Rose Bengal disodium

在2026年的今天，选择商品化的亲和填料变得很方便，比如抗体亲和的Protein A/Protein L、标签亲和的Ni Sepharose、GST捕获填料等。 但这些产品解决的是通用标签或通用识别问题。而真正头疼的，往往是那些“没有标准答案”的课题：没有标签的天然蛋白、特定抗原、多抗血清、自制抗体、功能肽、小分子探针、糖类配基、需要固定化的酶…… 这个时候，一种适配广泛的解法应运而生：预活化填料。预活化填料的价值就在于：它让我们能够自主定义亲和关系。 先说清楚：什么是预活化填料？ 亲和层析的关键，是利用“配基”和“目标分子”之间的特异性识别，把目标从复杂样本中识别出来。比如抗原识别抗体、抗体识别靶蛋白、凝集素识别糖蛋白，肽段或小分子也可以作为“诱饵”捕获相互作用蛋白。要实现这些应用，第一步就是：把配基稳定地固定到填料上。 预活化填料可以理解为“已经预装好接口的填料”。Cytiva 的NHS、CNBr、Epoxy等预活化填料，已经在Sepharose等基架表面引入了可反应的活性基团。用户只需要加入自己的抗体、抗原、蛋白、肽段、酶、小分子或糖类配基，就可以通过共价偶联，把配基接到填料上。 以NHS-activated Sepharose为例，具体的偶联步骤如下： 第一步 洗去保存液 NHS活化填料通常保存在100%异丙醇中，以保护活性基团。使用前，用冷的1 mM HCl等溶液快速洗去异丙醇，尽量减少NHS活性酯水解。 第二步 加入配基进行偶联 将抗体、抗原、蛋白或肽段等含伯氨基配基溶解在偶联缓冲液中，室温下约15–30 min即可完成偶联，也可在4 ℃反应约4 h。 第三步 封闭剩余活性基团 偶联完成后，填料表面可能还有未反应的NHS基团。可用乙醇胺或Tris等试剂封闭，避免后续样品中的蛋白发生非特异反应。 第四步 交替洗涤，去除残留配基 最后用高pH和低pH缓冲液交替洗涤，去除未偶联或非特异吸附的配基。 Cytiva预活化填料的种类 预活化填料的本质，是通过共价偶联把配基固定在Sepharose等层析介质表面。不同活性基团，就像不同类型的“接口”，适合连接不同配基。 NHS-activated Sepharose 主要偶联伯氨基，形成稳定酰胺键。 NHS活化填料表面带有N-羟基琥珀酰亚胺活化酯。当配基上有伯氨基时，例如蛋白N端氨基、赖氨酸侧链ε-氨基，伯氨基会进攻NHS活化酯，NHS作为离去基团释放，最终在配基和填料之间形成稳定的酰胺键。因此，该填料很适合偶联抗体、抗原、蛋白和肽段等含氨基配基。 图1.1 带有活化间隔臂的 NHS 活化 Sepharose的局部结构示意图 CNBr-activated Sepharose 主要偶联含氨基或类似亲核基团的配基，适合较大蛋白。 氰溴化物会活化琼脂糖基质上的羟基，生成可与配基中伯氨基反应的活性中间体。随后，蛋白配基上的氨基与活化基团反应，使配基共价连接到基架上。由于CNBr偶联常可实现蛋白配基的多点连接，因此比较适合较大蛋白配基的固定化，并有助于降低配基脱落。 图1.2 通过氰溴（CNBr）活化并与Sepharose进行偶联示意图 Epoxy-activated Sepharose 6B 可与羟基、氨基或巯基反应，适合糖类和小分子。 Epoxy活化填料表面带有环氧基团。环氧基可以被多种亲核基团打开，例如配基中的羟基、氨基或巯基。反应后，配基会通过稳定的共价键连接到基架上。由于很多糖类和小分子都带有羟基，该填料特别适合糖类、小分子以及其他多官能团配基的固定化。 图1.3 带有活化间隔臂的 Epoxy 活化 Sepharose的局部结构示意图 EAH Sepharose 4B 用于偶联含羧基的小分子化合物。 EAH Sepharose 4B是在 Sepharose 4B上通过环氧偶联方式接入1,6-二氨基己烷形成的介质，其特点是在填料表面带有游离氨基，并且这些氨基位于11原子亲水间隔臂的末端。它主要用于偶联含羧基的配基。在碳二亚胺的存在下，配基上的羧基可以与EAH Sepharose 4B表面的氨基发生偶联，形成稳定连接。 图1.4 带有活化间隔臂的 EAH 活化 Sepharose的局部结构示意图 预活化填料的应用场景 案例一 基于EAH填料的草酸亲和捕获---垂钓肾结石中草酸结合蛋白 利用EAH-Sepharose 4B预活化填料上的氨基间隔臂，通过EDC碳二亚胺偶联反应把草酸固定到填料上，制备成“草酸亲和层析柱”，用于从复杂样品中捕获和分离草酸结合蛋白。用已知草酸结合蛋白p62和非相关蛋白碳酸酐酶进行验证。（00046 Piyachat Roop-ngam, 2010） 图2.1 草酸与EAH预活化填料偶联示意图 证明该方法可高效富集草酸结合蛋白，为肾结石中草酸相关蛋白研究提供了简便有效的工具。 案例二 把Ulp1蛋白酶固定住，SUMO标签切割更省心 在重组蛋白表达中，SUMO标签经常用于提高蛋白可溶性，但后续去标签时往往需要加入游离Ulp1蛋白酶。游离酶用完后还要再去除，且不能便捷地重复使用。研究者将Ulp1蛋白酶固定化到NHS-activated Sepharose上，制备了一种可重复使用的固定化蛋白酶工具。 该研究显示，Ulp1在NHS-activated Sepharose上的固定化量约为1.68 mg/mL，固定化后仍保持约95%的底物切割能力，并显著提升了pH稳定性和热稳定性。 图2.2 对比固定后的Ulp1与游离的Ulp1的与底物的切割能力 更令人欣喜的是，固定化Ulp1可耐受pH 10.5、15% DMSO和20%乙醇，并且在超过15次批次反应后仍保留约90%活性。研究者还利用该固定化酶结合Ni-NTA体系，在约1 h内获得无标签重组蛋白。 图2.3 固定后的Ulp1与未固定的Ulp1在不同pH条件下的稳定性 这个案例说明，预活化填料不只可以用于亲和层析纯化使用，它还可以把一种消耗型酶变成可回收、可重复、流程更简洁的固定化工具。 案例三 利用TNF-α的抗体片段作为配基纯化TNF-α 这篇文献中，研究者将抗TNF-α的scFv抗体偶联到CNBr预活化Sepharose 4B填料上，制备亲和层析柱，用于从LPS诱导的Raji细胞裂解液中捕获并纯化TNF-α。结果显示，洗脱产物在SDS-PAGE上呈现约17 kDa的目标条带，纯度可达95%以上，证明该预活化填料可用于快速、高效制备高纯度TNF-α。（Adv Pharm Bull. 2013;3(1):19-23） 图2.4 采用CNBr活化Sepharose 4B填料纯化的TNF-α的电泳图谱。 泳道 1：层析前的粗蛋白提取物；泳道 2：纯化后的洗脱组分 所以，预活化填料并不只是一瓶填料或一根预装柱。它更像是一个自定义亲和工具箱。 当你问到： “这个靶标没有现成亲和填料怎么办？” Cytiva给了你一个肯定的答案： 那就把你的配基接上去，做一款独属于你自己研究所需的亲和介质。 声明：本文为作者原创首发，严禁私自转发或抄袭，如需转载请联系并注明转载来源，否则将追究法律责任

