The use of chitosan for cancer vaccine delivery

卡介苗(BCG)膀胱内注射治疗浅表性膀胱癌已有30年的历史。然而，20%-30%的患者会在初次卡介苗治疗中失败，30%-50%的患者会在5年内出现复发肿瘤。浅表性膀胱癌的治疗需要替代或补充策略。白介素12(IL-12)是一种强大的TH1细胞因子，具有强大的抗肿瘤活性和增强免疫记忆的能力。不幸的是，在最近对复发性浅表性膀胱癌患者的临床研究中，IL-12膀胱内注射并没有显示出抗肿瘤效果。我们推测，IL-12与生物相容的粘附性多糖壳聚糖共制剂可以改善IL-12在膀胱内的转运，为浅表性膀胱癌的治疗提供一种有效而持久的替代方案。在抗肿瘤研究中，携带原位膀胱肿瘤的小鼠经4次壳聚糖/IL-12膀胱内治疗后，88%至100%被治愈。相比之下，单独接受IL-12治疗的小鼠只有38%到60%被治愈，而接受卡介苗治疗的小鼠只有0%被治愈。壳聚糖/IL-12治疗后的抗肿瘤反应是持久的，并提供了对膀胱内肿瘤再攻击的完全保护。尿液细胞因子分析显示，壳聚糖/IL-12诱导的多种TH1细胞因子水平明显高于单独使用IL-12或卡介苗。免疫组织化学显示壳聚糖/IL-12治疗后T细胞和巨噬细胞呈中度至重度肿瘤浸润。治愈小鼠的膀胱粘膜下层含有残留的免疫细胞，几个月后恢复到基线水平。几丁糖/IL-12膀胱内注射是一种耐受性良好、有效的免疫疗法，值得进一步考虑在人类试验中用于浅表性膀胱癌的治疗。持续的、局部的免疫调节细胞因子递送正在接受研究，因为它能够增强疫苗和抗肿瘤临床反应的能力。该项目评估了壳聚糖(一种生物相容的多糖)在(1)控制细胞因子GM-CSF的扩散和(2)增强GM-CSF的免疫佐剂特性方面的能力。尽管细胞因子以前通过脂类佐剂和其他载体输送，但这些载体没有壳聚糖的临床安全性或独特的特性。我们发现壳聚糖溶液在皮下注射部位保持了可测量的重组GM-CSF(rGM-CSF)储备量长达9天。相反，当在生理盐水载体中给药时，RGM-CSF在1224小时内无法检测到。在壳聚糖溶液(壳聚糖/重组人粒细胞集落刺激因子)中注射重组人粒细胞集落刺激因子可使淋巴结瞬时扩张4.6倍，并使表达MHC II类的细胞和树突状细胞数量分别增加7.4倍和6.8倍。这些增加明显大于以标准的临床前剂量和程序在生理盐水中注射RGM-CSF时的测量结果，即每天4次皮下注射。此外，注射壳聚糖/重组人巨噬细胞集落刺激因子的小鼠的淋巴结细胞比单独注射重组人巨噬细胞集落刺激因子的小鼠的淋巴细胞诱导更大的同种异体T细胞增殖，表明抗原提呈能力增强。最后，在疫苗接种实验中，壳聚糖/RGM-CSF在促进抗原特异性的CD4+细胞增殖、多肽特异性CD8+五聚体染色和细胞毒T细胞杀伤方面优于单独使用的壳聚糖或RGM-CSF。总体而言，壳聚糖/RGM-CSF在树突状细胞募集、抗原呈递和疫苗增强方面优于标准RGM-CSF。我们认为壳聚糖溶液是一种很有前途的局部持续给药平台。在一项相关的研究中，壳聚糖溶液增强了皮下接种抗原时的体液和细胞免疫反应。为了最大限度地发挥新疫苗和/或现有疫苗的效力，开发安全的新型佐剂是必要的。壳聚糖是一种无毒、生物相容、可生物降解的天然多糖，来源于甲壳类和昆虫的外骨骼。壳聚糖的生物降解性、免疫学活性和高粘度使其成为肠外疫苗接种的理想仓库/佐剂。为此，我们探索了壳聚糖溶液作为佐剂，以模型蛋白抗原皮下接种小鼠。我们发现，壳聚糖可使抗原特异性抗体效价提高5倍以上，使抗原特异性脾组织中的CD4+细胞增殖能力提高6倍以上。抗体效价的显著增加和强健的迟发性超敏反应(DTH)表明，壳聚糖可诱导体液和细胞免疫反应。与传统疫苗佐剂相比，壳聚糖与不完全弗氏佐剂(IFA)相当，优于氢氧化铝。机理研究表明，壳聚糖至少表现出两个特性，可能使其作为免疫佐剂发挥作用。首先，粘性壳聚糖溶液创建了一个抗原库。更具体地说，当在生理盐水中输送蛋白质抗原时，8h后只有不到9%的蛋白质抗原留在注射部位。然而，在壳聚糖中输送的蛋白质抗原在注射部位停留7天的比例超过60%。其次，壳聚糖在引流淋巴结中诱导了67%的瞬时细胞扩张。壳聚糖注射后14~21d，膨胀度达到高峰，随着多糖的降解，膨胀度逐渐减弱。这些机制研究，再加上疫苗反应的增强，证明了壳聚糖是一种很有前途的安全的非肠道疫苗注射平台。

Intravesical bacillus Calmette-Guerin (BCG) has been used successfully to treat superficial bladder cancer for 3 decades. However, 20-30% of patients will fail initial BCG therapy and 30-50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. Interleukin-12 (IL-12) is a potent TH1 cytokine with robust antitumor activity and the ability to potentiate immunological memory. Unfortunately, intravesical IL-12 did not demonstrate anti-tumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that co-formulation of IL-12 with chitosan a biocompatible, mucoadhesive polysaccharide could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88 to 100% of mice bearing orthotopic bladder tumors were cured after 4 intravesical treatments with chitosan/IL-12. In contrast, only 38 to 60% of mice treated with IL-12 alone, and 0% treated with BCG, were cured. Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple TH1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosa from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well-tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer. Sustained, local delivery of immunomodulatory cytokines is under investigation for its ability to enhance vaccine and anti-tumor responses clinically. This project evaluated the ability of chitosan, a biocompatible polysaccharide, to (1) control the dissemination of a cytokine, GM-CSF, and (2) enhance the immunoadjuvant properties of GM-CSF. While cytokines have previously been delivered in lipid-based adjuvants and other vehicles, these do not have the clinical safety