Cimaglermin alfa

深度聚焦类器官与3D培养论坛，OTC2024论坛合作详询：王晨 180 1628 8769近期，国人团队发表重要综述 Organoids: opportunities and challenges of cancer therapy，仅需一张图简明扼要地总结了不同肿瘤类器官培养方法的关键。从肿瘤组织中通过机械或酶解获得肿瘤细胞，用几乎相同的基础培养基进行培养，而细胞因子是肿瘤类器官培养成功的决定因素之一。不同肿瘤类器官培养方法的简要概述（源自参考文献：Organoids opportunities and challenges of cancer therapy）接下来，我们将重点介绍几种重要的肿瘤类器官培养基组分及方案。1. 胃癌类器官通过构建源自胃癌患者的类器官（PDO），能够直接对临床样本进行研究。Vlachogiannis 等人为了成功培养胃癌类器官，除了使用 ADMEM/F12 培养基和基底膜基质外，还添加了 EGF、Noggin、R-Spondin 1、FGF-10 等细胞因子。胃癌患者的类器官（PDO）培养步骤：如下实验流程和数据均引用Vlachogiannis等人发表的文章（doi: 10.1126/science.aao2774），供参考。1，配置 PDO 培养基：改良的 DMEM/F12 基础培养基，添加 1x B27 additive、1x N2 additive、0.01% BSA、2 mM L-Glutamine、100 units/ml 青霉素-链霉素，以及如下表的细胞因子或其他添加物：*HGF 仅用于胆管癌类器官。2，样本收集：通过图像引导或内窥镜手术收集活检样品。收获后，样本置于冷 PBS 中，并在冰上运输到实验室，20 分钟～2 小时内将组织切片切碎。3，样本处理：组织切碎后，加入 5 ml 5 mM  的 PBS/EDTA，室温静置 15 分钟。用含有 2x TrypLe 的 1 mM PBS/EDTA 5 ml 在 37 ℃ 消化 1 小时。施加机械力（如吹液）以促进消化。4，接种细胞：用改良的 DMEM/F12 培养基收集分离的细胞，并在 4 ℃，1,200 转/分离心 5 分钟，使细胞成球状，用 120 µl GFR 基质胶重悬，接种到 24 孔或 48 孔的平底细胞培养板中。5，将基质在 37 ℃、5% CO2 的细胞培养箱中培养 20 分钟，进行固化。然后添加 500 µl 配置好的 PDO 培养基，要完全覆盖培养孔。6，每两天换一次培养基。2. 结直肠癌类器官患者来源的肿瘤类器官已成为结直肠癌临床前研究的一种出色的模型，原代上皮细胞可在特定生长因子的控制下和 3D 细胞外基质（ECM）中成功培养类器官。肠上皮细胞的自我更新由位于隐窝中的 Lgr5 干细胞驱动。Wnt 信号是维持隐窝干细胞活跃所必需的，Wnt 激动剂 R-Spondin 1 是 Lgr5 的配体，诱导隐窝增生。EGF 信号与肠道增殖有关，Noggin 诱导隐窝数量的急剧增加。因此 van de Wetering 等人开发的结直肠癌类器官培养体系如下：（如下实验流程和数据均引用 van de Wetering 等人发表的文章（doi: 10.1016/j.cell.2015.03.053），供参考。）1. 基础培养基：改良的 DMEM/F12 培养基，添加青霉素-链霉素、10 mM HEPES 和 Glutamax。2. 人肠干细胞培养基：Wnt 条件培养基、R-Spondin 1 条件培养基、Noggin 条件培养基和基础培养基按照 50%、20%、10% 和 20% 的体积比混合，然后加入 1x B27、1.25 mM n-Acetyl Cysteine、10 mM Nicotinamide、50 ng/ml 人 EGF、10 nM Gastrin、500 nM A83-01、3 uM SB202190、10 nM Prostaglandine E2 和 100 mg/ml Primocin。3. H&E 染色分析：通过 H&E 染色对培养后的原代肿瘤细胞和类器官进行分析，发现大多数类器官的特征是存在一个或多个管腔，与原代肿瘤的管状结构相似（如 P8 和 P19b），没有管腔的原代肿瘤会形成没有管腔的紧凑型类器官（P19a）。肿瘤类器官与原代肿瘤上皮相似3. 肝癌类器官肿瘤类器官的培养非常复杂，如肝癌类器。原发性肝细胞癌主要分为肝细胞癌（HCC）、胆管细胞癌（CC）和混合型（CHC）。在肝细胞癌类器官培养中需要使用地塞米松但是胆管细胞性肝细胞癌类器官培养则需加入 R-spondin 1 来维持细胞生长。Broutier 等人发现原发性肝癌（PLC）组织培养获得的类器官可以在体外真实地模拟人类 PLC 的遗传复杂性。构建的类器官包含 PLC 常见的三种亚型：HCC、CC 和 CHC，再现亲本肿瘤的组织结构和表达谱，同时保留患者及亚型之间的差异性，有利于肝癌相关基因和潜在生物标志物的鉴定。肝癌类器官分类培养基：如下实验流程和数据均引用 Broutier 等人发表的文章（doi: 10.1038/nm.4438），供参考。1，基础培养基：改良的 DMEM/F12 培养基，添加 1% 青霉素-链霉素、1% Glutamax、10 mM HEPES 和 1:50 B27 添加剂。2，经典人肝类器官分离培养基：Wnt 条件培养基、R-Spondin 1 条件培养基和基础培养基按照 30%、10% 和 70% 的体积比混合，然后加入 1:100 N2 添加剂、1.25 mM n-Acetyl Cysteine、10 mM Nicotinamide、10 nM 重组人 Gastrin I、50 ng/ml 重组人EGF、100 ng/ml 重组人 FGF10、25 ng/ml 重组人  HGF、25 ng/ml Noggin、5 uM A83-01、10 uM Forskolin、10 uM Y27632。