Long-term survivors in both oral PV-10 arms showed an absence of gross bladder tumor at necropsy, a finding not observed in any untreated, vehicle-control, or anti-PD-1 monotherapy animal. KNOXVILLE, Tenn., May 18, 2026 (GLOBE NEWSWIRE) -- Provectus Biopharmaceuticals, Inc. (“Provectus” or the “Company”) (OTCQB: PVCT) today reported data from a preclinical safety and efficacy evaluation of PV-10 — a formulation of the Company’s proprietary, pharmaceutical-grade rose bengal sodium (RBS) active pharmaceutical ingredient — administered by oral and intravesical routes as a single agent and in combination with anti-human PD-1 against an orthotopic bladder carcinoma tumor xenograft model in immunologically humanized mice. Translational Drug Development, LLC (TD2 Oncology) of Scottsdale, Arizona, an oncology contract research organization, conducted the study. TD2 Oncology was created from the Translational Genomics Research Institute (TGEN) in 2003. TGEN, a precision medicine research organization, is a part of City of Hope, one of the largest cancer research and treatment organizations in the U.S. Across the seven-arm study, oral PV-10 monotherapy was the top-ranked treatment arm under a scoring framework that assessed anti-tumor response, survival benefit, safety and tolerability, data quality, and translational potential. Notably, two animals treated with oral PV-10 — one from the monotherapy group and one from the group of PV-10 in combination with anti-PD-1 — survived to study end and showed an absence of gross bladder tumor at necropsy, a finding not observed in any untreated, vehicle-control, or anti-PD-1 monotherapy animal. Study Design and Context This preclinical study utilized the UMUC3-Luc luciferase-expressing bladder carcinoma cell line implanted orthotopically into the bladder of human peripheral blood mononuclear cell (PBMC)-engrafted NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) mice, an immunologically humanized model that allows evaluation of treatment effects in the presence of a functional human immune compartment. Tumor progression was monitored longitudinally by bioluminescence imaging (BLI). The study used 54 female mice across seven groups and ran for 45 days. As is inherent to human PBMC-engrafted NOG models, graft-versus-host disease (GvHD) independently contributed to morbidity across all engrafted groups; the scoring framework weighted survival accordingly and all arm-level comparisons are interpreted in this context. The seven study arms were: Group 1: No treatment (negative control, n=8), Group 2: Human PBMC engraftment + vehicle instillation (active vehicle control, n=7), Group 3: Human PBMC + PV-10 3 mg/dose intravesical (OB-IVS), once weekly ×4 weeks (n=7), Group 4: Human PBMC + PV-10 2 mg/dose oral (PO), 5 days on/2 days off to study end (n=8), Group 5: Human PBMC + anti-human PD-1 10 mg/kg intraperitoneal, twice weekly ×4 weeks (n=8), Group 6: Human PBMC + PV-10 3 mg/dose OB-IVS + anti-PD-1 10 mg/kg (n=8), and Group 7: Human PBMC + PV-10 2 mg/dose PO + anti-PD-1 10 mg/kg (n=8). To evaluate the study’s treatment arm (Groups 3-7) results, Provectus applied