FURIN

2021 年 1 月 29 号，Barney Graham 在推特饱含深情地写下这样一段话：明天就是 McLellan 四十岁生日。2017 年，在西班牙马拉加街头酷似病毒的圣诞装饰灯下，我们和故去的挚友 Jose Melero 拍下了这张照片。早在成功解析新冠 Spike 蛋白结构之前，我们三人就共同致力于破解 RSV F 蛋白结构。于是，我跑去查找 Jose Melero 逝世的确切时间点。结果看到，2018 年 3 月 5 号凌晨，McLellan 怀着无限感伤发推文：Jose Melero 于昨日逝世，他是一位伟大的病毒学家，在肺病毒领域，特别是 RSV，做出过开创性贡献。他是一个慷慨而又合群的人，一个绝佳的同行者。José，你将会被许多人缅怀。朋友们，如果你翻阅 RSV F 蛋白结构的解析历程，从粗糙的二级结构预测，到解析融合后构象，再到固定融合前构象，很难不被三人二十多年来不断的努力和最终的成就所打动，也更加能体会到他们对老友逝去的感伤。正道是，天之涯，地之角，知交半零落...图片来源：Barney Graham X 平台1956 年，人们从患有上呼吸道疾病的黑猩猩中分离出一种病毒，将其命名为黑猩猩鼻病毒。1957 年，研究者在患有呼吸道疾病的儿童中分离到相同的病毒，并且发现这种病毒极易感染婴幼儿。后来，该病毒被划归到副粘病毒科肺炎病毒亚科(Pneumovirinae)肺炎病毒属(Pneumovirus)，称为呼吸道合胞病毒 (RSV)。RSV 是一种有包膜的负链 RNA 病毒，病毒基因组长度为 15.2 kb，包含 10 个开放阅读框，编码 8 个结构蛋白和 3 个非结构蛋白。RSV 在多种病毒株和细胞系中呈丝状。病毒丝长度为 0.5 至 12um，平均丝直径约为 130nm。1966 年，在美国，有婴儿和儿童参与到福尔马林灭活 RSV 疫苗 (FIRSV)临床试验中。那些在接种疫苗前病毒血清呈阴性的儿童回到社区后，感染 RSV 的概率和感染后疾病的严重程度显著增加。这种 RSV 疾病的增强形式表现为发烧、喘息和支气管肺炎，并导致频繁住院。在一项研究中，FIRSV 接种者中感染 RSV 的儿童比例为 80%，而未接种疫苗的对照组仅有 5%的儿童感染 RSV。更为严重的是，两名免疫婴儿在幼儿时期就因随后感染 RSV 而死亡。这一事件使得 RSV 疫苗研发事业遭遇重创，更是成为疫苗安全性的警示牌。1   选择 F 蛋白作为 RSV 疫苗抗原RSV 病毒粒子表面有 2 种非常重要的糖蛋白：附着蛋白（G）与融合蛋白（F）。G 蛋白介导病毒与宿主细胞表面受体结合，而 F 蛋白促进病毒与宿主细胞膜融合。F 蛋白还可促进病毒感染细胞与临近细胞膜融合，从而形成典型的合胞体（syncytia）。此外，在病毒感染细胞表面还会大量表达一种小的疏水性糖蛋白，称为 SH，但是，这种蛋白只会插入到极小比例的病毒粒子表面。HRSV F 蛋白一直是疫苗和抗病毒药物开发的主要靶标，因为它介导病毒入侵宿主细胞，具有高度保守的序列和结构、强大的免疫原性以及在病毒粒子中的暴露位置。而且，相比其他副黏病毒，RSV F 蛋白还有一个独特之处：在没有附着 G 糖蛋白的情况下，RSV F 蛋白自身就足以介导膜融合和病毒感染。相比之下，G 蛋白在不同 HRSV 毒株之间差异很大 ，并且已发现 SH 蛋白在病毒生长中是可有可无的 。2   F 蛋白二级结构1998 年，Jose Melero 在Journal of Virology发表文章：Antigenic Structure of Human Respiratory Syncytial Virus Fusion Glycoprotein，在 F 蛋白特异性单抗存在下，他制备了一系列 RSV 突变体。然后，根据突变体所选择的序列鉴定出中和抗体识别的两个主要抗原区域位于 F1 上，一个位于半胱氨酸富集区前面，称为 siteⅡ，一个靠近半胱氨酸富集区的 C 端，包括 site Ⅳ,site Ⅴ,site Ⅵ。此外，在半胱氨酸富集区中部，还存在一个可被中和性抗体微弱识别的 Site I。基于这些抗原表位，Jose Melero 绘制出 RSV F 蛋白（Long 毒株）的粗略二级结构。RSV F 蛋白以无活性的前体 F0（574 氨基酸）合成，随后转运至内质网，形成三聚体，同时，发生 N-糖基化。进入高尔基体后，F0 前体在两个 Furin 位点处发生切割，变为 F1 和 F2 两条链，F1 和 F2 之间通过 2 个二硫键连接，同时释放出 27 个氨基酸的肽段 pep27。F 蛋白上存在 3 段疏水性序列：第 1 段是信号肽，位于 F2 的 N 端（1-22 氨基酸），将会从成熟的糖蛋白上移除；第 2 段是融合肽（Fusion peptide），位于 F1 N 端（1-19 氨基酸）；第 3 段靠近 F1 C 端（389-414），也就是膜锚定区。从大的分类上来讲，RSV F 蛋白属于 I 型病毒融合蛋白，此类蛋白还包括冠状病毒 Spike 蛋白(最大的 I 型病毒融合蛋白)、HIV 包膜糖蛋白（Env）、hMPV(人偏肺病毒)及 PIV3/5(人副流感病毒)。I 型病毒融合蛋白具有相似的结构特征（融合肽、HRA、HRB 及跨膜区）与融合机制，但是，序列相似性极其有限。所有这些蛋白初始合成时均为单链、无活性的前体 F0。它们需要在关键的酶切位点切割才能转变为活性形式。有些蛋白仅有一个酶切位点，而有些蛋白则有两个酶切位点，例如，RSV 或者冠状病毒。3   F 蛋白锥形杆柄与棒棒糖杆柄2000 年，Jose Melero 在Virology发表文章：Electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies。为探索 HRSV F 蛋白结构与免疫原性之间的关系， Jose Melero 设计出两种 F 蛋白序列，一种为全长 F 蛋白 FWT，另外一种是截掉 C 端 50 个氨基酸的无锚定突变体 FTM-。采用重组痘苗病毒（ vaccinia viruses）作为表达载体，感染 HEp-2 细胞，表达靶标蛋白。正如预期，FWT 表达于宿主细胞表面，无锚定突变体 FTM-分泌至培养基中。采用亲和层析和蔗糖密度梯度离心纯化分离 F 蛋白，然后，在负染电镜下，发现 FWT 和 FTM-呈现完全不同的形态结构：1. 当存在去污剂（Detergent）时，FWT 沉降条带集中分布于蔗糖梯度顶部附近，表面蛋白呈现均一性。当不存在去污剂时，FWT 会沿着蔗糖密度梯度连续沉降至底部，说明蛋白存在聚集体（aggregates）。在没有去污剂的情况下，FWT 负染电镜下呈现玫瑰花结形态（rosettes）。在玫瑰花结中，包含两种形态的杆柄：一种为圆锥形杆柄（cone-shaped rods），占到 70%，另一种为棒棒糖形杆柄(lollipop-shaped rods)，占到 30%。2. 无论是否存在去污剂，无锚定突变体 FTM-在蔗糖梯度中均沉降至相似位置，仅在底部观察到存在极小比例聚集体。在没有去污剂的情况下，无锚定突变体 FTM-在负染电镜下主要呈现单个的圆锥形杆柄，彼此之间不发生交联。有意思的是，在一些样品中，FTM-也可形成玫瑰花结。然而，与全长 FWT 形成鲜明对比的是，90%的 FTM 玫瑰花结杆柄的形状为棒棒糖状。此外，当无锚定突变体 FTM-在 4℃存储时间延长，融合肽暴露，引发玫瑰花结聚集体数量增加，杆柄也从圆锥状变为棒棒糖状。也就是说，FTM-一旦形成玫瑰花结，杆柄将由圆锥形变为棒棒糖状。3. 无锚定突变体 FTM-或者全长 FWT 均呈现三聚体。Jose Melero 推测圆锥形杆柄为激活前 F 蛋白结构，而棒棒糖杆柄为激活后 F 蛋白结构，也就是说 F 蛋白的两种杆柄形态可能代表的是该分子在膜融合过程中采用的不同结构形式。4   F 蛋白完全切割是膜融合必需的2001 年，Jose Melero 在 PNAS 发表文章：Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion，他通过对 F 蛋白中间体的 N 端测序，确定了 F 蛋白前体（F0）可在两个位点发生切割：位于 109 氨基酸之后的 Site I 与位于 136 氨基酸之后的 SiteII。这两个位点均有弗林蛋白酶识别位点，并且两个酶切位点之间是独立切割的。突变酶切位点 Site I 会降低酶切位点 SiteII 的切割效率，从而减少无锚定 F 蛋白的聚集比例。然而，突变 SiteII 会完全消除无锚定 F 蛋白的聚集，即便 Site I 仍旧高效切割。这说明 SiteII 位点的切割对于无锚定蛋白 F 的形态和聚集是必不可少的，而 Site I 的切割可提升 SiteII 位点的切割效率。重组牛痘-F 蛋白感染细胞后表达的无锚定 F 蛋白并非发生完全切割，而是存在一定比例的前体 F0 和切割中间体 F△1-109。一旦加入胰蛋白酶，随着酶量增加，F0/F△1-109 逐渐被完全切割成为 F1。在蔗糖密度梯度离心中，用胰蛋白酶处理的无锚定 F 蛋白，只会检测到 F1 条带，并且，相比胰蛋白酶处理之前，F1 沉降条带朝着梯度中部迁移。在电镜下， 胰蛋白酶处理后，无锚定蛋白形态由未聚集的圆锥形杆柄（cones）转变为棒棒糖杆柄（lollipops）聚集而成的玫瑰花结。此外，抑制无锚定 F 蛋白任意一个 Furin 酶切位点，都会消除 F 蛋白促进膜融合的能力。5   无锚定 F 蛋白玫瑰花结与融合肽暴露胰蛋白酶完全切割无锚定 FTM-蛋白，引发蛋白聚集，形成玫瑰花结。为搞清楚这种聚集是否由单体蛋白上的疏水性融合肽之间的相互作用引发，人们将融合肽前面的 10 个氨基酸删除，形成 FTM-(Δ137-146)。在蛋白印记胶图上，纯化得到的无锚定 FTM-蛋白存在一条主条带 F1，还存在未切割的前体 F0、仅在 site I 发生切割的 FΔ1-109 以及仅在 siteⅡ发生切割的 F2。纯化得到的 FTM-(Δ137-146)突变体也存在类似的酶切中间体，但是，这些中间体条带相对于主条带 F1 的比例与无锚定 F 蛋白又是不相同的，这说明删除融合肽前面的氨基酸会对 Furin 切割效率产生影响。无锚定 FTM-和 FTM-(Δ137-146)突变体经过胰蛋白酶处理后，FΔ1-109 和 F2 剩余位点发生切割。