A Novel Melanoma Vaccine Based on a Shared Neoantigen and a Liquid Polymer Platform

摘要 尽管免疫检查点抑制剂和靶向治疗在结果上有了显著改善， 大多数患有转移性BRAF突变的黑素瘤的患者最终经历其疾病的进展。一 对免疫检查点抑制剂耐药的关键原因是缺乏黑素瘤特异性T细胞， 不幸的是，大多数旨在扩增黑色素瘤特异性T细胞的早期黑色素瘤疫苗都失败了， 以证明其益处。下一代测序和计算机肽-HLA结合预测已经允许 研究人员为癌症患者接种新抗原疫苗，这些新抗原是任何正常人都不表达的肽。 组织，但只在肿瘤中发现。虽然新抗原疫苗具有很大的前景，但一个关键的限制是， 事实上，新抗原通常是私有的，因此不可能制造"现成的"新抗原疫苗 这将是适合一个大的病人群体。我们和其他人发现黑色素瘤患者可以 产生对突变的BRAF（在位置600处缬氨酸变为谷氨酸）的内源性T细胞应答。我们有 进一步证明了HLA-A2结合突变的BRAF肽（mBRAFp）在HLA-A2中具有高度免疫原性。 A2转基因小鼠。在这里，我们提出，免疫原性疫苗靶向V600E突变的BRAF在 在HLA-A2的背景下具有扩增黑素瘤特异性T细胞的潜力。作为共有的新抗原，mBRAFp是 约20 - 25%的黑素瘤患者（40 - 50%的黑素瘤患者）的相关肿瘤靶标 携带BRAF V600E的患者x 50%携带HLA-A2的患者）。这种现成的治疗性疫苗可以用于 作为高危切除黑色素瘤患者或肿瘤负荷最小患者的单药治疗。也可能是 联合抗PD-1治疗免疫检查点抑制剂治疗难治性黑色素瘤患者 和靶向治疗。我们的疫苗由mBRAF β和瑞喹莫特（RSQ）作为佐剂配制而成， CAPRO ™-一种新型的专有生物可降解液体聚合物，可局部控制释放 抗原和佐剂。先前我们发现（1）用卵清蛋白（OVA）和RSQ负载的疫苗接种的小鼠 在CAPRO中产生了持久的OVA特异性抗体和CD8 T细胞应答，以及（2）CAPRO负载的 mBRAF0和RSQ在HLA-A2转基因小鼠中引发抗原特异性T细胞应答。基于这些 我们已经启动了涉及疫苗CMC的临床前产品开发计划， 成分和配方开发，并已收到FDA的书面意见，以回应我们的 IND前调查。本项目的目标是定义疫苗产品，并为全面CMC和 用于IND申请的药理学/毒理学研究。为了实现这一目标，我们将首先评估 我们的疫苗产品在表达mBRAFp的HLA-T细胞中的预防性和治疗性抗肿瘤功效和安全性。 A2转基因小鼠模型，与使用Montanide（一种临床使用的疫苗）的制剂直接比较 佐剂系统）。我们还将探索将疫苗与免疫检查点抑制相结合的潜力 （抗PD1）。然后，我们将开发疫苗成分和最终疫苗产品的分析检测方法， 生成IND支持数据，以满足FDA的CMC要求。

Abstract Despite the dramatic improvement in outcomes afforded by immune checkpoint inhibitors and targeted therapies, most patients with metastatic BRAF-mutated melanoma ultimately experience progression of their disease. One key reason for resistance to immune checkpoint inhibitors is a paucity of melanoma-specific T cells, but unfortunately, the majority of early melanoma vaccines meant to expand melanoma-specific T cells have failed to demonstrate benefit. Next generation sequencing and in silico peptide-HLA binding prediction have allowed investigators to vaccinate cancer patients against neoantigens – peptides that are not expressed by any normal tissues but are only found in tumors. Although neoantigen vaccines hold great promise, a key limitation is the fact that neoantigens are generally private, thus it is not possible to make an “off-the-shelf” neoantigen vaccine that will be appropriate for a large group of patients. We and others have found that melanoma patients can mount an endogenous T cell response to a mutated BRAF (valine to glutamic acid at position 600). We have further demonstrated that an HLA-A2-binding mutated BRAF peptide (mBRAFp) is highly immunogenic in HLA- A2-transgenic mice. Here we propose that an immunogenic vaccine targeting V600E-mutated BRAF in the context of HLA-A2 has the potential to expand melanoma-specific T cells. As a shared neoantigen, mBRAFp is a relevant tumor target for approximately 20-25% of melanoma patients (40- 50% of melanoma patients harboring BRAF V600E x 50% of patients harboring HLA-A2). This off-the-shelf therapeutic vaccine may be used as a monotherapy for patients with high-risk resected melanoma or with minimal tumor burden. It may also be combined with anti-PD-1 for patients with melanoma that is refractory to immune checkpoint inhibitor therapy and targeted therapy. Our vaccine consists of mBRAFp and resiquimod (RSQ) as adjuvant formulated in CAPRO™ - a new class of proprietary biodegradable liquid polymers, providing local controlled release of both the antigen and adjuvant. Previously we found that (1) mice vaccinated with ovalbumin (OVA) and RSQ loaded in CAPRO developed long-lasting OVA-specific antibody and CD8 T cell responses, and (2) CAPRO-loaded mBRAFp and RSQ elicited antigen-specific T cell response in HLA-A2 transgenic mice. Based on these promising results, we have initiated preclinical product development program involving CMC of the vaccine ingredients and formulation development and have received written comments from the FDA in response to our pre-IND inquiry. The goal of this project is to define the vaccine product and set the stage for full CMC and pharmacology/toxicology studies toward an IND application. To accomplish this goal, we will first evaluate the prophylactic and therapeutic antitumor efficacy and safety of our vaccine product in a mBRAFp-expressing HLA- A2 transgenic mouse model with direct comparison with a formulation using Montanide (a clinically used vaccine adjuvant system). We will also explore the potential of combining vaccine with immune checkpoint inhibition (anti-PD1). We will then develop analytical assays for vaccine ingredients and final vaccine product, and generate IND-enabling data to meet CMC requirements of the FDA.