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的结果有了显著改善 治疗，大多数转移性BRAF突变黑色素瘤患者最终经历他们的进展 疾病。免疫检查点抑制剂耐药的一个关键原因是黑色素瘤特异性T细胞的缺乏 细胞，但不幸的是，大多数早期黑色素瘤疫苗旨在扩增黑色素瘤特异性T细胞 都没有表现出好处。下一代测序及多肽与人类白细胞抗原结合预测 使研究人员能够为癌症患者接种新抗原疫苗--新抗原不表达的多肽 在任何正常组织中都有，但只在肿瘤中发现。尽管新抗原疫苗前景看好，但一个关键 限制是这样一个事实，即新抗原通常是私有的，因此不可能制造“现成” 新抗原疫苗将适用于一大批患者。我们和其他人发现 黑色素瘤患者可以对突变的BRAF(缬氨酸到谷氨酸)产生内源性T细胞反应 位置600)。我们进一步证明了一个与HLA-A2结合的突变BRAF多肽(MBRAFp)是 在人类白细胞抗原A2转基因小鼠中具有高度的免疫原性。在这里，我们建议一种免疫原性疫苗靶向 在人类白细胞抗原A2背景下，V600E突变的BRAF具有扩增黑色素瘤特异性T细胞的潜力。作为一名 共有的新抗原，mBRAFp是大约20%-25%的黑色素瘤患者(40-25%)的相关肿瘤靶点。 携带BRAF V600E基因的黑色素瘤患者中有50%携带BRAF V600E基因(50%携带人类白细胞抗原A2基因的患者)。这是现成的 治疗性疫苗可作为高危黑色素瘤切除患者或 最小的肿瘤负担。它也可与抗PD-1联合用于治疗难治性黑色素瘤患者 免疫检查点抑制剂治疗和靶向治疗。我们的疫苗由mBRAFp和resquimod组成 (Rsq)作为CAPRO™-一种新型专利可生物降解液体聚合物-配制的佐剂， 提供抗原和佐剂的局部控制释放。之前我们发现(1)小鼠 CAPRO负载的卵清蛋白(OVA)和RSQ联合免疫可产生持久的OVA特异性抗体 和CD8 T细胞反应，以及(2)CAPRO负载的mBRAFp和RSQ诱导抗原特异性T细胞反应 在人类白细胞抗原A2转基因小鼠中。基于这些有希望的结果，我们推出了临床前产品 开发计划涉及CMC的疫苗成分和配方开发，并已 收到FDA的书面意见，回应我们的IND前询问。这个项目的目标是 确定疫苗产品，并为全面的CMC和药理学/毒理学研究奠定基础 申请。为了实现这一目标，我们将首先评估预防和治疗抗肿瘤的效果 我们的疫苗产品在mBRAFp表达的人类白细胞抗原A2转基因小鼠模型中的安全性 与使用Montanide(一种临床使用的疫苗佐剂系统)的配方进行比较。我们还将 探索将疫苗与免疫检查点抑制(抗PD1)相结合的可能性。然后我们将开发出 疫苗成分和最终疫苗产品的分析分析，并生成支持IND的数据以满足 美国食品及药物管理局的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.