Cancer Immunotherapy by Targeting 4-1BB with Novel Immunostimulatory Bispecific Antibodies

近几十年来，免疫疗法彻底改变了癌症治疗，并使癌症免疫学领域重新焕发活力。这种成功的一个关键因素是单克隆抗体（mAb）的开发，其通过结合称为免疫检查点的T细胞抑制分子来促进针对肿瘤的细胞毒性T细胞应答。尽管如此，免疫检查点抑制剂mAb仅在一小部分肿瘤类型中有效，大多数癌症患者未能做出反应。因此，需要额外的治疗方法被强调。靶向T细胞上的共刺激受体的免疫刺激性mAb有望作为靶向不同但互补的免疫调节途径的组合剂。值得注意的是，靶向肿瘤坏死因子受体超家族成员4-1BB的抗体显示出潜力，两种抗人4-1BB（h4-1BB）mAb（称为Urelumab和Utomilumab）已经在临床上使用。然而，激动剂4-1BB抗体的进一步临床开发目前受到与靶向脱肿瘤活性相关的免疫相关毒性的阻碍。此外，目前缺乏对什么赋予抗h4 -1BB抗体共刺激潜力和激动活性的了解。为了试图显示抗癌功效可以与靶向脱肿瘤毒性分离以及提高对赋予抗h4 -1BB抗体共刺激潜力和激动活性的因素的理解，我们开发了一组靶向h4-1BB和人B7-H3（hB 7-H3）的新型双特异性抗体（BsAb）构建体; B7-H3是一种肿瘤相关抗原，通常在肿瘤细胞和肿瘤基质上过度表达。设计所有BsAb构建体以靶向不同的富含半胱氨酸的结构域中的不同h4-1BB表位，以允许我们研究表位、与膜的结合距离和结合亲和力对共刺激潜力和激动活性的影响。使用体外测定来测量与h4-1BB和hB 7-H3的细胞结合以及h4-1BB下游的核因子κ B途径活化，我们迄今为止已经表明，所有我们的新型BsAb构建体都具有共刺激潜力和激动活性，这是由它们交联hB 7-H3的能力促进的。共刺激潜力和激动活性似乎是不依赖于表位的，并且不受与膜的结合距离和结合亲和力的影响。总之，这项工作突出了使用这种形式的新型抗h4 -1BB抗hB 7-H3 BsAb构建体在肿瘤微环境中提供4-1BB共刺激和诱导T细胞活化的潜力。此外，它提出了一个点，从继续BsAb的发展和表征。

In recent decades immunotherapy has revolutionised cancer treatment and rejuvenated the field of cancer immunology. A key factor to this success has been the development of monoclonal antibodies (mAbs) that promote cytotoxic T cell responses against tumours by binding T cell inhibitory molecules known as immune checkpoints. Nonetheless, immune checkpoint inhibitor mAbs are only effective in a small fraction of tumour types with most cancer patients failing to respond. Thus, the need for additional therapeutic approaches is highlighted. Immunostimulatory mAbs targeting co-stimulatory receptors on T cells hold promise as combination agents targeting different yet complementary immune regulatory pathways. Notably, antibodies targeted towards the tumour necrosis factor receptor superfamily member 4-1BB show potential, with two anti-human 4-1BB (h4-1BB) mAbs known as Urelumab and Utomilumab already in the clinic. However, further clinical development of agonist 4-1BB antibodies is currently being hampered by immune-related toxicities associated with on-target off-tumour activity. Moreover, insight into what confers co-stimulatory potential and agonistic activity to anti-h4-1BB antibodies is currently lacking. In attempt to show anti-cancer efficacy can be decoupled from on-target off-tumour toxicity as well as improve understanding of what confers co-stimulatory potential and agonistic activity to anti-h4-1BB antibodies, we developed a panel of novel bispecific antibody (BsAb) constructs targeted towards h4-1BB and human B7-H3 (hB7-H3); B7-H3 is a tumour-associated antigen that is commonly over-expressed on both tumour cells and tumour stroma. All BsAb constructs were designed to target different h4-1BB epitopes across different cysteine-rich domains to allow us to investigate the effects of epitope, binding distance from the membrane and binding affinity on co-stimulatory potential and agonistic activity. Using in vitro assays to measure cellular binding to h4-1BB and hB7-H3 as well as nuclear factor kappa B pathway activation downstream from h4-1BB, we have so far shown that all our novel BsAb constructs have co-stimulatory potential and agonistic activity, facilitated by their ability to cross-link hB7-H3. Co-stimulatory potential and agonistic activity appear to be epitope-independent and unaffected by both binding distance from the membrane and binding affinity. Overall, this work highlights the potential for using novel anti-h4-1BB anti-hB7-H3 BsAb constructs of this format in providing 4-1BB co-stimulation and inducing T cell activation in a tumour microenvironment. Furthermore, it presents a point from which to continue BsAb development and characterisation.