profile or unique properties of chitosan. We found that chitosan solution maintained a measurable depot of recombinant GM-CSF (rGM-CSF) at a subcutaneous injection site for up to 9 days. In contrast, when delivered in a saline vehicle, rGM-CSF was undetectable in 1224 h. Furthermore, a single s.c. injection of 20 μg rGM-CSF in chitosan solution (chitosan/rGM-CSF(20 μg)) transiently expanded lymph nodes up to 4.6-fold and increased the number of MHC class II expressing cells and dendritic cells by 7.4-fold and 6.8-fold, respectively. These increases were significantly greater than those measured when rGM-CSF was administered in saline at the standard preclinical dose and schedule, i.e. 4 daily s.c. injections of 20 μg. Furthermore, lymph node cells from mice injected with chitosan/rGM-CSF(20 μg) induced greater allogeneic T cell proliferation, indicating enhanced antigen presenting capability, than lymph node cells from mice injected with rGM-CSF alone. Finally, in vaccination experiments, chitosan/rGM-CSF was superior to either chitosan or rGM-CSF alone in enhancing the induction of antigen-specific CD4+ proliferation, peptide-specific CD8+ pentamer staining and cytotoxic T cell lysis. Altogether, chitosan/rGM-CSF outperformed standard rGM-CSF administrations in dendritic cell recruitment, antigen presentation and vaccine enhancement. We conclude that chitosan solution is a promising delivery platform for the sustained, local delivery of rGM-CSF. In a related study, chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination of an antigen. The development of safe, novel adjuvants is necessary to maximize the efficacy of new and/or available vaccines. Chitosan is a non-toxic, biocompatible, biodegradable, natural polysaccharide derived from the exoskeletons of crustaceans and insects. Chitosans biodegradability, immunological activity and high viscosity make it an excellent candidate as a depot/adjuvant for parenteral vaccination. To this end, we explored chitosan solution as an adjuvant for subcutaneous vaccination of mice with a model protein antigen. We found that chitosan enhanced antigen-specific antibody titers over five-fold and antigen-specific splenic CD4+ proliferation over six-fold. Strong increases in antibody titers together with robust delayed-type hypersensitivity (DTH) responses revealed that chitosan induced both humoral and cell-mediated immune responses. When compared with traditional vaccine adjuvants, chitosan was equipotent to incomplete Freunds adjuvant (IFA) and superior to aluminum hydroxide. Mechanistic studies revealed that chitosan exhibited at least two characteristics that may allow it to function as an immune adjuvant. First, the viscous chitosan solution created an antigen depot. More specifically, less than 9% of a protein antigen, when delivered in saline, remained at the injection site after 8 h. However, more than 60% of a protein antigen delivered in chitosan remained at the injection site for 7 days. Second, chitosan induced a transient 67% cellular expansion in draining lymph nodes. The expansion peaked between 14 and 21 days after chitosan injection and diminished as the polysaccharide was degraded. These mechanistic studies, taken together with the enhancement of a vaccine response, demonstrate that chitosan is a promising and safe platform for parenteral vaccine delivery.