在蔗糖密度梯度离心中，无锚定 F 蛋白经过胰蛋白酶处理，沉降条带将会向更高密度梯度迁移。在电镜下，无锚定蛋白 FTM-以未聚集的圆锥杆存在，但是，胰蛋白酶处理后的无锚定 FTM-蛋白将会聚集成棒棒糖杆状的玫瑰花结。与此形成鲜明对比的是，胰蛋白酶切割前后的 FTM-(Δ137-146)在蔗糖密度梯度离心中的沉降位置始终未发生变化，同时，在电镜下，胰蛋白酶切割前后的 FTM-(Δ137-146)突变体均处于未聚集的单体。正是基于以上数据，2004 年，Jose Melero 在 Journal of General Virology 发表文章：Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism，他们认为 F 蛋白激活后发生的一个关键事件便是融合肽的暴露，无锚定 FTM-蛋白的未切割和切割形式可能代表着 RSV F 蛋白的融合前和融合后构象。在 RSV 病毒入侵靶细胞表面时，F 蛋白发生最后的酶切位点切割，触发膜融合和感染。2005 年，有研究者在 PNAS 发表文章：Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein，研究人员采用杆状病毒表达载体，在昆虫细胞中表达切除跨膜锚定区、未发生切割的副粘病毒、人副流感病毒 PVI3 solF0 蛋白（I 型病毒融合蛋白）。解析未发生切割的 solF0 蛋白结构，发现其由三部分组成：一个球形的、含有β 折叠片组成的头部结构域，由β折叠片和α螺旋组成的颈部（neck），α螺旋占主导的茎部构成（stalk）。切除跨膜锚定区、未发生切割的分泌型 hPIV3 F 蛋白最为典型的特征是存在由 HRA 和 HRB 形成的 6HB 结构，这代表着该蛋白处于融合后构象，同时，说明跨膜锚定区对于蛋白实现融合前亚稳态构象极其重要。6   F 蛋白构象转变与膜融合2006 年，有学者在 Nature 发表文章：Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation，通过在副流感病毒 PIV5 F 蛋白羧基末端添加三聚化结构域，稳定其融合前构象，完成 PIV5 F 蛋白晶体结构的解析。在 PIV5 sF-GCNt 三聚体中，HRB 形成的 3 个α螺旋卷曲螺旋附着于一个大球状头部。GCNt 三聚体结构域位于茎部的 C 末端，使得 PIV5 sF 头部远离病毒膜。F-GCNt 每个亚基的头部区域包含 3 个结构域（DI-DIII），它们围绕三聚体轴延伸，形成广泛的亚基间接触。头部底部有一个大空腔，由 DII 形成的侧面和 DI 形成的底部组成。在头部区域存在 3 个突出的尖峰，每个尖峰由两对环构成（残基 60-65 和 残基 178-185），这些环从每个亚基的球状结构域向上突出。DIII（残基 42-278）覆盖腔体顶部，包括突出的尖峰、HRA 和融合肽。融合肽采用部分延伸、部分β折叠和部分α螺旋构象，夹在其自身亚基的 DIII 和另一个亚基的 DII 之间。PIV5 sF 蛋白处于一种非常特殊的、明显的 pretriggered 构象，表明 sF 蛋白 C 端结构域对于维系此融合前构象是至关重要的。此外，热激可触发 PIV5 sF 蛋白融合前构象发生改变。通过比较 PIV5 F-GCNt 融合前构象与 PIV3 solF0 融合后构象，研究者提出了 F 蛋白介导的膜融合与其构象变化相互偶联的模型：HRB 螺旋融化，破坏底部相互作用，使得 F 蛋白从预融合构象转变为“开茎”构象。接着，DIII 重新折叠形成预发夹中间体，促进 HRA 形成卷曲螺旋并将融合肽插入靶细胞膜。牵拉病毒膜与宿主细胞膜，F 蛋白 6HB 组装完成，转变为融合后构象，完成膜融合。通过 PIV5 与 PIV3 F 蛋白结构的解析， 我们可以看出，副粘病毒融合糖蛋白 F 以亚稳态（metastable）、预触发(pretriggered)形式锚定于病毒粒子膜上。一经触发，F 蛋白经历剧烈的构象延伸，将其疏水融合肽插入到靶细胞膜，自身回折，将两种膜拉在一起，启动膜融合。7   首次表达融合前 RSV F 蛋白2011 年 4 月 15 号，Mark E. Peeples 在 Journal of Virology 发表文章：Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer，为研究 RSV F 蛋白触发机制，他使用 6His tag 标签替换跨膜区和胞质区，生成一种可溶性的 F 蛋白（sF）。sF 蛋白可从 293T 细胞中以未切割形式，高效释放。这种 sF 蛋白可被中和抗体识别，在电镜下呈现球形，且未聚集，这些特征说明与天然的、未触发的三聚体相一致。F 蛋白在 Ni +2 柱上纯化并用含有 500mM NaCl 和 250mM 咪唑的 50mM 磷酸盐缓冲液洗脱。透析至 10 mM 缓冲液中，将会触发 sF 蛋白构象改变，形成“帽针”（hat pin-shaped）状分子，聚集成玫瑰花结，这是典型的触发后特征。触发效率与缓冲液摩尔浓度降低息息相关。稀释缓冲液摩尔浓度导致融合肽暴露，与脂质体融合，证实 sF 蛋白已被触发。突变紧邻融合肽的 furin 切割位点会阻止脂质体融合，更进一步证实膜融合需要融合肽。Jose Melero 对 RSV F 蛋白结构的一系列相关研究，一直采用的是 Long 毒株 F 蛋白，而 Mark E. Peeples 采用的是存在 6 个氨基酸差异的 D53 毒株 F 蛋白。因此，Mark E. Peeples 认为 Jose Melero 观察到的 RSV F 蛋白电镜下的形态结构均处于融合后构象。因为 Jose Melero 推测认定的融合前（未切割）和融合后（已切割）的无锚定 F 蛋白具有非常相似的外形：一个小而圆的头，连着相对较长的茎。关键 Bug 在于，这两种形式的 F 蛋白均表现出非常耐热，在温度达到约 90°C 之前均没有显示出构象变化。极端热稳定性并不是亚稳态蛋白 F 蛋白的典型特征，相反，极度热稳定是触发后、处于 6HB 形式的 F 蛋白特征。Mark E. Peeples 还认为 Jose Melero 观察到的融合构象的 F 蛋白换可能是由于纯化处理过程触发的。Mark E. Peeples 发现 10 mM 磷酸盐、100 mM NaCl 缓冲液可将大部分 sF 蛋白转化为触发后形式的玫瑰花结。此外，分离和储存过程中的差异也可能触发 F 蛋白构象的转变。冷冻和解冻会触发 F 蛋白构象改变。如果将融合前、预触发形式的 F 蛋白在 4°C 条件下储存在 50 mM 磷酸盐 （pH 8.0）、500 mM NaCl、250 mM 咪唑缓冲液，那么直到三周后才会转变为触发后形式。Mark E. Peeples 构建的 RSV sF 蛋白可被完全切割，但是，依然表达产生融合前构象的 F 蛋白，这说明 Furin 位点的完全切割并不会触发 F 蛋白形成融合后构象。在解析其他副粘病毒 F 蛋白晶体结构时，为避免触发构象变化，研究者均采用突变 F 蛋白切割位点，该突变 F 蛋白不能被弗林蛋白酶切割。然而，令人惊讶的是，未切割的 PIV3 sF 蛋白/新城疫病毒 F 蛋白均处于融合后构象。胰蛋白酶处理已触发的 PIV3 sF 蛋白，引发 Furin 位点切割，形成玫瑰花结。显然，玫瑰花结的形成是融合肽暴露的结果，而不是触发蛋白构象改变的结果，因为 PIV3 sF 蛋白在胰蛋白酶处理之前已经处于触发后形式。F 蛋白 Furin 位点的完全切割显然不能像 Jose Melero 提出的那样作为 F 蛋白构象改变的触发机制 。移除 PIV3 F 蛋白的膜锚定区会触发构象改变。为避免这种情况，研究者将来自外源蛋白 GCNt 的三聚体化结构域融合到 PIV5 sF 蛋白的 C 末端。在电镜下，这种携带三聚体化结构域 PIV5 sF 蛋白表现为一个球体，其短茎由其 HRB 和 GCNt α螺旋延伸组成，处于融合前构象。GCNt-PIV5 sF 蛋白 F1 与 F2 之间的 Furin 位点是完全切割的，但是，并没有触发构象变化，依然处于融合前构象，再次证实酶切位点的切割并不是 F 蛋白构象变化的触发因素。加热后的 PIV5 sF 蛋白具有更小的头部和更长的茎，类似于 PIV3 sF 蛋白触发后的 6-HB 形式，形成玫瑰花结，说明加热可触发 PIV5 sF 蛋白从融合前构象转变为融合后构象。总的来说，对于 RSV F 蛋白来说，膜锚定区和三聚化结构域均不是维系其融合前构象所必需的。Mark E. Peeples 首次制备了完全切割的、分泌型的、融合前构象的 RSV F 蛋白，并且，无需添加外源三聚体化结构域。8   首次解析 RSV F 蛋白融合后构象RSV F 的融合前和融合后形式作为疫苗抗原均具有潜在的缺点。在负染电镜下，棒棒糖形的融合前 F 三聚体和拐杖形（Crutch）融合后 F 三聚体之间存在明显的结构差异，这表明融合前和融合后 F 三聚体可能具有不同的抗原属性。为了防止病毒进入，在病毒包膜与细胞膜融合之前，F 特异性中和抗体可能必须结合病毒粒子上 F 的融合前构象。因此，研究人员推测 RSV F 必须以融合前构象存在才能有效引发中和抗体。关键的问题是，融合前 F 是一种“亚稳态”结构，处于较高的能级，很容易重新排列成较低能量的融合后状态，加上疏水性融合肽的暴露而聚集。2011 年 5 月 17，诺华公司研发团队在PNAS发表文章：Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers，他们删除融合肽、跨膜区和胞质结构域，保留 Furin 切割位点，构建表达稳定的、非聚集的 RSV F 蛋白。在电镜下，这种无锚定的 F 蛋白突变体形成非聚集的均质拐杖状分子（nonaggregated, homogeneous crutch-shaped molecules），与融合后的 F 三聚体一致。用两剂 5 μg 剂量的明矾吸附 RSV F 可保护棉鼠免受鼻内 RSV 攻击，触发高水平的中和抗体滴度。他们解析了融合后构象 F 蛋白的晶体结构，发现融合后 RSV F 的总体结构与融合后副流感病毒 F 糖蛋白总体相同。糖蛋白由三个紧密缠绕的亚基组成，形成球状头和细长的茎。每个亚基包含三个结构域，分别指定为 I、II 和 III。结构域 I 和 II 位于三聚体头部的顶部，形成三角形冠。