PDF(Pubmed)
Abstract：
UNASSIGNED: Multiple myeloma (MM) remains incurable, despite the advent of chimeric antigen receptor (CAR)-T cell therapy. This unfulfilled potential can be attributed to two untackled issues: the lack of suitable CAR targets and formats. In relation to the former, the target should be highly expressed and reluctant to shedding; two characteristics that are attributed to the CS1-antigen. Furthermore, conventional CARs rely on scFvs for antigen recognition, yet this withholds disadvantages, mainly caused by the intrinsic instability of this format. VHHs have been proposed as valid scFv alternatives. We therefore intended to develop VHH-based CAR-T cells, targeting CS1, and to identify VHHs that induce optimal CAR-T cell activation together with the VHH parameters required to achieve this.
UNASSIGNED: CS1-specific VHHs were generated, identified and fully characterized, in vitro and in vivo. Next, they were incorporated into second-generation CARs that only differ in their antigen-binding moiety. Reporter T-cell lines were lentivirally transduced with the different VHH-CARs and CAR-T cell activation kinetics were evaluated side-by-side. Affinity, cell-binding capacity, epitope location, in vivo behavior, binding distance, and orientation of the CAR-T:MM cell interaction pair were investigated as predictive parameters for CAR-T cell activation.
UNASSIGNED: Our data show that the VHHs affinity for its target antigen is relatively predictive for its in vivo tumor-tracing capacity, as tumor uptake generally decreased with decreasing affinity in an in vivo model of MM. This does not hold true for their CAR-T cell activation potential, as some intermediate affinity-binding VHHs proved surprisingly potent, while some higher affinity VHHs failed to induce equal levels of T-cell activation. This could not be attributed to cell-binding capacity, in vivo VHH behavior, epitope location, cell-to-cell distance or binding orientation. Hence, none of the investigated parameters proved to have significant predictive value for the extent of CAR-T cell activation.
UNASSIGNED: We gained insight into the predictive parameters of VHHs in the CAR-context using a VHH library against CS1, a highly relevant MM antigen. As none of the studied VHH parameters had predictive value, defining VHHs for optimal CAR-T cell activation remains bound to serendipity. These findings highlight the importance of screening multiple candidates.
UNASSIGNED: CS1-specific VHHs were generated, identified and fully characterized, in vitro and in vivo. Next, they were incorporated into second-generation CARs that only differ in their antigen-binding moiety. Reporter T-cell lines were lentivirally transduced with the different VHH-CARs and CAR-T cell activation kinetics were evaluated side-by-side. Affinity, cell-binding capacity, epitope location, in vivo behavior, binding distance, and orientation of the CAR-T:MM cell interaction pair were investigated as predictive parameters for CAR-T cell activation.
UNASSIGNED: Our data show that the VHHs affinity for its target antigen is relatively predictive for its in vivo tumor-tracing capacity, as tumor uptake generally decreased with decreasing affinity in an in vivo model of MM. This does not hold true for their CAR-T cell activation potential, as some intermediate affinity-binding VHHs proved surprisingly potent, while some higher affinity VHHs failed to induce equal levels of T-cell activation. This could not be attributed to cell-binding capacity, in vivo VHH behavior, epitope location, cell-to-cell distance or binding orientation. Hence, none of the investigated parameters proved to have significant predictive value for the extent of CAR-T cell activation.
UNASSIGNED: We gained insight into the predictive parameters of VHHs in the CAR-context using a VHH library against CS1, a highly relevant MM antigen. As none of the studied VHH parameters had predictive value, defining VHHs for optimal CAR-T cell activation remains bound to serendipity. These findings highlight the importance of screening multiple candidates.
摘要：
■多发性骨髓瘤（MM）仍然无法治愈，尽管出现了嵌合抗原受体（CAR）-T细胞疗法。这种未实现的潜力可归因于两个未解决的问题：缺乏合适的CAR目标和格式。相对于前者,目标应该是高表达和不愿意脱落;归因于CS1抗原的两个特征。此外，传统的CAR依赖于scFvs进行抗原识别，然而，这隐瞒了不利之处，主要是由这种格式的内在不稳定性造成的。已提出VHH作为有效的scFv替代物。因此，我们打算开发基于VHH的CAR-T细胞，靶向CS1，并鉴定诱导最佳CAR-T细胞激活的VHH以及实现此目的所需的VHH参数。
■生成了CS1特定的VHH，识别和充分表征，在体外和体内。接下来,它们被整合到第二代CAR中,这些CAR的抗原结合部分仅不同.用不同的VHH-CAR慢病毒转导报告T细胞系,并且并排评估CAR-T细胞活化动力学。亲和力，细胞结合能力，表位位置，体内行为，绑定距离,研究了CAR-T:MM细胞相互作用对的方向作为CAR-T细胞活化的预测参数。
■我们的数据表明，VHHs对其靶抗原的亲和力对其体内肿瘤示踪能力具有相对预测性，因为在MM的体内模型中，肿瘤摄取通常随着亲和力的降低而降低。这并不适用于他们的CAR-T细胞激活潜力，因为一些中间亲和力结合VHHs被证明非常有效，而一些较高亲和力的VHH未能诱导相等水平的T细胞活化。这不能归因于细胞结合能力，体内VHH行为，表位位置，细胞间距离或结合方向。因此，没有一个研究的参数证明对CAR-T细胞活化的程度具有显著的预测价值。
■我们使用针对高度相关的MM抗原CS1的VHH文库深入了解了CAR背景下VHH的预测参数。由于研究的VHH参数都没有预测价值，定义最佳CAR-T细胞激活的VHHs仍然与偶然性有关。这些发现强调了筛选多个候选人的重要性。
■生成了CS1特定的VHH，识别和充分表征，在体外和体内。接下来,它们被整合到第二代CAR中,这些CAR的抗原结合部分仅不同.用不同的VHH-CAR慢病毒转导报告T细胞系,并且并排评估CAR-T细胞活化动力学。亲和力，细胞结合能力，表位位置，体内行为，绑定距离,研究了CAR-T:MM细胞相互作用对的方向作为CAR-T细胞活化的预测参数。
■我们的数据表明，VHHs对其靶抗原的亲和力对其体内肿瘤示踪能力具有相对预测性，因为在MM的体内模型中，肿瘤摄取通常随着亲和力的降低而降低。这并不适用于他们的CAR-T细胞激活潜力，因为一些中间亲和力结合VHHs被证明非常有效，而一些较高亲和力的VHH未能诱导相等水平的T细胞活化。这不能归因于细胞结合能力，体内VHH行为，表位位置，细胞间距离或结合方向。因此，没有一个研究的参数证明对CAR-T细胞活化的程度具有显著的预测价值。
■我们使用针对高度相关的MM抗原CS1的VHH文库深入了解了CAR背景下VHH的预测参数。由于研究的VHH参数都没有预测价值，定义最佳CAR-T细胞激活的VHHs仍然与偶然性有关。这些发现强调了筛选多个候选人的重要性。
