Directing Cytokine Specificity Through Co-translational Carrier Coupling

通过共翻译载体偶联指导细胞因子特异性

• 批准号: 10581947
• 负责人: Yina Hsing Huang
• 金额: $ 20.4万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-08 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10581947
• 关键词: Address; Affinity; Alpha Interleukin 2 Receptor; Amino Acids; Autoimmune Diseases; Autoimmunity; Benchmarking; Binding; Blood; C-terminal; Cancer Model; Cancer Patient; Cells; Circulation; Clinic; Clinical; Clinical Treatment; Clinical Trials; Complex; Continuous Infusion; Coupling; Cytokine Receptors; Cytokine Signaling; DNA Sequence Alteration; Dependence; Disease; Dissociation; Disulfides; Dose; Drug Kinetics; Engineering; Exhibits; Fc Receptor; Filtration; Fluorescence-Activated Cell Sorting; Future; Generations; Genetic; Geometry; Goals; Half-Life; Human; IL2 gene; Immune response; Immunologic Deficiency Syndromes; Immunosuppressive Agents; Immunotherapy; Infection Control; Inflammatory; Insulin-Dependent Diabetes Mellitus; Interferon-beta; Interleukin 2 Receptor; Interleukin-2; Kidney; Label; Leukocytes; Libraries; Link; Longevity; Lupus Nephritis; Lymphocyte Activation; Malignant Neoplasms; Measures; Mediating; Metastatic Melanoma; Modeling; Molecular; Multiple Sclerosis; Mus; Mutation; Natural Killer Cells; Patients; Progression-Free Survivals; Proliferating; Property; Protein Secretion; Proteins; Psoriatic Arthritis; Quality Control; Recombinant Cytokines; Recycling; Regimen; Regulatory T-Lymphocyte; Relapse; Renal carcinoma; Role; Saccharomyces cerevisiae; Safety; Scanning; Serum Albumin; Site; Specificity; Surface; Symptoms; Systemic Lupus Erythematosus; T cell response; T-Lymphocyte; Therapeutic; Therapeutic Uses; Toxic effect; Tumor Immunity; Variant; cancer cell; cancer immunotherapy; cell type; chemokine; chronic autoimmune disease; combinatorial; cost effective; crosslink; cytokine; cytokine therapy; cytotoxic CD8 T cells; design; dimer; disulfide bond; effector T cell; efficacy evaluation; experience; immune activation; immune stimulatory agent; immunogenicity; improved; in vitro Assay; in vivo; interest; interleukin-23; manufacture; melanoma; neonatal Fc receptor; patient subsets; pleiotropism; polypeptide; prevent; protein purification; prototype; receptor; receptor recycling; recurrent infection; response; success

Recombinant cytokines are used in the clinic to treat both chronic autoimmune diseases such as multiple sclerosis and malignancies such as metastatic melanoma, for the purpose of either suppressing autoimmunity or activating tumor immunity. Their considerable success in ameliorating established disease, measured by decreased clinical symptoms of autoimmunity and prolonged progression free survival of cancer patients, support the clinical importance of cytokine therapies, particularly in comparison to broadly immunosuppressive or immunostimulatory drug regimens. Yet, additional improvements are needed because in both clinical settings, cytokine-based therapies suffer from two major challenges, a) tendency of cytokines to induce pleiotropic effects through multiple target cells, which can result in severe toxicities, and b) short cytokine half- lives, which require frequent administration or high doses. We propose to address both issues by disulfide crosslinking cytokines of interest with different surfaces of HSA. We hypothesize that this approach can be generalized to small bioactive proteins to yield precision engineered variants with high receptor selectivity, by virtue of HSA-mediated steric blocking, with extended circulation half-life, by virtue of the fusions’ increased size and potential for neonatal Fc receptor (FcRn) recycling, and with ease in manufacturing and purification of a single molecular entity. In this application, we specifically chose IL-2 as the prototypic cytokine for HSA- disulfide design due to its central role in regulating and activating immune responses through differential binding to trimeric and dimeric IL-2 receptors expressed on regulatory T cells (Tregs) and CD8 cytotoxic cells (CTLs). Treg cells constitutively express all three components of the high affinity IL-2 receptor (IL-2R), which renders them sensitive to relatively low concentrations of IL-23. In contrast, CTLs and natural killer (NK) cells only transiently express the high affinity IL-2R during activation, but more generally express the medium affinity receptor comprised of just IL-2Rβ and γc (IL-2Rβγ). For this reason, high doses of IL-2 are required to activate CTL for the treatment of metastatic renal cancer (mRC) and metastatic melanoma (MM). Low IL-2 doses target Treg and are being explored for the treatment of autoimmune diseases such as type I diabetes (T1D) and systemic lupus erythematosus (SLE). Although current dose differentiated regimens of native IL-2 have shown some promise, the intrinsic overlapping activity toward both CTLs and Treg cells has thus far proved a serious barrier to effective IL-2 immunotherapy. Moreover, as a small 133 amino acid residue protein, native IL-2 is cleared from circulation quickly, requiring continuous infusion to maintain efficacious blood concentrations. To address these critical limitations of IL-2, both its receptor specificity and pharmacokinetics (PK) must be optimized. To achieve these goals, we propose to engineer disulfide cross-linked HSA-cc-IL2 complexes that preferentially activate 1) effector T cells to promote their tumor immunity and 2) regulatory T cells to promote suppression of inflammatory diseases.

