Localizing Immunotherapy to Improve Therapeutic Index

局部免疫治疗以提高治疗指数

• 批准号: 8835080
• 负责人: Karl Dane Wittrup
• 金额: $ 39.54万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-04 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8835080
• 关键词: Affect; Affinity; Antibodies; Antigen Targeting; Antigens; Artificial nanoparticles; Benchmarking; Biological Assay; Biomedical Engineering; Biotinylation; Bispecific Antibodies; Blood Circulation; C57BL/6 Mouse; CD8B1 gene; CTLA4 gene; Cancer Model; Carcinoembryonic Antigen; Cell Surface Receptors; Cells; Chelating Agents; Clinical Trials; Collaborations; Combined Modality Therapy; Complex; Dendritic Cells; Development; Diffuse; Distal; Docking; Dose; Dose-Limiting; Drug Formulations; Effector Cell; Fc domain; Freedom; Genetic Engineering; Genetically Engineered Mouse; Human; Immune; Immune response; Immune system; Immunity; Immunoglobulin G; Immunosuppression; Immunotherapeutic agent; Immunotherapy; Injection of therapeutic agent; Interleukin-12; Interleukin-2; KRAS2 gene; Knock-out; Label; Learning; Ligands; Liposomes; MC38; Malignant Neoplasms; Mediating; Metals; Methods; Modeling; Mus; Myelogenous; Natural Killer Cells; Nature; Pharmaceutical Preparations; Primary Neoplasm; Process; Protein Engineering; Proteins; Protocols documentation; Radioisotopes; Regulatory T-Lymphocyte; Research Personnel; Safety; Signal Transduction; Site; Specificity; Suppressor-Effector T-Lymphocytes; Surface; System; T-Lymphocyte; TNF gene; Testing; Therapeutic; Therapeutic Index; Therapy trial; Time; Tissues; Toxic effect; Transgenic Mice; Transgenic Organisms; Tumor Antibodies; Tumor Antigens; Tumor Tissue; Tumor-Infiltrating Lymphocytes; base; cancer immunotherapy; cell killing; cytokine; design; improved; knowledge base; lung sarcoma; lymph nodes; macrophage; melanoma; mimicry; mutant; nanoparticle; neoplastic cell; novel; novel strategies; public health relevance; recombinase; response; scaffold; small molecule; subcutaneous; tumor; tumor microenvironment; uptake; vector

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).

描述（由申请人提供）：这个新的R01提案是Wittrup和Irvine两位研究者之间的合作，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，利用新的方法在肿瘤组织中局部集中强效免疫刺激分子，可以显著提高癌症免疫治疗的治疗指标，从而提高疗效，降低脱靶毒性。我们将开发两种互补和潜在协同的局部递送方法用于癌症的免疫治疗：预靶向和肿瘤内纳米颗粒注射。这两种方法将被优化，以在同基因和基因工程小鼠肿瘤模型中联合使用。我们将阐明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，该方案提供了螯合剂DOTA的高度肿瘤特异性定位。我们将位点特异性地将DOTA附着在有效载荷IL-2、IL-12、TNF-、ctla4 scFv和cd137 scFv上。之所以选择这些分子，是因为它们在临床试验中具有免疫治疗潜力，同时也存在显著的毒性问题。我们的dota放射性金属螯合物验证方案将适用于dota标记有效载荷的特定递送。我们设计了脂质体和稳定胶束载体用于免疫刺激分子的表面锚定，并证明了它们在肿瘤内注射到B16F10同基因黑色素瘤的有效性和安全性。用于预靶向的相同双特异性抗体将锚定在这些载体的表面，因此完全相同的dota标记有效载荷可以进行模块化测试，而无需重新优化偶联方法。bsAb是一种支架，通过与dota标记的有效载荷的非共价偶联，可以直接模仿免疫细胞因子、双特异性抗体和Fc偶联物。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，使用相同的抗体靶向肿瘤和相同的免疫刺激分子。我们将在表达CEA的转基因小鼠中测试这些方案，皮下接种表达人CEA的B16F10肿瘤。最成功的方案将在皮下MC38-CEA肿瘤中进行进一步测试，然后在肺部和肉瘤的基因工程KP肿瘤中进行测试（通过病毒传递的Cre重组酶敲除flxed p53和停止flxed活化的KRAS表达）。我们将通过最有效的治疗方案仔细检查肿瘤微环境和肿瘤引流淋巴结，以寻找Tregs、tam或MDSCs逆转免疫抑制的证据。我们还将使用缺乏bsAb （CEA）靶向抗原的同基因肿瘤检测保护性免疫和抗原扩散。