The use of chitosan for cancer vaccine delivery

膀胱内BCG已成功用于治疗浅表性膀胱癌30年。然而，20%至30%的患者将在初始BCG治疗中失败，30%至50%的患者将在5年内复发肿瘤。浅表性膀胱癌的管理需要替代或补充策略。白细胞介素-12（IL-12）是一种有效的TH 1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在最近的复发性浅表性膀胱癌患者的临床研究中没有显示出抗肿瘤疗效。我们假设，IL-12与壳聚糖（一种生物相容的粘膜粘附多糖）的共制剂可以改善膀胱内IL-12的递送，并为浅表性膀胱癌的治疗提供有效且持久的替代方案。在抗肿瘤研究中，88%至100%的荷原位膀胱肿瘤的小鼠在用壳聚糖/IL-12进行四次膀胱内治疗后治愈。相比之下，仅38%至60%的单独用IL-12治疗的小鼠和0%用BCG治疗的小鼠被治愈。壳聚糖/ IL-12治疗后的抗肿瘤反应是持久的，并提供了完整的保护膀胱内肿瘤再激发。尿细胞因子分析表明，壳聚糖/IL-12诱导多种TH 1细胞因子的水平显着高于单独的IL-12或BCG。免疫组织化学显示，在壳聚糖/IL-12治疗后，T细胞和巨噬细胞中度至强烈的肿瘤浸润。治愈小鼠的膀胱粘膜下层含有残留的免疫细胞群，这些免疫细胞群在几个月后恢复到基线水平。膀胱内注射壳聚糖/IL-12是一种耐受性良好、有效的免疫疗法，值得进一步考虑用于人类浅表性膀胱癌的治疗。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。我们假设肿瘤内（i.t.）施用与可生物降解的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。非侵入性成像研究监测了i.t.注射单独的IL-12和与壳聚糖共配制的IL-12（壳聚糖/IL-12）的抗肿瘤功效在具有已建立的结肠直肠（MC 32a）和胰腺（Panc 02）肿瘤的小鼠中评估。设计了涉及免疫细胞亚群耗竭、肿瘤再激发和CTL活性的其他研究，以阐明消退和肿瘤特异性免疫的机制。与壳聚糖的共制剂使局部IL-12保留从1至2天增加至5至6天。每周i.t.单独注射IL-12根除了小于10%的已建立的MC 32a和Panc 02肿瘤，而i.t.壳聚糖/IL-12免疫疗法在80%至100%的小鼠中引起完全肿瘤消退。CD 4+或Gr-1+细胞的消耗对壳聚糖/IL-12介导的肿瘤消退没有影响。然而，CD 8+或NK细胞耗竭完全消除了抗肿瘤活性。I.T.壳聚糖/IL-12免疫疗法产生了全身肿瘤特异性免疫，因为80%的小鼠用i.t.壳聚糖/IL-12免疫疗法至少部分地保护免于肿瘤再攻击。此外，来自治愈小鼠脾脏的CTL裂解负载MC 32a和gp 70肽的靶标。壳聚糖/IL-12免疫治疗增加了IL-12在肿瘤微环境中的局部保留，根除了已建立的侵袭性小鼠肿瘤，并产生了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以产生Th 1型免疫应答。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，Th 1诱导细胞因子。壳聚糖溶液被设计为在皮下注射部位产生抗原和IL-12的贮库。我们测量了在1.5%壳聚糖谷氨酸盐溶液中配制的含有0.25、1或4 mg鼠IL-12和75 mg卵清蛋白（OVA）的疫苗的体内免疫应答。壳聚糖/IL-12/OVA疫苗，与壳聚糖/OVA，IL-12/OVA，或单独的OVA相比，引起更大的抗原特异性CD 4+和CD 8 + T细胞反应，如通过CD 4+脾细胞增殖，Th 1细胞因子释放，CD 8 + T细胞干扰素-γ释放，和MHC I类肽五聚体染色所确定的。壳聚糖和IL-12的组合也增强了对OVA的IgG 2a和IgG 2b抗体应答。因此，壳聚糖和IL-12的共制剂促进了对模型蛋白疫苗的Th 1免疫应答的产生。

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.