The use of chitosan for cancer vaccine delivery

膀胱内卡介苗治疗浅表性膀胱癌已有三十年的历史。然而，20% - 30%的患者最初的卡介苗治疗失败，30% - 50%的患者在5年内肿瘤复发。浅表性膀胱癌的治疗需要替代或补充策略。白细胞介素-12 （IL-12）是一种有效的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，膀胱内IL-12在最近的一项复发浅表性膀胱癌患者的临床研究中没有显示出抗肿瘤功效。我们假设IL-12与壳聚糖（一种生物相容性、黏附性的多糖）的联合制剂可以改善IL-12在膀胱内的递送，并为浅表性膀胱癌的治疗提供有效和持久的替代方案。在抗肿瘤研究中，经壳聚糖/IL-12四次膀胱内治疗后，88%至100%的原位膀胱肿瘤小鼠治愈。相比之下，单独用IL-12治疗的小鼠只有38%至60%治愈，而用BCG治疗的小鼠只有0%治愈。壳聚糖/ IL-12治疗后的抗肿瘤反应是持久的，并对膀胱内肿瘤的再攻击提供了完全的保护。尿细胞因子分析显示，壳聚糖/IL-12诱导的多种TH1细胞因子水平显著高于单独IL-12或BCG。免疫组化结果显示，壳聚糖/IL-12治疗后，肿瘤有中重度T细胞和巨噬细胞浸润。治愈小鼠的膀胱粘膜下层含有残留的免疫细胞群，几个月后恢复到基线水平。膀胱内壳聚糖/IL-12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浅表性膀胱癌的治疗中进行试验。IL-12是一种有效的抗肿瘤细胞因子，在全身给药后表现出显著的临床毒性。我们假设瘤内注射与生物可降解多糖壳聚糖共配制的IL-12可以增强IL-12的抗肿瘤活性，同时限制其全身毒性。无创成像研究监测IL-12注射后，有无壳聚糖共制剂的局部滞留。在结直肠（MC32a）和胰腺（Panc02）肿瘤小鼠中，研究了单独使用IL-12和壳聚糖共配制IL-12（壳聚糖/IL-12）的抗肿瘤效果。其他涉及免疫细胞亚群消耗、肿瘤再挑战和CTL活性的研究旨在阐明消退和肿瘤特异性免疫的机制。与壳聚糖共配可使局部IL-12滞留从1 ~ 2天增加到5 ~ 6天。每周单独注射IL-12可根除不到10%的MC32a和Panc02肿瘤，而壳聚糖/IL-12免疫治疗可使80%至100%的小鼠肿瘤完全消退。CD4+或Gr-1+细胞的消耗对壳聚糖/ il -12介导的肿瘤消退无影响。然而，CD8+或NK细胞耗尽完全取消抗肿瘤活性。壳聚糖/IL-12免疫治疗可产生全身肿瘤特异性免疫，80%经壳聚糖/IL-12免疫治疗的小鼠至少部分免受肿瘤再攻击。此外，来自治愈小鼠脾脏的ctl可裂解MC32a和gp70肽负载靶标。壳聚糖/IL-12免疫治疗增加肿瘤微环境中IL-12的局部滞留，根除已建立的侵袭性小鼠肿瘤，并产生全身肿瘤特异性保护性免疫。壳聚糖/IL-12是一种耐受性良好、有效的免疫疗法，具有相当大的临床转化潜力。基于重组蛋白的疫苗需要佐剂系统才能产生th1型免疫反应。我们开发了一种疫苗佐剂系统，使用粘性壳聚糖溶液和白细胞介素(IL)-12，一种诱导th1的细胞因子。壳聚糖溶液的设计目的是在皮下注射部位建立抗原和IL-12的储存库。我们测量了含有0.25、1或4 mg小鼠IL-12和75 mg卵清蛋白（OVA）的疫苗的体内免疫应答，这些疫苗在1.5%的壳聚糖谷氨酸溶液中配制。通过CD4+脾细胞增殖、Th1细胞因子释放、CD8+ t细胞干扰素γ释放和MHC类肽五聚体染色，壳聚糖/IL-12/OVA疫苗与壳聚糖/OVA、IL-12/OVA或单独OVA相比，引起了更大的抗原特异性CD4+和CD8+ t细胞反应。壳聚糖和IL-12联合使用也增强了IgG2a和IgG2b抗体对OVA的应答。因此，壳聚糖和IL-12的共同配方促进了对模型蛋白疫苗产生Th1免疫应答。

Intravesical BCG has been used successfully to treat superficial bladder cancer for three decades. However, 20% to 30% of patients will fail initial BCG therapy and 30% to 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 immunologic memory. Unfortunately, intravesical IL-12 did not show antitumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that coformulation 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 four 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. IL-12 is a potent antitumor cytokine that exhibits significant clinical toxicities after systemic administration. We hypothesized that intratumoral (i.t.) administration of IL-12 coformulated with the biodegradable polysaccharide chitosan could enhance the antitumor activity of IL-12 while limiting its systemic toxicity. Noninvasive imaging studies monitored local retention of IL-12, with and without chitosan coformulation, after i.t. injection. Antitumor efficacy of IL-12 alone and IL-12 coformulated with chitosan (chitosan/IL-12) was assessed in mice bearing established colorectal (MC32a) and pancreatic (Panc02) tumors. Additional studies involving depletion of immune cell subsets, tumor rechallenge, and CTL activity were designed to elucidate mechanisms of regression and tumor-specific immunity. Coformulation with chitosan increased local IL-12 retention from 1 to 2 days to 5 to 6 days. Weekly i.t. injections of IL-12 alone eradicated less than 10% of established MC32a and Panc02 tumors, while i.t. chitosan/IL-12 immunotherapy caused complete tumor regression in 80% to 100% of mice. Depletion of CD4+ or Gr-1+ cells had no impact on chitosan/IL-12-mediated tumor regression. However, CD8+ or NK cell depletion completely abrogated antitumor activity. I.t. chitosan/IL-12 immunotherapy generated systemic tumor-specific immunity, as >80% of mice cured with i.t. chitosan/IL-12 immunotherapy were at least partially protected from tumor rechallenge. Furthermore, CTLs from spleens of cured mice lysed MC32a and gp70 peptide-loaded targets. Chitosan/IL-12 immunotherapy increased local retention of IL-12 in the tumor microenvironment, eradicated established, aggressive murine tumors, and generated systemic tumor-specific protective immunity. Chitosan/IL-12 is a well-tolerated, effective immunotherapy with considerable potential for clinical translation. Vaccines based on recombinant proteins require adjuvant systems in order to generate Th1-type immune responses. We developed a vaccine adjuvant system using a viscous chitosan solution and interleukin (IL)-12, a Th1-inducing cytokine. The chitosan solution is designed to create a depot of antigen and IL-12 at a subcutaneous injection site. We measured the in vivo immune response of a vaccine containing 0.25, 1, or 4 mg murine IL-12 and 75 mg ovalbumin (OVA), formulated in a 1.5% chitosan glutamate solution. The chitosan/IL-12/OVA vaccine, in comparison to chitosan/OVA, IL-12/OVA, or OVA alone, elicited greater antigen-specific CD4+ and CD8+ T-cell responses, as determined by CD4+ splenocyte proliferation, Th1 cytokine release, CD8+ T-cell interferon-gamma release, and MHC class I peptide pentamer staining. The combination of chitosan and IL-12 also enhanced IgG2a and IgG2b antibody responses to OVA. Co-formulation of chitosan and IL-12 thus promoted the generation of a Th1 immune response to a model protein vaccine.