3，肿瘤细胞特异性分离培养基：经典人肝类器官分离培养基中添加 3 mM 地塞米松。4，肝癌类器官培养基：基础培养基、10% （体积比）R-Spondin 1 条件培养基、1:100 N2 添加剂、1.25 mM n-Acetyl Cysteine、10 mM Nicotinamide、10 nM 重组人 Gastrin I、50 ng/ml 重组人 EGF、100 ng/ml 重组人 FGF10、25 ng/ml 重组人 HGF、5 uM A83-01、10 uM Forskolin。4. 乳腺癌类器官乳腺癌类器官的培养基与其他肿瘤的略有不同。Seino 等人在类器官培养基优化过程中发现，Neuregulin 1 与乳腺发育和肿瘤发生有关，将其添加到培养基中可维持类器官有效再生和长期增殖（超过 20 代）。Wnt-3A 对类器官培养条件改善不明显。EGF 对乳腺癌类器官培养具有双重作用，高于 5 ng/ml 促进细胞增殖，但会导致类器官解体，失去自组织功能，低浓度则相反。Hans Clevers 等构建乳腺癌类器官的培养基组分：（如下实验流程和数据均引用 Hans Clevers 等人发表的文章（ doi 10.1016/j.cell.2017.11.010 ），供参考。）a：类器官出现解体时减少或不添加，b：浓度超过 1 μM 会降低类器官生成效率5. 肺癌类器官培养肺癌是全球常见的恶性实体肿瘤，但是肺癌早期临床表现不明显，容易与其他感染混淆而被忽视，一般确诊时已发展到中晚期，因此，及时准确地筛选合适的抗肿瘤药物对肺癌患者非常重要。1 份从原发性肿瘤组织中衍生出肺腺癌类器官（成功率达到 80%）的培养基组分：（如下实验流程和数据均引用Zhichao Li等人发表的文章（10.1016/j.xpro.2020.100239 ），供参考。）试剂详细配制指南关键：所有操作都应在二级生物安全柜中进行，以保持无菌状态。1200 μg/mL FGF-10 (10,000 X)a. 取100 μg溶于0.5 mL 无菌的 PBS（加 0.1% BSA，质量/体积）。b. 每管 5 μL，将溶液分装到 1.5 mL 的离心管中，可在 -20 ℃ 保持 3 个月。2250 μg/mL FGF-7 (12,500 X)a. 取 100 μg 溶于 0.4 mL 无菌的 PBS（加 0.1% BSA，质量/体积）。b. 每管 4 μL，将溶液分装到 1.5 mL 的离心管中，可在 -20 ℃ 保持 3 个月。3100 μg/mL R-spondin 1 (200 X)a. 取 250 μg 溶于 2.5 mL 无菌的 PBS（加 0.1% BSA，质量/体积）。b. 每管 250 μL，将溶液分装到 1.5 mL 的离心管中，可在 -20 ℃ 保持 1 个月。4100 μg/mL Noggin (1,000 X)a. 取 100 μg 溶于 1 mL 无菌的 PBS（加 0.1%  BSA，质量/体积）。b. 每管 50 μL，将溶液分装到 1.5 mL 的离心管中，可在 -20 ℃ 保持 2 个月。5100 mM Y-27632 dihydrochloride (10,000 X)a. 取 50 mg 溶于 1.56 mL 的超纯水中。b. 用 0.22 μm 的过滤器过滤溶液。c. 每管 5 μL，将溶液分装到 1.5 mL 的离心管中，可在 -20 ℃ 保持 6 个月。6500 mM N-Acetylcysteine (400 X)a. 取 408 mg 溶于超纯水，至终体积为 5 mL。b. 用 0.22 μm 的过滤器过滤溶液。c. 每管 125 μL，将溶液分装到 1.5 mL 的离心管中，可在 -20 ℃ 保持 3 个月。72 M Nicotinamide (200 X)a. 取 6.1 g 溶于 1x PBS，至终体积为 25 mL。b. 用 0.22 μm 的过滤器过滤溶液。c. 每管 250 μL，将溶液分装到 1.5 mL 的离心管中，可在 -20 ℃ 保持 3 个月。830 mM SB202190 单盐酸盐水合物 (3,000 X)a. 取 5 mg 溶于 0.453 mL 的二甲基亚砜（DMSO）中b. 每管 16.67 μL，将溶液分装到 1.5 mL 的黑色离心管中，可在 -20 ℃ 保持 3 个月。关键：不要用聚偏二氟乙烯（PVDF）膜过滤含有 DMSO 的溶液，因为 DMSO 可以溶解这种膜。关键：使用黑色离心管，防止该溶液受到光照。建议：过滤 DMSO 溶液，可以使用 DMSO 安全过滤器或尼龙膜过滤器。925 mM A83-01 (50,000 X)a. 取 5 mg 溶于 0.474 mL 的二甲基亚砜（DMSO）中b. 每管 2 μL，将溶液分装到 1.5 mL 的离心管中，可在 -20 ℃ 保持 3 个月。10分装基质胶a. 取一瓶新的基质胶，放于 4 ℃ 冰箱中解冻 8 小时。b. 将瓶子倒置几次，使其充分混合。c. 取 1.5 mL 的离心管置于冰上冷却。d. 将基质胶和 1.5 mL 的离心管置于冰上，按实验所需体积将基质胶分装到 1.5 mL 的离心管中，并在 -20 ℃ 保存。关键：移液器枪头应在抽吸基质胶之前，为了防止基质胶黏在枪头上，应通过多次抽吸和排出冷的无菌 PBS（含 01.% BSA）进行预湿和预冷。注意：基质胶是一种重组基膜制剂，富含胞外蛋白，应避免反复冻融。6. 卵巢癌类器官目前，培养卵巢癌类器官的方法有多种，但成功率较高的是使用基质胶和改良 DMEM/F12 基础培养基的组合，并辅以 EGF、FGF2、FGF10、Noggin、Rspondin1、p38 抑制剂等，可使类器官培养成功率达到 80%～90%。