域 III 形成头部的基部。长螺旋 HRA 从结构域 III 延伸并在茎的中心形成三聚体卷曲线圈。HRB 螺旋与结构域 II 相连，向下到达茎的头部远端，在那里它与 HRA 内部卷曲线圈形成六螺旋束的外部线圈。在全长 F 中，疏水性融合肽（HRA 的 N 端）和跨膜区（HRB 的 C 端）将并置在茎的底部并插入靶细胞膜中。融合前和融合后 RSV F 中 A 和 C 位点的保留合理地解释了融合后 RSV F 抗原在免疫动物中引发高效价中和抗体的能力。与这一假设一致，竞争 ELISA 表明，用明矾吸附的融合后 RSV F 抗原免疫的小鼠的混合血清（而非未免疫小鼠的血清）抑制帕利珠单抗结合。RSV F 融合后三聚体的晶体结构表明，帕利珠单抗/莫他珠单抗表位（位点 A）在融合后三聚体结构上暴露且可接近，因此，用 RSV F 融合后三聚体免疫的小鼠血清竞争性抑制帕利珠单抗竞争性与 F 蛋白的结合。位点 C（被中和抗体（如 101F）识别的另一个重要中和表位）也很好地暴露在融合后 RSV F 上三聚体。因此，融合后 RSV F 三聚体的晶体结构表明，关键的中和结合位点均可暴露出来，为引发高效价中和抗体提供了基础。2008 年，McLellan 进入位于美国国立卫生研究院（NIH）疫苗研究中心四楼的 Peter Kwong 实验室，接受博士后期间的训练。由于实验室空间紧张，McLellan 在疫苗研究中心二楼毗邻病毒学大佬 Barney Graham 房间的位置办公学习。没过多久，Barney Graham 邀请 McLellan 共同从事 RSV 疫苗相关的研究，自此开启两人长达多年的友情。McLellan 为得到 RSV F 蛋白晶体结构，最初将跨膜结构域删除，构建无锚定突变 RSV F ΔTM（1-513 氨基酸）。该突变体蛋白在 HEK293F 细胞中表达良好，可高效切割为 F1/F2。但是，由于融合肽暴露，该无锚定突变体 F 蛋白会聚集成为玫瑰花结。后来，McLellan 借鉴 Jose Melero 2004 年发表的文章，又将融合肽前 9 个氨基酸（137 至 146 氨基酸）删除，构建 RSV F ΔFP，可避免形成聚集体，形成的是分散的、单个的、融合后三聚体，从而能够进行结晶并确定其结构。2011 年 8 月，McLellan 在 Journal of Virology 发表文章：Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes。在电镜下，观察到 RSV F ΔFP 呈现锥形杆状形态，具有典型的 6 螺旋束，证实其处于融合后构象。莫维珠单抗和 101F 表位（位点 II 和 IV）暴露于溶剂中，其构象与抗体结合肽结构中观察到的相似。此外，Pro389 位于 F 蛋白顶部环的顶端，其突变会导致靶向靶向抗原位点 I 的抗体失去结合能力。这三个抗原位点的位置、暴露、构象以及表面等离子表明融合后 F 蛋白与靶向三种不同中和表位的抗体发生紧密结合。9   融合后 RSV F 蛋白疫苗相继失利自从融合后 RSV F 蛋白结构被解析出来，并且，有研究团队在绵鼠中验证了其免疫原性，以 Novavax 和 Medlmmune 为代表的多家公司开始以融合后 F 蛋白为抗原进行开发 RSV 疫苗。然而，不幸的是，融合后 F 蛋白 RSV 疫苗均在临床试验期间遭遇失败。此后，McLellan将目光转向融合前F蛋白，即将迎来自己的高光时刻...识别微信二维码，添加生物制品圈小编，符合条件者即可加入生物制品微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

Emerging research shows that targeting initial mutations expressed on a tumor cell surface, called clonal mutations, is critical in achieving a durable clinical benefitFor example, lung cancer patients with high levels of clonal neoantigens treated with checkpoint inhibitors experienced significantly better clinical outcomes as shown in multiple retrospective analysesMost immunotherapies have not been designed explicitly to directly target clonal neoantigens which may explain their limitations in solid tumorsIn a Phase 2b clinical study, Gradalis demonstrated that its clonal neoantigen targeting immunotherapy, Vigil®, works best when used in patients with a high level of clonal neoantigen rich tumorsA biomarker for measuring clonal neoantigens may expand the use of certain immunotherapies and support a pan-cancer application DALLAS, Dec. 05, 2023 (GLOBE NEWSWIRE) -- Gradalis, Inc., a clinical-stage biotechnology company developing a personalized anti-cancer therapy for patients with ovarian and other cancers, announced a peer-reviewed publication in Cancers, highlighting the newly discovered critical clinical role of clonal neoantigens in the durable efficacy of immunotherapies in oncology and the potential to use clonal neoantigens as a new biomarker to better identify patients who will best respond to certain immunotherapies. The publication, entitled “Clonal Neoantigen: Emerging ‘Mechanism Based’ Biomarker of Immunotherapy Response”, provides insight into the science and implications of this key discovery. It reviews the potential limitations of certain immunotherapies designed without explicit consideration of clonal neoantigens. This may also explain why clonal neoantigen targeting immunotherapies like Gradalis’ Vigil have shown longer-term overall survival benefits in a randomized controlled trial of patients with solid tumors. The full text of the article can be found here. “It is now possible to identify the external structures, or neoantigens, found on the surface of millions of cells of a patient’s tumor. Using this information and proprietary algorithms, we can identify the neoantigens related to initiating tumor mutations. These are called clonal neoantigens. These clonal neoantigens can be targets for guiding new therapies to eliminate tumor cells. Since these targets can vary in structure and number from patient to patient, targeting them needs to be custom designed, or personalized, for each patient to have the most durable clinical effect. Research shows the more personalized, the better for ensuring long-lasting efficacy,” Gradalis’ Chief Executive Officer Steven Engle stated. “The human immune system has evolved to eliminate errant cells like tumor cells if it can identify them. Vigil is the only therapy in late-stage clinical testing that has been shown in clinical studies to