Design of highly immunogenic immunogens for development of monoclonal antibodies to selfpeptides: a combined simulation and experimental study

单克隆抗体（mAb）疗法是一种免疫疗法，它使用高度特异性的抗体与特定的细胞或蛋白质结合，以刺激免疫系统破坏这些细胞。因此它可以用来治疗癌症。这个过程包括将来自肿瘤的小成分（肽）结合到纳米颗粒上。用这些被肽包被的纳米颗粒免疫会刺激脾细胞产生一系列抗体，这些抗体有可能摧毁生长中的肿瘤。利用杂杂瘤技术，单个分离和克隆的脾脏细胞可以产生仅针对一个靶点（单克隆抗体）的高度特异性抗体，并可以在工业规模上分离和培养，用于开发单克隆抗体治疗药物。然而，需要进行大量的研究，不仅要找到正确的肽，还要确保它们以正确的方向与纳米颗粒结合，以便免疫系统随后识别“自肽”作为目标。我们将首先使用模型自肽（促性腺激素释放激素，GnRH），我们之前已经通过模拟和实验进行了研究，在那里我们研究了如何最好地将GnRH结合到二氧化硅纳米颗粒（SiNPs）。我们模拟了GnRH（天然）和半胱氨酸标记的修饰类似物（cys-GnRH）在二氧化硅上的吸附，发现天然肽通过其精氨酸残基吸附，使其N（氨基）和C（羧基）端松散结合，可用于进一步的相互作用。相反，吸附的cyys - gnrh仅将其n端暴露于溶液中。我们还模拟了cys-GnRH共价偶联到BSA（在碳二亚胺化学反应后实现交联），发现这导致更好的GnRH表位呈现到溶液中。我们还利用吸附在sinp上的肽和bsa -肽- sinp系统进行了一系列免疫学研究。我们发现GnRH-SiNP由于其两个末端残基的可用性而诱导药物作用，而cys-GnRH-SiNP则没有。我们还发现BSA偶联系统有效地诱导抗体产生。这种免疫和激素反应的范围是通过模拟结果和多肽溶液的呈现来解释的。因此，这项工作不仅为在分子尺度上深入了解治疗作用铺平了道路，更重要的是，为在分子模拟的指导下合理设计药物输送和疫苗系统铺平了道路；我们将在这个项目中采用这种新策略。为了演示该技术的实用性，我们将在项目中使用一个癌症应用程序。建模将用于选择最佳候选者，并对其进行修改以与sinp结合，并设计最佳呈现条件。设计的材料将用于实验，随后的抗体和细胞因子将用于评估肿瘤细胞杀伤潜力。最佳候选药物将被推进单克隆抗体治疗。该项目将汇集来自斯特拉斯克莱德大学和TAC的多学科监督团队。该学生的监督将来自化学和过程工程系、斯特拉斯克莱德制药和生物医学科学研究所以及阿奇西超级计算机中心。我们还将与格拉斯哥卡利多尼亚大学和格拉斯哥大学密切合作，他们将提供患者的临床样本，并提供患者信息。工业主管将是埃里克·瓦格纳（TAC）。里程碑1：肽修饰和计算建模（1-12个月，Mulheran博士，Kubiak-Ossowska实验室）里程碑2：免疫原制备和免疫增强评估（13-18个月，Ferro博士实验室，TAC）里程碑3：癌症研究（19-36个月，Mulheran博士，Ferro与Williams和Souter实验室合作）里程碑4：传播和撰写（43-48个月）；包括知识产权受到保护后的出版物。

Monoclonal antibody (mAb) therapy is an immunotherapy that uses highly specific antibodies to bind to particular cells or proteins, in order to stimulate the immune system to destroy those cells. It can therefore be used to target cancers. The process involves binding small components (peptides) derived from tumours to nanoparticles. Immunisation with these peptide-coated nanoparticles results in stimulation of spleen cells to produce a range of antibodies that have the potential to destroy growing tumours. Using hybridoma technology, individually isolated and cloned spleen cells that produce highly specific antibodies to only one target (mAbs) can be isolated and grown on an industrial scale for the development of mAb therapeutics. However, a lot of research is required to not only find the right peptides, but to then ensure they bind to the nanoparticle with the correct orientation so that the immune system subsequently identifies the "self-peptides" as targets.We will initially use a model self-peptide (gonadotrophin releasing hormone, GnRH), that we have previously studied using simulation and experiment, where we investigated how best to conjugate GnRH to silica nanoparticles (SiNPs). We simulated the adsorption of GnRH(native) and a modified analogue that was cysteine-tagged (cys-GnRH) to silica, and found that the native peptide adsorbed via its arginine residue to leave its N (amino) and C (carboxyl) termini loosely bound and available for further interactions. In contrast, the adsorbed cys-GnRH exposed only its N-terminus to solution. We also simulated cys-GnRH covalently conjugated to BSA (following a carbodiimide chemical reaction to enable crosslinking), finding that this led to better GnRH epitope presentation to solution. A series of immunological studies was also conducted with the peptides adsorbed to SiNPs and with the BSA-peptide-SiNP systems. We found that the GnRH-SiNP induced a drug effect due to its availability of both terminal residues, whereas the cys-GnRH-SiNP did not. We also found that the BSA conjugate systems effectively induced antibody production. This range of immunological and hormonal response is explained by the simulation results and the presentation of the peptides to solution. Hence, this work paves the way for not only a molecular scale insight into therapeutic action, but more significantly, for a rational design of drug delivery and vaccine systems guided by molecular simulation; it is this new strategy that we will exploit in this project.To demonstrate the utility of the technology, we will use a cancer application in the project. Modelling will be used to select the best candidates with modification to bind onto SiNPs and design conditions for optimum presentation. The designed materials will be deployed experimentally, and the subsequent antibodies and cytokines will be evaluated for tumour cell killing potential. The best candidate(s) will be taken forward into a mAb therapeutic(s).The project will bring together a multi-disciplinary supervisory team from the University of Strathclyde and TAC. Supervision of the student will come from the Department of Chemical and Process Engineering, the Strathclyde Institute of Pharmacy and Biomedical Sciences, and the ARCHIE-WeSt supercomputer centre. We will also work in close collaboration with Glasgow Caledonian University and University of Glasgow who will supply clinical samples from patients and also provide access to patient information. The industrial supervisor will be Eric Wagner (TAC).Milestone 1: Peptide Modification and Computational Modelling (Months 1-12; Drs Mulheran, Kubiak-Ossowska labs)Milestone 2: Immunogen Preparation and Immune Enhancement Evaluation (Months 13-18; Dr Ferro labs, TAC)Milestone 3: Cancer studies (Months 19-36; Drs Mulheran, Ferro in collaboration with Williams and Souter labs)Milestone 4: Dissemination and Write-up (Months 43-48); including publications once IP has been protected.