a scoring framework of five weighted domains: anti-tumor response (30%), survival benefit (25%), safety and tolerability (25%), data quality and interpretability (12%), and translational and development potential (8%). Each domain was scored on a zero-to-ten scale using a pre-specified rubric grounded exclusively in observed study data, including Day 23 BLI tumor burden, time-to-morbidity, body weight trajectory, evaluable animal counts at key timepoints, and long-term survivor necropsy findings. The scoring framework was then validated through a sensitivity analysis across five weighting scenarios: base case, safety-first, efficacy-heavy, survival-dominant, and equal weight configurations. Notably, the rank order of all five active treatment arms was invariant across every scenario tested. Key Study Findings Oral PV-10 monotherapy (Group 4): The top-ranked arm: Day 23 BLI tumor burden approximately 40% lower than the vehicle control on a geometric mean basis (log₁₀ mean 10.39 vs. 10.61), Body weight nadir of −1.9% across all eight animals, the best tolerability pro all PBMC-engrafted groups, One long-term survivor (Day 45) whose gross necropsy did not record bladder tumor, compared to the near-universal “tumor throughout bladder” finding in untreated, vehicle-control, and anti-PD-1 monotherapy animals, Top scores in survival, safety, and data quality domains of the scoring framework, each at 10.0 out of 10.0, and Weighted total score of 9.24 out of 10. Oral PV-10 + anti-PD-1 combination (Group 7): The second-ranked arm: Highest translational potential score, reflecting the established clinical rationale for combining checkpoint blockade with novel immunomodulatory agents in bladder cancer, Body weight nadir of −14.4%; anti-PD-1-associated GvHD burden limited safety score relative to Group 4, One long-term survivor (Day 45) whose gross necropsy similarly did not record bladder tumor, and Weighted score of 7.84 out of 10. Intravesical PV-10 (Groups 3 and 6): The intravesical arms at 3 mg/dose (30–60 mg/mL concentration) were not tolerated at the instillation parameters tested: Six of seven Group 3 mice and three of eight Group 6 mice were lost by Day 12, following the first intravesical dosing event, consistent with acute mucosal toxicity at the concentrations employed. These findings represent a maximum tolerated concentration failure at the doses tested, not a negative efficacy signal. The intravesical route remains scientifically open at lower concentrations; clinical intravesical agents such as BCG and mitomycin-C are administered at substantially lower concentrations than those employed in this study. Drug Development Evaluation Framework Domain scores and sensitivity analysis results are presented in Tables 1 and 2 below. Table 1. Domain Scores by Arm Group Treatment Response (30%) Survival (25%) Safety (25%) Data Quality (12%) Translation (8%) Weighted Total 1 No Treatment 4.5 10.0 10.0 10.0 5.0 7.95/10 2 PBMC + Vehicle 6.0 10.0 9.0 9.0 5.0 8.03/10 3 PV-10 OB-IVS 0.0 0.0 5.0 1.0 1.0 1.45/10 4 