重组细胞因子在临床上用于治疗慢性自身免疫性疾病 硬化症和恶性疾病，如转移性黑色素瘤，目的是抑制自身免疫 或者激活肿瘤免疫力。他们在改善既定疾病方面取得了相当大的成功，衡量标准是 减少自身免疫的临床症状，延长癌症患者的无进展生存期， 支持细胞因子治疗的临床重要性，特别是与广泛的免疫抑制相比 或免疫刺激性药物疗法。然而，还需要进一步的改进，因为在临床上 背景下，基于细胞因子的治疗面临两大挑战，a)细胞因子诱导 通过多个靶细胞的多效性效应，这可能导致严重的毒性，以及b)短细胞因子一半- 生命，这需要经常给药或高剂量。我们建议通过二硫化物来解决这两个问题 目的细胞因子与人血清白蛋白不同表面的交联。我们假设这种方法可以是 推广到小的生物活性蛋白，以产生具有高受体选择性的精密工程变体，通过 由于融合增加，HSA介导的空间位阻延长了循环半衰期 新生儿Fc受体(FcRN)回收的大小和潜力，以及易于制造和纯化 一个单一的分子实体。在这个应用中，我们特别选择了IL-2作为HSA的原型细胞因子- 二硫键设计，因其通过差异调节和激活免疫反应的中心作用 与调节性T细胞和CD8细胞毒细胞上表达的三聚体和二聚体IL-2受体的结合 (CTL)。Treg细胞结构性地表达高亲和力IL-2受体(IL-2R)的所有三种成分，这三种成分 使它们对相对较低浓度的IL-23敏感。相比之下，CTL和自然杀伤(NK)细胞 在激活过程中只短暂地表达高亲和力的IL-2R，但更普遍地表达中等亲和力 受体仅由IL-2Rβ和γc(IL-2Rβγ)组成。因此，需要高剂量的IL-2才能激活 CTL治疗转移性肾癌(MRC)和转移性黑色素瘤(MM)。低剂量IL-2靶点 Treg和正在探索用于治疗自身免疫性疾病，如I型糖尿病(T1D)和 系统性红斑狼疮(SLE)。尽管目前天然IL-2的不同剂量方案已经显示 一些人承诺，到目前为止，针对CTL和Treg细胞的内在重叠活性被证明是一个严重的 有效的IL-2免疫治疗的障碍。此外，作为一种133个氨基酸残基的小蛋白，天然的IL-2是 迅速从循环中清除，需要持续输液以维持有效的血液浓度。至 要解决IL-2的这些关键限制，其受体特异性和药代动力学(PK)都必须 最优化。为了实现这些目标，我们建议设计二硫键交联的hsa-cc-IL2络合物，以 优先激活1)效应T细胞促进其肿瘤免疫和2)调节性T细胞促进 抑制炎症性疾病。