safely enable the immune system to identify tumor cells, and as a result, to generate customized effector cells, which include T cells, and as many types and number as needed, to target the clonal neoantigens. This allows us for the first time go after the root cause of cancer, specifically the mutations that cause it,” Mr. Engle continued. A few of the key findings in this area include: A meta-analysis in 1008 patients across 12 landmark trials of factors associated with Overall Survival response to Checkpoint Inhibitors (CPIs) showed that clonal Tumor Mutational Burden (clonal TMB) was the leading biomarker of clinical benefit with high clinical significance (p = 2.9x10-7)1 when compared to subclonal TMB and other factors.Levels of clonal neoantigens have been shown to predict disease free survival in untreated early-stage lung cancer patients (p ≤ 0.025), not subclonal neoantigens (p = n. s.)2Greater numbers of clonal TMB and clonal neoantigens have been found in tumor cells with stable DNA repair capacity, the homologous recombination proficient (HRP) genetic profile.3 Clonal TMB is a likely surrogate for clonal neoantigen expression on the surface of the tumor cell. “Immunotherapy has improved the outlook for many cancer patients, but its benefit has been limited to a fraction of the total patients in each tumor type. Targeting clonal neoantigens may be key to improving this situation. Clonal neoantigens are tumor initiating mutations and therefore found on the surface of all cells of a patient’s tumor. Clonal mutations expressed on the cancer surface provide a distinguishing difference between cancer cells and normal non-cancer cells. Emerging research shows that targeting clonal mutations may be critical in achieving a durable clinical benefit,” stated John Nemunaitis, MD, Chief Scientific Officer of Gradalis and co-author of the paper. “For example, cancers displaying high levels of clonal neoantigens have been shown to be more responsive to checkpoint inhibitors, a type of immunotherapy used in several tumor types. In multiple retrospective analyses of studies of lung cancer patients treated with CPIs, higher levels of clonal neoantigens are highly correlated with better patient outcomes.” As described in the paper, Vigil is a personalized neoantigen immunotherapy that is designed to enable the immune system to recognize each patient’s unique tumor clonal neoantigens and generate effector cells, including T-cells, to target the clonal neoantigens and thereby provide durable clinical benefit. In a Phase 2b trial, the benefit seen as measured by Recurrence Free Survival (RFS) and Overall Survival (OS) was highest in patients of the HRP type. Patients with the HRP genetic profile have an enhanced ability to maintain their DNA sequences which research indicates results in a higher level of clonal neoantigens. These patients represent approximately 50% of ovarian cancer patients and up to 80% of other cancer types. “There is a major unmet medical need for new therapies in HRP ovarian cancer patients because they do not respond well to standard-of-care therapies like chemotherapy and PARP inhibitors. This represents a major gap in treatment, especially in recurrent ovarian patients of the HRP type where multiple PARPi therapies were withdrawn in 2022,” observed Dr. Nemunaitis. “In its study, Gradalis discovered that Vigil works best when used in clonal neoantigen rich tumors as identified by the HRP genetic test. One of Vigil’s main strengths as a therapy is that it enables the immune systems to “see” the tumor cells and decide on the most effective targets. Vigil’s ability to increase clonal neoantigen targeting effector cells may represent a major breakthrough in achieving more consistent clinical benefit not only with Vigil as monotherapy, but also in combination with other immune therapeutic approaches like checkpoint inhibitors. The company is preparing to initiate a Phase 2 clinical study designed for potential accelerated approval opportunity to evaluate Vigil in platinum-sensitive recurrent ovarian cancer patients with the HRP profile.” David Willoughby, PhD, VP of Genomic Services of Frontage Laboratories Inc. and a co-author, stated “Molecular genomic assessment utilizing next generation and whole exome sequencing is now being increasingly applied to biomarker determination to refine use of targeted immune