PV-10 PO 8.0 10.0 10.0 10.0 8.0 9.24/10 5 anti-PD-1 5.0 10.0 6.0 10.0 7.0 7.26/10 6 PV-10 OB-IVS + anti-PD-1 5.0 0.0 1.0 3.0 2.0 2.27/10 7 PV-10 PO + anti-PD-1 7.4 8.8 6.0 10.0 9.0 7.84/10 Groups 1 and 2 are control arms of the study and are included in the above table for scoring framework calibration. They are not ranked against the treatment arms of Groups 3 to 7. Table 2. Sensitivity Analysis Weighting scenario Group 3 PV-10 OB-IVS Group 4 PV-10 PO Group 5 anti-PD-1 Group 6 PV-10 OB-IVS + anti-PD-1 Group 7 PV-10 PO + anti-PD-1 G4 lead over G7 Base case (30/25/25/12/8) 1.45 (#5) 9.24 (#1) 7.26 (#3) 2.27 (#4) 7.84 (#2) +1.40 Safety-first (20/20/40/12/8) 2.20 (#4) 9.44 (#1) 7.16 (#3) 1.92 (#5) 7.56 (#2) +1.88 Efficacy-heavy (40/30/15/10/5) 0.90 (#5) 9.10 (#1) 7.25 (#3) 2.55 (#4) 7.95 (#2) +1.15 Survival-dominant (20/40/25/10/5) 1.40 (#5) 9.50 (#1) 7.85 (#3) 1.65 (#4) 7.95 (#2) +1.55 Equal weight (20/20/20/20/20) 1.40 (#5) 9.20 (#1) 7.60 (#3) 2.20 (#4) 8.24 (#2) +0.96 In Table 2, the narrowest Group 4–Group 7 margin occurs under equal weight (+0.96), driven by Group 7’s high translation domain score (9.0 vs Group 4’s 8.0). Group 4’s lead widens under safety-first weighting (+1.88), reflecting Group 4’s perfect safety score versus Group 7’s GvHD-penalized score of 6.0. Dominic Rodrigues, Provectus’s President and Vice Chairman of the Board of Directors, said “This preclinical study marks three firsts for Provectus: the first evaluation of PV-10 in bladder cancer, the first evaluation of any PV-10 route in an orthotopic tumor model, and the first evaluation of oral PV-10 against a solid tumor cancer. Historical Company and research collaborator preclinical work employed solid tumor cancer flank mouse models to evaluate PV-10 administered by intratumoral injection.” He added, “These oral PV-10 findings are encouraging. The long-term survivor necropsy findings — two animals treated with oral PV-10 that showed an absence of gross bladder tumor at Day 45 — are an important signal. PV-10’s mechanism’s capacity to perturb the tumor microenvironment and drive antitumor immune activity is not bladder cancer-specific. That is precisely what makes this result scientifically interesting and developmentally promising beyond the indication in which it was observed.” Ed Pershing, Provectus’s Chairman and Chief Executive Officer, said, “The path from this preclinical signal to the clinic requires FDA acceptance of an expanded Investigational New Drug application, which we currently have for intratumoral administration of PV-10, to permit oral PV-10 human testing. That is our next goal and regulatory milestone. As we consider which indication to pursue first in a Phase 1 study, we are drawn to cancers where the gap between what standard of care offers and what patients need remains widest. Bladder cancer is one such disease. Pancreatic cancer and glioblastoma are others we are watching closely. Oral PV-10’s tolerability profile — demonstrated here in a tumor model with no dose-limiting events and a body weight nadir below