Therapeutic Self-Assembly of Recognition Elements on Cell Surfaces

描述(由申请人提供)：我们为个性化抗癌治疗提出了以下范例：医生确定他/她想要从患者身上消除的细胞类型的独特的基于表面的特征(或“指纹”)。根据指纹与健康细胞指纹的比较，医生将现成的试剂结合起来，给患者服用，特别是杀死这种细胞类型(同时保护类似的非靶细胞和组织)。这个建议有多现实？淋巴瘤和白血病的免疫表型指纹可以很容易地用单抗(MAb)来识别。当前应用的主题是我们范式的第二个组成部分：工程模块化现成试剂，这些试剂可以组合在一起，以触发带有这些独特指纹的目标细胞的死亡，同时保护健康细胞。在这一资助期结束时，我们将为完整的范例提供概念验证实验，并根据免疫表型指纹消除或保护三个模型细胞系(CCRF-CEM、Raji、HL-60)中的任何一个或两个。我们的目标是在未来三年内证明分子混合物可以对细胞表面进行AND而不是逻辑运算，从而产生治疗上适用的结果(例如，细胞死亡或存活)。我们将首先构建模块化试剂，这些试剂可以结合成针对细胞表面标记的寡核苷酸-Fab(抗体片段)结合物。为了选择性地靶向CCRF-CEM、Raji和HL-60细胞系，我们将合成能够靶向CD71、CD5、CD52和CD33的试剂。然后将追求以下具体目标：目标1和逻辑演示：在每个模型细胞系的表面上基于选择性寡核苷酸的Fab-寡核苷酸偶联物对的交联。在这个目标中，我们还将研究在药物释放的情况下和不在药物释放的情况下，由交联剂引发的细胞凋亡/坏死过程。目标2，非逻辑演示：选择性地保护每个模型细胞系免受在所有三种细胞系上发现的药物-寡核苷酸结合靶向标记的作用。在这一资助期结束时，我们将准备在患者的实际样本上测试这种方法，并启动白血病和淋巴瘤小鼠模型的自我组装研究。我们将为个性化抗癌治疗提供以下范例的概念验证：医生确定他/她想要从患者身上消除的细胞类型的独特表面特征(或“指纹”)。根据指纹与健康细胞指纹的比较，医生将现成的试剂结合起来，给患者服用，特别是杀死这种细胞类型(同时保护类似的非靶细胞和组织)。这个建议有多现实？淋巴瘤和白血病的免疫表型指纹可以很容易地用单抗(MAb)来识别。当前应用的主题是我们范式的第二个组成部分：工程模块化现成试剂，这些试剂可以组合在一起，以触发带有这些独特指纹的目标细胞的死亡，同时保护健康细胞。在本资助期结束时，我们将为完整的范例提供概念验证实验，并根据免疫表型指纹消除或保护三个模型细胞系中的任何一个或两个。

DESCRIPTION (provided by applicant): We propose the following paradigm for personalized anti-cancer therapy: a physician determines the unique surface-based characteristic (or "fingerprint") of a cell type that he/she would like to target for elimination from a patient. Guided by the comparison of that fingerprint to the fingerprints of healthy cells, the physician combines off-the-shelf reagents and administers them to the patient, specifically killing that cell type (while protecting similar non-target cells and tissues). How realistic is this suggestion? Immunophenotype fingerprints for lymphomas and leukemias could be readily identified using monoclonal antibodies (MAb's). The topic of the current application is the second component of our paradigm: engineering modular off-the-shelf reagents that may be combined in order to trigger the death of target cells bearing these unique fingerprints, while protecting healthy cells at the same time. At the end of this funding period, we will provide proof-of-concept experiments for the complete paradigm, and either eliminate or protect any one or two out of three model cell lines (CCRF-CEM, Raji, HL-60), based on their immunophenotype fingerprints. It is our goal over the next three years to demonstrate that mixtures of molecules can perform AND and NOT logical operations on cell surfaces with therapeutically applicable outcomes (e.g., cell death or survival). We will first construct modular reagents that can be combined into oligonucleotide-Fab (antibody fragment) conjugates targeting cell- surface markers. In order to target selectively each of CCRF-CEM, Raji, and HL-60 cell lines, we will synthesize the reagents capable of targeting CD71, CD5, CD52, and CD33. The following specific aims will be then pursued: Aim 1, AND Logic Demonstration: Selective oligonucleotide-based crosslinking of pairs of Fab-oligonucleotide conjugates on the surface of each one of the model cell lines. In this aim we will also study apoptotic/necrotic processes triggered by crosslinking with and without drug release. Aim 2, NOT Logic Demonstration: Selective protection of each one of the model cell lines from the action of drug-oligonucleotide conjugate targeting markers found on all three cell lines. At the end of this funding period we will be ready to test this approach on actual samples from patients, and to initiate studies of self-assembly in murine models of leukemia and lymphoma. We will provide a proof-of-concept for the following paradigm for personalized anti- cancer therapy: a physician determines the unique surface-based characteristic (or "fingerprint") of a cell type that he/she would like to target for elimination from a patient. Guided by the comparison of that fingerprint to the fingerprints of healthy cells, the physician combines off-the-shelf reagents and administers them to the patient, specifically killing that cell type (while protecting similar non-target cells and tissues). How realistic is this suggestion? Immunophenotype fingerprints for lymphomas and leukemias could be readily identified using monoclonal antibodies (MAb's). The topic of the current application is the second component of our paradigm: engineering modular off- the-shelf reagents that may be combined in order to trigger the death of target cells bearing these unique fingerprints, while protecting healthy cells at the same time. At the end of this funding period, we will provide proof-of-concept experiments for the complete paradigm, and either eliminate or protect any one or two out of three model cell lines, based on their immunophenotype fingerprints.