Senkowski 等人为了获得高级别浆液性卵巢癌（HGSC）类器官的长期培养方案，对培养基添加组分进行优化，获得两种成功率更高的配方。并且体外培养的 HGSC 类器官保留了原始肿瘤的基因组和表型特征，其药物反应与临床结果具有相关性。培养基优化主要涉及添加物种类和浓度，尤其是细胞因子类。研究人员在改良型 DMEM/F12 培养基中加入 Glutamax、Primocin、N-acetyl-cysteine 和 B27 添加剂构成基础培养基。通过分析添加剂对 HGSC 原代细胞短期类器官形成和生长的影响，使用添加 FGF-10、SB202190 和 A83-10 的基础培养基（M 0.1）进行优化。但 M 0.1 培养基能促进生长，但在传代后并不能维持生长。进一步确定 HGSC 类器官培养基配方中加入了 5 mM 烟酰胺，形成培养基 M1。在培养基 M1 中添加 EGF、Heregulin-β-1、Hydrocortisone 和 Forskolin，形成培养基 M2。对于不同的样品，M2 可能具有促进或抑制类器官生长的「矛盾」结果，因此为了最大限度地提高类器官形成，每个 HGSC 样品应在两种不同的培养基 M1 和 M2 中平行培养。类器官形成和长期培养测试的主要因子及其影响概述7. 其他肿瘤类器官膀胱癌类器官利用 CTOS 培养基进行培养，主要成分为改良型 DMEM/F12、B27、A83-01，需补充添加 FGF10、FGF7、HER3。培养前列腺类器官时，一般会加入 FGF10、FGF2、p38 抑制剂 SB202190、Rho 激酶抑制剂等，而小鼠前列腺癌类器官无需添加 FGF10、FGF2、PGE2、烟酰胺和 SB202190。文章来源：生物学霸END深度聚焦类器官与3D培养论坛，OTC2024论坛合作详询：王晨 180 1628 8769戳“阅读原文”立即领取限量免费参会名额!

– Completed Second Cohort Showed an Average 30% Relative Increase in Ejection Fraction (EF) at 90 Days Following a Single Administration of JK07 – – Cohort 3 Sentinel Subject Demonstrated a >70% Relative EF Increase at Day 30 – – Company also Announces the First CTA Approval for JK08, the First Investigational IL15-CTLA4 Antibody Fusion for Solid Tumors – GAITHERSBURG, Md.--(BUSINESS WIRE)-- Salubris Biotherapeutics Inc. (SalubrisBio), a clinical-stage biotechnology company dedicated to discovering and developing novel complex biologic therapeutics, today announced positive interim data from the ongoing Phase 1b study of JK07 in heart failure with reduced ejection fraction (HFrEF). The interim data includes eleven NYHA II/III subjects across three single-ascending dose cohorts. Single doses were administered intravenously and changes from baseline values of left ventricular EF were measured for each patient. Following completion of enrollment in the first two cohorts, administration of JK07 led to dose-dependent improvements in EF, with an average improvement of 30% observed at day (D) 90 in Cohort 2. The unblinded sentinel subject in Cohort 3 has shown a 70% improvement from baseline at D30. “JK07’s antibody-fusion design is demonstrating its potential to deliver the durable therapeutic effect of neuregulin, without the associated toxicity, to regenerate heart function and restore quality of life for heart failure patients,” said Sam Murphy, Chief Executive Officer of SalubrisBio. “The consistent, dose-dependent trends we’re observing in EF following a single dose, in conjunction with a favorable safety pro concordant changes