therapies. Recent research discussed in the article provided several case examples followed by retrospective study assessment that have convincingly demonstrated clonal neoantigens are relevant predictors of response to checkpoint inhibitor therapy. A meta-analysis of over 1000 cancer patients from 12 landmark trials referred to in the publication showed no clinical benefit to CPI therapy in targeting patients with a high level of subclonal TMB, whereas targeting patients with a high level of clonal TMB was found to be highly correlated with better overall survival. The publication summarizes these findings and describes advantages of immunotherapy treatment utilizing clonal neoantigens as a biomarker to define the likelihood of cancer response to immunotherapy.” Rodney Rocconi, MD, Director of Cancer Center and Research Institute at the University of Mississippi Medical Center, co-author and lead Principal Investigator in Gradalis’ Phase 2b study, commented, “There are multiple biomarkers used to predict potential patient response to immunotherapies, but they are not currently focused on the level of clonal neoantigen expression. It may be that use of immunotherapies that help the immune system identify clonal neoantigens will broaden the clinical application of immunotherapies such as checkpoint inhibitors. Work outlined in our review article support characterization of clonal neoantigen as an advancement in defining checkpoint inhibitor activity and logically suggest combination of Vigil with checkpoint inhibitor as a fruitful direction in further clinical testing.” About VigilVigil® is a novel, triple function immunotherapy platform that modifies a patient’s tumor by using bi-shRNA to reduce furin, an enzyme which facilitates immunosuppressive TGF beta protein production, and to maximize DNA expression of GM-CSF, which stimulates the immune system and attracts key immune system effector cells, including T-cells. By utilizing the patient's own tumor as the antigen source, Vigil is designed to elicit an immune response that is specifically targeted and broadly relevant to each patient's unique “clonal” tumor neoantigens. Vigil therapy has been well tolerated in Phase 1, 2a and 2b clinical studies. The company is preparing to initiate a Phase 2 clinical study in platinum-sensitive recurrent ovarian cancer patients with the HRP profile. In VITAL, a multicenter, randomized, double-blind, placebo-controlled Phase 2b trial in Stage III/IV newly diagnosed, frontline ovarian cancer patients, Vigil showed a positive trend in the primary endpoint of recurrence free survival (RFS) in the overall population and a statistically significant improvement in the secondary endpoint of recurrence free survival and overall survival (OS), with a median survival time of three years to date, in patients with the BRCAwt molecular profile. Most importantly in patients with tumors of the HRP type, where there is a high unmet medical need, a greater statistically significant improvement was seen in RFS and OS. Additionally, Phase 1 results in an “all-comer” clinical trial have shown positive signals of activity in 19 different tumor types and some patients treated with Vigil remain in the study 48 months later. Vigil has also demonstrated safety and benefit in two separate pilot studies when administered concurrently or in sequence prior to treatment with check point inhibitor therapy. The sequential treatment results support the hypothesis that Vigil administration prior to starting checkpoint inhibitor therapy may focus the immune effector population to the cancer, potentially enhancing activity and limiting off target side effects. The Vigil manufacturing process takes two days. It does not require the bioreactors and other time consuming and costly processes required by other cell therapies. Following the initial removal of the tumor, a small amount of tumor tissue is shipped to the company’s manufacturing plant in Dallas, Texas where its genetics are modified to produce the immune system enhancing effects. It is then put in vials and stored for in-office intradermal administration by the patient’s physician every three to four weeks for an average four to six month course of treatment. About Gradalis, Inc.Founded in 2006, Gradalis is a privately held, clinical-stage biotechnology company developing a new category of personalized immunotherapy