

Sustained in vivo local activity of UC-1V150/MSA conjugates without systemic effect. C57BL/6 mice were anesthetized and administered i.t. with 3 nmol of UC-1V150/MSA. At the indicated time points, mice were killed and both BALF and sera were collected for multiplex immunoassay of cytokine levels. The data were combined from two separate experiments with at least six mice per group. The results show the mean values ± SEM.

Fig. 7.

Preclinical efficacy of UC-1V150/MSA in pulmonary infectious diseases. (A) Age-matched female A/J mice were administered i.n. saline only or saline containing MSA (amount equivalent to UC-1V150/MSA), UC-1V150, or UC-1V150/MSA at 0.75 nmol per mouse 1 day before B. anthracis infection, and survival was followed for 13 days. (B) BALB/c mice were administered i.n. saline or UC-1V-150/MSA at 5 nmol per mouse 1 day before influenza infection. Survival followed up to 21 days. In each model, Kaplan–Meier survival curves and log-rank tests were performed to determine significance. At least eight mice were tested in each group.