Directing Cytokine Specificity Through Co-translational Carrier Coupling

重组细胞因子在临床上用于治疗慢性自身免疫性疾病，如多发性骨髓瘤和慢性自身免疫性骨髓瘤。 硬化和恶性肿瘤如转移性黑色素瘤，用于抑制自身免疫 或激活肿瘤免疫。他们在改善既定疾病方面取得了相当大的成功， 降低自身免疫的临床症状和延长癌症患者的无进展生存期， 支持细胞因子疗法的临床重要性，特别是与广泛的免疫抑制疗法相比， 或免疫刺激药物方案。然而，需要额外的改进，因为在两个临床 在这种情况下，基于精氨酸的疗法遭受两个主要挑战，a）细胞因子诱导 通过多个靶细胞的多效性效应，这可导致严重的毒性，和B）短的细胞因子半衰期， 生命，这需要频繁的管理或高剂量。我们建议通过二硫化物解决这两个问题 将感兴趣的细胞因子与HSA的不同表面交联。我们假设这种方法可以 一般化到小的生物活性蛋白质，以产生具有高受体选择性的精确工程化变体， 由于HSA介导的空间阻滞，具有延长的循环半衰期，由于融合物的增加， 新生儿Fc受体（FcRn）再循环的大小和潜力，并且易于制造和纯化 一个单一的分子实体在本申请中，我们特别选择IL-2作为HSA的原型细胞因子。 二硫键设计，因为其在通过差异表达调节和激活免疫应答中的中心作用， 与在调节性T细胞（T细胞）和CD 8细胞毒性细胞上表达的三聚体和二聚体IL-2受体的结合 （CTL）。Treg细胞组成型表达高亲和力IL-2受体（IL-2 R）的所有三种组分， 使它们对相对低浓度的IL-23敏感。相反，CTL和自然杀伤（NK）细胞 在活化过程中仅瞬时表达高亲和力IL-2 R，但更普遍地表达中等亲和力IL-2 R IL-2 R βγ是由IL-2 R β和γc组成的受体。因此，需要高剂量的IL-2来激活 CTL用于治疗转移性肾癌（mRC）和转移性黑色素瘤（MM）。低IL-2剂量目标 Treg和正在探索用于治疗自身免疫性疾病，如I型糖尿病（T1 D）和 系统性红斑狼疮（SLE）。尽管目前天然IL-2的剂量分化方案已经显示 尽管有一些希望，但迄今为止，对CTL和Treg细胞的内在重叠活性已被证明是严重的免疫缺陷。 有效的IL-2免疫治疗的障碍。此外，作为一种小的133个氨基酸残基的蛋白质，天然IL-2是 快速从循环中清除，需要持续输注以维持有效的血药浓度。到 为了解决IL-2的这些关键限制，必须同时考虑其受体特异性和药代动力学（PK）。 优化为了实现这些目标，我们提出工程化二硫键交联的HSA-a-IL 2复合物， 优先激活1）效应T细胞以促进其肿瘤免疫和2）调节T细胞以促进其肿瘤免疫。 抑制炎性疾病。

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.