two percent — is directly relevant to patients in those settings. This preclinical study tells us oral PV-10 belongs in that conversation.” About Provectus Provectus Biopharmaceuticals, Inc. is a clinical-stage biotechnology company developing a pipeline of immunotherapy medicines based on rose bengal sodium, a first-in-class synthetic small molecule from the halogenated xanthene family. The Company’s clinical programs span oncology, dermatology, and ophthalmology, with additional proof-of-concept programs in hematology, wound healing, infectious diseases, and tissue repair. For more information, visit . Forward Looking Statements The information in this press release may include “forward-looking statements,” within the meaning of the Private Securities Litigation Reform Act of 1995, relating to the business of Provectus and its affiliates, which are based on currently available information and current assumptions, expectations, and projections about future events and are subject to a variety of risks and uncertainties and other factors that could cause actual events or results to differ materially from those projected in the forward-looking statements. Forward-looking statements are often, but not always, identified by the use of words such as “aim,” “likely,” “outlook,” “seek,” “anticipate,” “budget,” “plan,” “continue,” “estimate,” “expect,” “forecast,” “may,” “will,” “would,” “project,” “projection,” “predict,” “potential,” “targeting,” “intend,” “can,” “could,” “might,” “should,” “believe,” and similar words suggesting future outcomes or statements regarding an outlook. The safety and efficacy of the agents and/or uses under investigation have not been established. There is no guarantee that the agents will receive health authority approval or become commercially available in any country for the uses being investigated or that such agents as products will achieve any particular revenue levels. Due to the risks, uncertainties, and assumptions inherent in forward-looking statements, readers should not place undue reliance on these forward-looking statements. The forward-looking statements contained in this press release are made as of the date hereof or as of the date specifically specified herein, and Provectus undertakes no obligation to update or revise any forward-looking statements, whether as a result of new information, future events or otherwise, except in accordance with applicable securities laws. The forward-looking statements are expressly qualified by this cautionary statement. Risks, uncertainties, and assumptions include those discussed in the Company’s filings with the Securities and Exchange Commission, including those described in Item 1A of the Company’s Annual Report on Form 10-K for the period ended December 31, 2025 and Quarterly Report on Form 10-Q for the period ended March 31, 2026 . Contacts: Provectus Biopharmaceuticals, Inc. Heather Raines, CPA Chief Financial Officer hraines@pvct.com (866) 594-5999 Investor Relations & Media Susan Xu sxu@allianceadvisors.com (778) 323-0959