called Vigil, that has been tested in multiple studies in ovarian and other cancer tumor types. Vigil is the first cellular immunotherapy to demonstrate longer-term survival benefits in a randomized controlled trial of patients with solid tumors. The results of the company’s Phase 2b study have been published in Lancet Oncology and presented at the American Society of Clinical Oncology. Vigil is being studied in other women’s cancer types and has shown positive results in combination with checkpoint inhibitors. Gradalis’ Vigil platform uses the patient’s immune system to target the entire tumor. Based on multiple clinical studies, Gradalis has developed a pioneering oncology platform that is designed to decloak the full repertoire of a patient’s tumor antigens, including all clonal neoantigens, reactivate the immune system, and summon key effector cells to deliver a durable clinical response. When combined, these are a powerful Trifecta of anti-cancer activities, potentially eliminating even the elusive metastatic cells, and as shown in Phase 2 clinical studies in ovarian cancer, a potential gamechanger in oncology. Our clinical trials have also demonstrated that Gradalis’ platform is better tolerated compared to standard cancer treatments since Vigil uses the patient’s immune system operating within its natural state of balance rather than in an artificial overdrive as with some technologies. Vigil utilizes proprietary bi-shRNA technology that has been proven to silence multiple genes in a variety of cancers and has the potential to be used in other diseases. Forward-Looking Statements This press release contains forward-looking statements, including, without limitation, statements regarding the success, cost, and timing of our product development activities and clinical trials, our plans to research, develop, and commercialize our product candidates, and our plans to submit regulatory filings and obtain regulatory approval of our product candidates. These forward-looking statements are based on Gradalis’ current expectations and assumptions. Because forward-looking statements relate to the future, they are subject to inherent uncertainties, risks, and changes in circumstances that may differ materially from those contemplated by the forward-looking statements, which are neither statements of historical fact nor guarantees or assurances of future performance. Important factors that could cause actual results to differ materially from those in the forward-looking statements include but are not limited to: (a) the timing, costs, and outcomes of our clinical trials and preclinical studies, (b) the timing and likelihood of regulatory filings and approvals for our product candidates, and (c) the potential market size for our product candidates. These forward-looking statements speak only as of the date made and, other than as required by law, we undertake no obligation to publicly update or revise any forward-looking statements. This press release does not constitute an offer to sell, or a solicitation of an offer to buy, any securities. Gradalis ContactMark Early(214) 442-8161mearly@gradalisinc.com LifeSci Advisors ContactJoyce Allaire+1 (617) 435-6602jallaire@lifesciadvisors.com 1 Litchfield et al Cell 20212 Russo et al Science 20193 Wu et al Seminars in Cancer Biology 2022; Wolf et al Cell 2019

Gradalis, Inc., a leader in the development of personalized anti-cancer cellular therapies for patients with solid tumors, announced today that it has been awarded a $9.9 million Product Development Research grant from the Cancer Prevention and Research Institute of Texas (CPRIT). The funds will be used to support the company's Phase 2 clinical study of Vigil® in platinum-sensitive patients who have recurrent ovarian cancer with a homologous recombination proficient (HRP) molecular profile. These patients face an acutely high unmet medical need due to the removal of PARP inhibitor therapies in 2022. The study is designed as a pivotal trial to confirm the positive survival results seen in ovarian cancer patients with the HRP profile tumors in Gradalis’ earlier VITAL study in frontline maintenance therapy. The company intends to pursue accelerated approval based on positive results. The company’s pioneering immuno-oncology therapy, Vigil, decloaks the full repertoire of tumor antigens, including the patient’s clonal neoantigens, reactivates