A novel nanobody-based agonist-redirected checkpoint (ARC) molecule, aPD1-Fc-OX40L, for cancer immunotherapy

免疫检查点抑制剂（ICI）在广泛的恶性肿瘤中显示出前所未有的临床活性。 然而，由于原发性或获得性耐药，它们在许多恶性肿瘤中的疗效仍然有限。针对性 激活肿瘤坏死因子受体超家族（TNFRSF）成员（如OX 40和4-1BB）的疗法， 目前正在探索增加ICI的临床疗效。在本研究中，我们构建了一种新型的纳米抗体 (Nb)基于激动剂的重定向检查点（ARC）平台，可巩固免疫检查点阻断（ICB） 和TNFRSF激动作用。我们的长期目标是利用这个基于Nb的ARC平台开发 治疗犬和人类癌症患者的新型免疫治疗剂。本R15提案的目标是 研究我们的第一个基于Nb的ARC分子aPD 1-Fc-OX 40 L对局部和全身抗肿瘤作用的影响 口腔恶性黑色素瘤（OM）宠物犬免疫应答。aPD 1-Fc-0X 40 L利用抗PD 1抗体 纳米抗体以破坏PD 1/PD-L1轴和OX 40配体的细胞外结构域（ECD）以激活PD 1/PD-L1轴。 OX 40途径。我们的初步数据显示，aPD 1-Fc-0X 40 L折叠正确结合同源受体， 高亲和力，激活OX 40通路，并阻断犬PD-L1与PD 1的结合。我们的具体目标是：（目标 1)评估aPD 1-Fc-0X 40 L的安全性和药代动力学（PK）特征;（目的2）进行II/III期临床试验， aPD 1-Fc-OX 40 L在患有口腔黑素瘤（OM）的犬患者中的试验;以及（目的3）评估功能活性 aPD 1-Fc-0X 40 L的治疗效果。我们将进行临床相关研究（使用流式细胞术和 免疫组织化学（IHC））对给药前和给药后收集的肿瘤和外周血样品进行分析 aPD 1-Fc-OX 40 L的细胞和分子机制，以了解决定抗肿瘤免疫的细胞和分子机制。 反应在得出结论后，我们将获得对安全性、PK特征和临床疗效的有价值的见解， aPD1-Fc-0X40L。这一贡献是重要的，因为这将是第一项评价伴随PD 1的研究。 阻断和OX 40激动在高度相关的大型动物模型的人粘膜黑色素瘤。数据 从这项研究中收集的信息将为采用类似的治疗策略治疗人类癌症创造一个蓝图 患者该研究具有创新性，因为我们开发了一种高度灵活的Nb基ARC 该平台可以容易地适应于工程化靶向其他免疫系统的新型免疫系统组合， 检查点和TNFRSF受体。这些物种特异性免疫治疗剂的可用性将允许 将患有自发性肿瘤的犬患者纳入免疫肿瘤学研究，这将使双方受益 人类和宠物狗。它将为狗提供尖端的癌症治疗，同时确保 人们接受的治疗更有可能成功。

Immune checkpoint inhibitors (ICIs) have shown unprecedented clinical activity in a wide range of malignancies. However, their efficacy remains limited in many malignancies due to primary or acquired resistance. Targeted therapies that activate tumor necrosis factor receptor superfamily (TNFRSF) members, like OX40 & 4-1BB, are currently explored to augment the clinical efficacy of ICIs. In this study, we have constructed a novel-nanobody (Nb) based Agonist-Redirected Checkpoint (ARC) platform that consolidates immune checkpoint blockade (ICB) and TNFRSF agonism in a single biologic. Our long-term goal is to utilize this Nb-based ARC platform to develop novel immunotherapeutics to treat canine and human cancer patients. The objective of this R15 proposal is to investigate the impact of our first Nb-based ARC molecule, aPD1-Fc-OX40L, on local and systemic antitumor immune response in pet dogs with oral malignant melanoma (OM). aPD1-Fc-OX40L utilizes an anti-PD1 nanobody to disrupt the PD1/PD-L1 axis and the extracellular domain (ECD) of the OX40 ligand to activate the OX40 pathway. Our preliminary data show that aPD1-Fc-OX40L folds properly binds to cognate receptors with high affinity, activates the OX40 pathway, and blocks canine PD-L1 binding to PD1. Our specific aims will: (Aim 1) assess the safety and pharmacokinetic (PK) profile of aPD1-Fc-OX40L; (Aim 2) conduct a phase II/III clinical trial of aPD1-Fc-OX40L in canine patients with oral melanoma (OM); and (Aim 3) assess the functional activities of aPD1-Fc-OX40L over monotherapies. We will perform clinical correlative studies (using flow cytometry and immunohistochemistry (IHC)) on tumor and peripheral blood samples collected before and after administration of aPD1-Fc-OX40L to understand the cellular and molecular mechanisms that determine the antitumor immune response. Upon conclusion, we will gain valuable insights into the safety, PK profile, and clinical efficacy of aPD1-Fc-OX40L. This contribution is significant since this will be the first study to evaluate concomitant PD1 blockade and OX40 agonism in a highly relevant large animal model of human mucosal melanoma. The data gathered from this study will create a blueprint to employ a similar therapeutic strategy to treat human cancer patients. The proposed research is innovative because we have developed a highly flexible Nb-based ARC platform that can be easily adapted to engineer novel immunotherapeutic combinations targeting other immune checkpoints and TNFRSF receptors. The availability of these species-specific immunotherapeutics will allow the integration of canine patients with spontaneous tumors into immune-oncology research, which will benefit both humans and pet dogs. It will provide dogs with access to cutting-edge cancer treatments while ensuring that people are given treatments that are more likely to succeed.