Localizing Immunotherapy to Improve Therapeutic Index

描述(由申请人提供)：这个新的R01提案是两个研究人员Wittrup和Irvine之间的合作，结合了蛋白质工程和纳米颗粒合成的专业知识。我们的中心假设是，通过使用新的方法在肿瘤组织中局部聚集强大的免疫刺激分子，可以显著提高肿瘤免疫治疗的治疗指数，从而提高疗效并减少靶外毒性。我们将开发两种互补和潜在协同的局部给药方法用于癌症的免疫治疗：预靶向和肿瘤内纳米颗粒注射。这两种方法将在同基因和基因工程小鼠肿瘤模型中进行联合应用的优化。我们将阐述证明有效的方案的免疫治疗机制。我们已经开发了一种基于双特异性抗体的预靶向方案，它提供了高度肿瘤特异性的螯合剂DOTA的定位。我们将特定站点地将DOTA附加到有效载荷IL-2、IL-12、TNF-？、-CTLA4 scFv和？-CD137 scFv。选择这些分子是因为它们在临床试验中证明了免疫治疗潜力，以及显著的毒性问题。我们验证了DOTA-放射性金属络合物的方案将适用于DOTA标记的有效载荷的特定递送。我们设计了脂质体和稳定的胶束载体用于免疫刺激分子的表面锚定，并通过瘤内注射B16F10同基因黑色素瘤证明了它们的有效性和安全性。用于预靶向的相同双特异性抗体将被锚定在这些载体的表面，因此可以对完全相同的DOTA标记的有效载荷进行模块化测试，而无需重新优化连接方法。BsAb是一种支架，通过与DOTA标记的有效载荷进行非共价连接，可以直接模拟免疫细胞因子、双特异性抗体和Fc结合物。这将使我们能够对我们的新方法的安全性和有效性进行基准测试，以对抗这些更常用的载体，使用相同的肿瘤靶向抗体和相同的免疫刺激分子。我们将在表达CEA的转基因小鼠身上测试这些方案，这些转基因小鼠皮下接种表达人CEA的B16F10肿瘤。最成功的方案将在皮下MC38-CEA肿瘤中进一步测试，然后在肺和肉瘤的基因工程KP肿瘤中进行测试(通过Cre重组酶病毒传递的FLOXED P53基因敲除和Stop-FLOXED激活的KRAS表达)。我们将密切观察经最有效方案治疗后的肿瘤微环境和肿瘤引流淋巴结，以寻找Tregs、TAMs或MDSCs逆转免疫抑制的证据。我们还将使用缺乏bsAb(CEA)靶向抗原的同种肿瘤来测试保护性免疫和抗原扩散。

DESCRIPTION (provided by applicant): This new R01 proposal is a collaboration between two investigators, Wittrup and Irvine, combining protein engineering and nanoparticle synthesis expertise. Our central hypothesis is that the therapeutic index of cancer immunotherapy can be improved significantly by using novel methods to locally concentrate potent immunostimulatory molecules in tumor tissue for increased efficacy and decreased off-target toxicity. We will develop two complementary and potentially synergistic localized delivery methods for immunotherapy of cancer: pretargeting, and intratumoral nanoparticle injection. The two methods will be optimized for combined utility in syngeneic and genetically engineered mouse tumor models. We will explicate the immune therapeutic mechanisms of protocols that demonstrate efficacy. We have developed a bispecific antibody-based pretargeting protocol that provides highly tumor-specific localization of the chelator DOTA. We will site-specifically attach DOTA to the payloads IL-2, IL-12, TNF-¿, ¿-CTLA4 scFv, and ¿-CD137 scFv. These molecules were chosen due to their demonstrated immuno-therapeutic potential in clinical trials, together with significant toxicity issues. Our protocol validated for DOTA-radiometal chelates will be adapted for specific delivery of the DOTA-labeled payloads. We have devised liposomal and stabilized micellar vehicles for surface anchoring of immunostimulatory molecules, and demonstrated their efficacy and safety from intratumoral injection into B16F10 syngeneic melanoma tumors. The same bispecific antibody used for pretargeting will be anchored on the surface of these vehicles, so that the exact same DOTA-labeled payloads can be modularly tested without re-optimization of conjugation methods. The bsAb is a scaffold that enables straightforward mimicry of immunocytokines, bispecific antibodies, and Fc conjugates by noncovalent conjugation with DOTA-labeled payloads. This will enable us to benchmark safety and efficacy of our novel approaches against these more commonly used vehicles, using the same antibody for tumor targeting and identical immunostimulatory molecules. We will test these protocols in transgenic mice expressing CEA, inoculated subcutaneously with B16F10 tumors expressing human CEA. The most successful protocols will be further tested in subcutaneous MC38-CEA tumors, and then in genetically engineered KP tumors in lung and sarcoma (floxed p53 knockout and stop-floxed activated KRAS expression via Cre recombinase delivered virally.) We will closely examine the tumor microenvironment and tumor draining lymph nodes following treatment by the most efficacious protocols, for evidence of reversal of immunosuppression by Tregs, TAMs, or MDSCs. We will also test for protective immunity and antigen spreading using syngeneic tumors lacking the antigen targeted by the bsAb (CEA).