the immune system, and summons key effector cells to deliver a durable clinical response. Vigil’s approach enables the very powerful immune system, which has been honed over eons to specifically identify the optimal targets on tumor cells required to destroy the tumor cells. The result is a dramatic expansion of the effector cell population targeting clonal neoantigens throughout the body and destroying metastasized tumor cells. The company has shown in multiple studies that Vigil therapy causes the immune system to generate a reliable increase in T-cells that specifically target the tumor cells. In Phase 1, 2A and 2B studies, Vigil has been shown to be well tolerated – no Grade 3 or 4 treatment related adverse events were seen and no dose modifications were required. “We are honored to have been approved by CPRIT for this award which provides additional capital to support the clinical development of Vigil and external validation of our technology by the world class experts who conducted medical, scientific and commercial diligence on behalf of CPRIT,” said Steve Engle, CEO of Gradalis. Clonal neoantigens are tumor initiating mutations, and research by others has shown that the targeting of clonal neoantigens predicts survival in cancer patients. This targeting is critical to achieving durable efficacy. By utilizing the patient’s own tumor as the source for immunologic targets, Vigil is designed to elicit an immune response that is specifically targeted and broadly relevant to each patient's unique “clonal” tumor neoantigens. Other engineered therapeutic approaches including CAR-T, TILs and some cancer vaccines may be limited by an inability to identify or target the key clonal neoantigens. “This award will accelerate the start-up of our Phase 2 study in recurrent ovarian cancer patients who face a very difficult path due to the limited effectiveness of chemotherapy and the recent withdrawals of PARP inhibitors. We expect the study will confirm Vigil’s ability to target each patient’s unique clonal neoantigens, the elimination of which has been shown by other research to be critical in achieving a durable overall survival benefit,” commented John Nemunaitis, MD, Gradalis’ Chief Scientific Officer and co-founder. “Following the extensive review of several drug development opportunities, CPRIT was impressed with Gradalis' Vigil product activity and clinical benefit results. We look forward to continued relationship with Gradalis as results of the Phase 2 trial unfold,” said Ken Smith, Ph.D., Chief Product Development Officer at CPRIT. Since its founding in 2007, CPRIT has awarded more than $3 billion in grants to Texas research institutions and organizations through its academic research, prevention, and product development research programs. CPRIT has also recruited 293 distinguished researchers to Texas, supported the establishment, expansion, or relocation of 56 companies to Texas, and supported nearly 9 million prevention services reaching all 254 counties in Texas. In 2019, Texas voters approved a constitutional amendment to provide an additional $3 billion to CPRIT for a total $6 billion investment in cancer research and prevention. About Ovarian Cancer In the US, over 20,000 patients are diagnosed with ovarian cancer each year. The majority of women (65%) diagnosed with ovarian cancer present in an advanced stage (Stage III/IV). Even with current therapies, 65% will recur within two years and 14,000 patients will succumb to the disease each year. Over 230,000 patients are living with ovarian cancer in the U.S. Ovarian cancer patients are composed of two groups with differing levels of DNA repair capability, each representing about 50% of ovarian cancer patients: the HRP group has good DNA repair capability and the HRD/BRCA-mutant group has poor DNA repair capability. Virtually all ovarian cancer patients are tested for HRP/HRD and reimbursement is covered by payors. Because HRP tumors are better able to repair DNA, the clonal neoantigens are better preserved. Patients with the HRD/BRCA-mutant profile have an impaired DNA repair mechanism that is associated with a smaller proportion of clonal neoantigens compared to the patients with the HRP profile. As a result, HRP patients’ tumors would be expected to respond better to Vigil therapy than HRD/BRCA-mutant patients. Results of Vigil in a Phase 2b study in HRP ovarian