Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization

Abstract

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund's adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund's adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund's adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.

Figure 1

Comparison of anti-Id titers following immunization using CpG ODN or DNA as an adjuvant. 38C13 Id IgM was conjugated to KLH using gluteraldehyde to produce Id-KLH. A total of 50 μg Id-KLH was mixed with adjuvant in aqueous solution and injected subcutaneously into naive 6- to 8-week-old female C3H mice in a volume of 0.2 ml. Each mouse received a single immunization. Blood was obtained weekly, and serum was evaluated for the presence of anti-Id IgG by ELISA. Normal mouse serum supplemented with a known concentration of monoclonal anti-Id was used as a standard. Three mice were included in each group.

Figure 2

Route of immunization. Mice were immunized and anti-Id levels determined as in Fig. 1. Development of anti-Id was determined after subcutaneous, intradermal, or intraperitoneal immunization.

Figure 3

Dose response to CpG ODN. Mice were immunized and anti-Id levels determined as in Fig. 1. All mice received 50 μg Id-KLH. The dose of CpG ODN 1758 was varied to assess dose response to adjuvant.

Figure 4

Comparison of ipsilateral and contralateral injection. Mice were immunized and anti-Id levels determined as in Fig. 1. All mice received 50 μg Id-KLH and 50 μg CpG ODN 1758. Mice were injected with Id-KLH and CpG ODN 1758 mixed together (ipsilateral) or were injected on the right flank with Id-KLH and CpG ODN 1758 on the left flank (contralateral) to assess whether the adjuvant effect of CpG ODN is systemic or local.

Figure 5

Comparison of anti-Id titers following immunization with CpG ODN and CFA. Id-KLH was mixed with either CpG ODN 1758 in PBS or homogenized with CFA and injected subcutaneously in a volume of 0.2 ml. Mice were boosted with a second injection at 2 weeks with Id-KLH and CpG ODN 1758 or Id-KLH and incomplete Freund’s adjuvant.

Figure 6

Tumor protection in C3H mice. Mice were immunized subcutaneously with a single dose of Id-KLH and CpG ODN 1758, Id-KLH, and CFA or were unimmunized. Two weeks later they were challenged with 1,000 38C13 cells intraperitoneally. Survival was followed for 100 days. All mice that were alive after 55 days remained tumor-free for the entire observation period. Ten mice were included in each group.