The effect of targeting strategies for improving the interaction of liposomal PorA with dendritic cells (DC) on the immunogenicity of PorA was investigated. PorA, a major antigen of Neisseria meningitidis, was purified and reconstituted in different types of (targeted) liposomes, i.e., by using mannose or phosphatidylserine as targeting moieties, or with positively charged liposomes. We studied the efficiency of liposome uptake and its effect on the maturation of and interleukin 12 (IL-12) production by murine DC. Moreover, mice were immunized subcutaneously to study the localization and immunogenicity of PorA liposomes. Uptake of liposomes by DC was significantly increased for targeted liposomes and resulted in the maturation of DC, but to various degrees. Maturation markers (i.e., CD80, CD86, major histocompatibility complex class II, and CD40) showed enhanced expression on DC incubated with targeted PorA liposomes relative to those incubated with nontargeted PorA liposomes. Moreover, only the uptake of targeted PorA liposomes induced production of IL-12 by DC, with levels similar to those produced by lipopolysaccharide (LPS)-pulsed DC. Mannose-targeted PorA liposomes administered subcutaneously had an increased localization in draining lymph nodes compared to nontargeted PorA liposomes. Liposomes in draining lymph nodes interacted preferentially with antigen-presenting cells, an effect that was enhanced with targeted PorA liposomes. Immunization studies showed an improvement of the bactericidal antibody response (i.e., increased number of responders) generated by targeted PorA liposomes compared to that generated by nontargeted ones or LPS-containing outer membrane vesicles. In conclusion, the use of targeted PorA liposomes results in an improved uptake by and activation of DC and an increased localization in draining lymph nodes. These effects correlate with an enhanced immune response toward the vaccine.

The aim of this work was to improve radiotherapy results by immune stimulation. We tested the effects of a combination of radio‐ and immunotherapy, i.e., local low dose recombinant interleukin‐2 (rIL‐2) treatment, in two murine tumor models. Syngeneic tumors (SL2 lymphoma or M8013 mammary carcinoma) were induced subcutaneously on one or both flanks of mice. Irradiation was given either as a single dose (20 Gy) or fractionated (25 Gy) in 2 weeks. One or two cycles of rIL‐2 were given concurrent with or subsequent to radiotherapy. One cycle of rIL‐2 consisted of peritumoral injections administered on 5 consecutive days. Treatment effects were measured in terms of local tumor response and disease‐free survival (DFS). The combined treatment modality was significantly better than treatment with either irradiation alone or rIL‐2 alone. When tumors were inoculated on both flanks of the mice, combined radioimmunotherapy of one of the tumors also resulted in regression of the contralateral untreated tumor, indicating that a systemic anti‐tumor immune reaction was induced. Additional rIL‐2 injections did not enhance radiation toxicity. In conclusion supplementing irradiation with locally administered low doses of rIL‐2 results in better local anti‐tumor responses and DFS rates than either treatment alone without enhanced treatment toxicity. Furthermore, the local treatment induces a systemic anti‐tumor reaction, influencing the growth patterns of a second, untreated tumor. Radiat. Oncol. Invest. 5:54–61, 1997. © 1997 Wiley‐Liss, Inc.

Tumor recurrence and outgrowth of metastases limit the therapeutical effect of radiotherapy. We have tested whether these problems can be overcome by supplementing radiotherapy with locoregional interleukin‐2 (IL‐2) treatment. The SL2 lymphoma and the M8013 mammary carcinoma were used. Mice bearing a 10‐day‐old s.c. tumor were locally irradiated and were treated daily with IL‐2 peritumorally for 5 or 10 days. Low‐dose IL‐2 therapy improved local response (LR) and increased disease‐free survival (DFS) in both tumor models following either single‐dose irradiation or fractionated irradiation. For example, 93% of SL2‐bearing mice treated with single‐dose irradiation and 10 days of IL‐2 experienced long‐term DFS, compared with 17% for irradiation alone (p < 0.0001). Additionally, treatment of one tumor with irradiation +IL‐2 led to anti‐tumor effects in a second, untreated tumor in 80% of SL2‐bearing mice. LR was increased to 100% and DFS to 70% when the second, non‐irradiated tumor was also treated with peritumoral IL‐2. We conclude that supplementing local radiotherapy with low doses of IL‐2 results in increased local tumor control and regression of distant, non‐irradiated tumors. This type of radioimmunotherapy is a promising new approach for the clinic. Int. J. Cancer 72:1003–1007, 1997. © 1997 Wiley‐Liss, Inc.

SummaryDuring the last decades, an increase is apparent in the use of analgesics for laboratory animals in situations where this was previously considered unnecessary. Mice with advanced tumours often show clear signs of discomfort which may be a result of chronic pain or a result of general ill-being. The syngeneic murine tumour model most frequently used in our experiments was used to investigate whether this discomfort can be reduced with an analgesic. Twenty DBA/2 mice bearing SL2 lymphoma were given 0.5 mg/kg buprenorphine (Temgesic®) in food gel twice daily, 20 tumour-bearing mice were given control food gel at the same times. Indicators of well-being were monitored daily. These included behavioural parameters such as exploration, grooming, and posture; food and water consumption and fur quality. All mice showed a clear increase of discomfort with time: explorative behaviours and grooming decreased, while sitting in hunched posture increased. Food and water consumption and fur quality also decreased. Major significant differences between the buprenorphine treated group and the control group were not apparent. In conclusion, we could not document a positive effect or buprenorphine on discomfort in mice as evaluated by our scoring system. It remains possible that pain itself was not the primary cause of the discomfort in mice bearing these tumours, or that the analgesic effect of buprenorphine was insufficient under these circumstances.

T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4 + T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134 + T cells showed a specific proliferative response to the diseaseassociated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4 + CD134 + T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'-fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134 + T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4 + T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development.