cancer patients are consistent with these findings. Vigil is a novel, triple function immunotherapy platform that modifies a patient’s tumor by using bi-shRNA to reduce furin, an enzyme which facilitates immunosuppressive TGF beta protein production, and to maximize DNA expression of GM-CSF, which stimulates the immune system and attracts key immune system effector cells, including T cells. By utilizing the patient's own tumor as the antigen source, Vigil is designed to elicit an immune response that is specifically targeted and broadly relevant to each patient's unique “clonal” tumor neoantigens. Vigil therapy has been well tolerated in Phase 1, 2a and 2b clinical studies. The company is preparing to initiate a Phase 2 clinical study in platinum-sensitive recurrent ovarian cancer patients with the HRP profile. In VITAL, a multicenter, randomized, double-blind, placebo-controlled Phase 2b trial in Stage III/IV newly diagnosed, frontline ovarian cancer patients, Vigil showed a positive trend in the primary endpoint of recurrence free survival (RFS) in the overall population and a statistically significant improvement in the secondary endpoint of RFS and overall survival (OS), with a median survival time of three years to date, in patients with the BRCAwt molecular profile. Most importantly in patients with tumors of the HRP molecular profile where there is a high unmet medical need, a statistically significant improvement was seen in RFS and OS. Additionally, Phase 1 results in an “all-comer” clinical trial have shown positive signals of activity in 19 different tumor types and some patients treated with Vigil remain in the study 48 months later. Vigil has also demonstrated safety and benefit in two separate studies when administered concurrently or in sequence prior to treatment with check point inhibitor therapy. The sequential treatment results support the hypothesis that Vigil administration prior to starting checkpoint inhibitor therapy may focus the immune effector population to the cancer, potentially enhancing activity and limiting off target side effects. The Vigil manufacturing process takes two days. It does not require the bioreactors and other time consuming and costly processes required by other cell therapies. Following the initial removal of the tumor, a small amount of tumor tissue is shipped to the company’s manufacturing plant in Dallas, Texas where its genetics are modified to produce the immune system enhancing effects. It is then put in vials and stored for in-office intradermal administration by the patient’s physician every three to four weeks for an average four to six month course of treatment. Founded in 2006, Gradalis is a privately held, clinical stage biotechnology company developing a new category of personalized immunotherapy called Vigil, that has been tested in multiple studies in ovarian and other cancer tumor types. Vigil is the first cellular immunotherapy to demonstrate longer-term overall survival benefits in a randomized controlled trial of patients with solid tumors. The results of the company’s Phase 2b study have been published in Lancet Oncology and presented at the American Society of Clinical Oncology. Vigil has shown positive results in combination with checkpoint inhibitors. Gradalis’ Vigil platform uses the patient’s immune system to target the entire tumor. Based on multiple clinical studies, Gradalis has developed a pioneering oncology platform that is designed to decloak the full repertoire of a patient’s tumor antigens, including all clonal neoantigens, reactivate the immune system, and summon key effector cells to deliver a durable clinical response. When combined, these are a powerful Trifecta of anticancer activities, potentially eliminating even the elusive metastatic cells, and as shown in Phase 2 clinical studies in ovarian cancer, a potential gamechanger in oncology. Our clinical trials have also demonstrated that Gradalis’ platform is better tolerated compared to standard cancer treatments since Vigil uses the patient’s immune system operating within its natural state of balance rather than in an artificial overdrive as with some technologies. Vigil utilizes proprietary bi-shRNA technology that has been proven to silence multiple genes in a variety of cancers and has the potential to be used in other diseases. 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