in established biomarkers, are highly encouraging and we look forward to further results from our third cohort and beyond. We are also excited to now be recruiting for the first clinical trial with our IL15-CTLA4 antibody fusion, JK08, and we expect to share initial data in 2023.” Summary of JK07 Data At D30, Cohorts 1 (dose: 0.03 mg/kg) and 2 (dose: 0.09 mg/kg) demonstrated similar average improvements in EF, with Cohort 1 (n=4) achieving a 20% improvement and Cohort 2 (n=4) achieving a 19% improvement. Early data from the sentinel subject in Cohort 3 (dose: 0.27 mg/kg) demonstrated a robust response to a single administration of JK07 with a >70% increase in EF at D30 (EF improvement from 22% to 38% absolute). At D90 in the first two cohorts, average EF improvement in Cohort 1 receded to 9% above baseline, while Cohort 2 further increased to 30%. In contrast, placebo subjects (n=2) showed a 4% average improvement in EF at D30 and a decline of 14% on D90. Moreover, dose-dependent increases in biomarker surrogates of target engagement were observed in each JK07-treated subject across the first two cohorts, and again in the sentinel subject in Cohort 3, indicating that target engagement has not yet been saturated. JK07 has been generally well-tolerated in the study, with no serious adverse events (SAEs) reported to date and enrollment of the randomized portion of Cohort 3 ongoing. Detailed findings will be presented at a future medical conference. The JK07 Cohort 2 EF improvement of 30% at D90 is comparable to a precedent neuregulin study which demonstrated 33% improvement at D90 following single-dose administration1. However, in the precedent study, dose limiting toxicities were observed at those same dose levels (0.19 – 1.51 mg/kg, aggregated in analysis)1. JK07 Cohort 3, therefore, represents the potential for new levels of activity not seen before in neuregulin clinical studies. Initiation of JK08 Clinical Trial in Europe The Company’s first clinical trial application to begin a Phase 1/2 study of JK08 as a monotherapy treatment for solid tumors in adults has been approved in Europe, and recruiting is now ongoing. The study will assess the safety, pharmacokinetics, and anti-tumor activity of JK08 at multiple dose levels and will include multiple expansion cohorts following selection of the optimal biologic dose. About JK07 JK07 is a recombinant fusion protein consisting of a fully human immunoglobulin IgG1 monoclonal antibody