Targeting Effector Immune cells to Cancer with Chemically Self-Assembled Nanorings (CSANs)

使用化学自组装纳米环 (CSAN) 将效应免疫细胞靶向癌症

• 批准号: 10600820
• 负责人: CARSTON R. WAGNER
• 金额: $ 55.91万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10600820
• 关键词: Affinity; Antibiotics; Antibodies; Antigen Targeting; Antigens; Avidity; B lymphoid malignancy; Binding; Bispecific Antibodies; Bite; Breast Cancer Cell; CD3 Antigens; Cancer Model; Cancer Patient; Cell Adhesion Molecules; Cell membrane; Cell surface; Cells; Chemicals; Chimeric Proteins; Clinical; Consumption; Continuous Infusion; Development; Dihydrofolate Reductase; Dose; Effector Cell; Engineering; Epithelial Cells; FDA approved; Genetic Engineering; Heterogeneity; Immune; Immunotherapy; In Vitro; Incubated; Inflammatory; Libraries; Ligands; Malignant Neoplasms; Methodology; Methods; Methotrexate; Molecular; Mus; Natural Killer Cells; Normal tissue morphology; Oligonucleotides; Patients; Pharmaceutical Preparations; Primary Neoplasm; Progression-Free Survivals; Proliferating; Prosthesis; Resistance; Resistance development; Safety; Solid Neoplasm; Specificity; Surface Antigens; System; T cell therapy; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Time; Toxic effect; Trimethoprim; Tumor Antigens; Tumor Markers; Tumor-infiltrating immune cells; arm; bi-specific T cell engager; cancer cell; cancer immunotherapy; cancer stem cell; cancer therapy; chemical addition; chimeric antigen receptor; chimeric antigen receptor T cells; clinical development; clinically relevant; combat; cost effective; crosslink; cytokine; cytotoxicity; fluorophore; genetic approach; in vivo; method development; neoplastic cell; non-genetic; patient derived xenograft model; pharmacologic; pre-clinical; radiotracer; self assembly; success; triple-negative invasive breast carcinoma; trispecific killer engager; tumor; tumor eradication; tumor initiation

Of the many immunotherapy approaches under development, the ability to use bispecific antibodies or chimeric antigen receptors (CARs) to direct T-cells to selectively kill tumor cells has demonstrated significant early success. Typically, bispecific antibodies or bispecific T-cell engagers (i.e., BiTes) cross-link T-cells by binding to CD3 and to a target cancer cell surface antigen, usually through a monovalent interaction. An alternative approach is to genetically engineer a cancer patient’s T-cells to express a single chain antibody (scFv)-CD3ζ fusion protein that can target the cancer cell surface antigen. After re-introduction into the patient, CAR-expressing T-cells have been able to selectively eliminate the target cancer cells. While successful, the genetic engineering of cell surfaces is time consuming and irreversible and the use of BiTes requires continuous infusion. Furthermore, the resistance to both approaches due to antigen loss has been observed. Our group has shown that two dihydrofolate reductase molecules (DHFR2) fused to an αCD3 single chain antibody (scFv) can be engineered to spontaneously self-assemble upon the addition of the chemical dimerizer, bis-methotrexate (BisMTX), into either highly stable octavalent chemically self-assembled nanorings (CSANs). CSANs have been prepared with BisMTX containing a third arm, thus enabling it to be conjugated to oligonucleotides, fluorophores, radiolabels and drugs. Recently, we have prepared αEpCAM/αCD3 CSANs and αCD133/αCD3-CSANs. The bispecific CSANs rapidly (min) and stably (days) bind to CD3 on T-cell membranes, thus forming chemically self assembled prosthetic antigen receptor (PAR) T-cells. Upon incubation of the PAR T-cells with EpCAM+ and/or CD133+ cancer cells, rapid and selective killing of primary and tumor initiating cancer stem cells (CSC) was observed. We have also demonstrated with an orthotopic murine cancer model that αEpCAM and αCD133 PAR T-cells are non-toxic and able in combination to eradicate tumors in vivo. A unique safety feature of our approach is the ability to remove the CSANs from the T-cells by dosing with the FDA-approved non-toxic antibiotic trimethoprim at clinically relevant concentrations, thus allowing us to deactivate the cells pharmacologically and reduce cytokine release. Consequently, as an alternative to current approaches, we determine the generality T-cell induced killing and eradication of TNBC tumors with αEpCAM/α-CD3-CSANS and αCD133/αCD3-CSANS. In addition, we will develop a tripspecific αEpCAM/αCD133/αCD3 CSANs that will allow the simultaneous elimination of TNBC primary tumor cells and CSC. The successful completion of the project milestones should result in the elucidation of the key features governing a chemical biologically based non-genetic and reversible method for T-cell targeting, as well as the importance of CD133 on TNBC proliferation. These rules will be applicable to the clinical development of anti-cancer immunotherapy with greater selectivity, lower toxicity and a reduced ability for the development of resistance.

在开发的许多免疫疗法方法中，使用双特异性抗体或 嵌合抗原受体（CAR）直接杀死肿瘤细胞的T细胞已显示出显着的 早期成功。通常，双特异性抗体或双特异性T细胞探测器（即叮咬）交联的T细胞通过 通常通过单价相互作用与CD3和目标癌细胞表面抗原结合。一个 替代方法是在基因上设计癌症患者的T细胞以表达单链抗体 （SCFV）-CD3ζ融合蛋白，可以靶向癌细胞表面抗原。重新引入患者后， 表达汽车的T细胞能够选择性地消除目标癌细胞。虽然成功， 细胞表面的基因工程是耗时且不可逆转的，叮咬的使用需要 连续输注。此外，已经观察到由于抗原损失引起的两种方法的抗性。 我们的小组表明，两个二氢叶酸还原分子（DHFR2）融合到αCD3单链 抗体（SCFV）可以在添加化学品后设计为自我组装 二聚体，双甲状腺素（BISMTX），将任何一个高度稳定的八度八度纳米组装成化学自我组装的纳米 （CSAS）。 CSAS已使用包含第三臂的BISMTX制备，从而使其能够共轭到 寡核苷酸，荧光团，放射性标记和药物。最近，我们准备了αepcam/αcd3csans 和αCD133/αCD3-CSAN。双特异性CSAS迅速（最小）和稳定（天）与T细胞上的CD3结合 膜，从而形成化学自我组装的假体抗原受体（PAR）T细胞。之上 将PAR T细胞与EPCAM+和/或CD133+癌细胞一起孵育，初级迅速而有选择性地杀死 观察到肿瘤引发的癌症干细胞（CSC）。我们还用原位演示 αEPCAM和αCD133PAR T细胞无毒的鼠类癌模型，可以结合使用 根除体内肿瘤。我们方法的独特安全功能是能够从 通过与FDA批准的非毒性抗生素三甲氧苄啶的剂量，以临床相关的浓度， 从而使我们能够停用药物的细胞并减少细胞因子释放。 因此，作为当前方法的替代方法，我们确定了一般性T细胞诱导的杀戮 TNBC肿瘤和αepCAM/α-CD3-CSAS和αCD133/αCD3-CSAS的辐射。此外，我们将 开发TripspificαEPCAM/αCD133/αCD3CSAS，该CSAS将允许简单消除TNBC 原发性肿瘤细胞和CSC。项目里程碑的成功完成应导致 阐明负责化学上基于化学生物学的非遗传和可逆方法的关键特征 T细胞靶向以及CD133对TNBC增殖的重要性。这些规则将适用于 抗癌免疫疗法的临床发展具有更大的选择性，较低的毒性和降低 抵抗发展的能力。