and an active polypeptide fragment of the human growth factor neuregulin [NRG-1]. NRG-1 is a clinically validated growth factor that has shown promising activity in HFrEF, but also undesirable side effects. Research has shown that NRG-1 induces signaling through interaction with two different receptors – HER3/ErbB3 and HER4/ErbB4. The HER4/ErbB4 pathway appears to be responsible for the regenerative effects in the heart, while the HER3/ErbB3 pathway appears primarily responsible for safety and tolerability limitations of recombinant NRG-1. By blocking HER3/ErbB3 signaling with an antibody fusion design, JK07 selectively stimulates the HER4/ErbB4 pathway with a favorable pharmacokinetic profile, which has the potential to significantly widen the therapeutic window of NRG-1 and yield better clinical effects. About JK08 JK08 is a recombinant fusion protein consisting of a CTLA-4-specific antibody and a C-terminal IL-15/sushi domain. JK08 was designed based on clinical studies from both individual molecules, which together portend synergistic effects in an antibody fusion construct. The CTLA-4-specific antibody ipilimumab validated CTLA-4 as a target for cancer therapy, but response rates are limited. Analysis of clinical samples demonstrated that NK cell activity signatures and ADCC biomarkers correlate with ipilimumab responses. Recombinant IL-15 has demonstrated potent stimulation of NK cell expansion and activation in clinical studies. JK08 channels the potent immune stimulation of IL-15 through the CTLA-4 antibody domain towards T-regulatory cell depletion and reversing immunosuppression which may contribute to cancer progression. About SalubrisBio SalubrisBio is a clinical-stage biotechnology company dedicated to discovering and developing complex biologics for cardiovascular, oncology, and neurodegenerative diseases. SalubrisBio was founded in August 2016 as a wholly-owned subsidiary of the China-based pharmaceutical company Shenzhen Salubris Pharmaceuticals Co. Ltd. Headquartered in the US, SalubrisBio reflects Shenzhen Salubris Pharmaceuticals’ commitment to innovation and expansion into the global market and retains the core philosophy of developing therapeutics for large patient populations with significant unmet needs. 1 Lenihan et al. A Phase I, Single Ascending Dose Study of Cimaglermin Alfa (Neuregulin 1β3) in Patients With Systolic Dysfunction and Heart Failure. JACC Basic Transl Sci. 2016 Dec 26;1(7):576-586.