Botulinum Toxin Type B

Net sales of Qelbree® increased 92% in the second quarter of 2024, compared to the same period in 2023. $59.4 million and $104.5 million of net sales in the second quarter and first six months of 2024, respectively. Net sales of GOCOVRI® increased 10% in the second quarter of 2024, compared to the same period in 2023. $31.7 million and $58.3 million of net sales in the second quarter and first six months of 2024, respectively. Total revenues were $168.3 million in the second quarter of 2024, an increase of 24% compared to the same period in 2023. Total revenues excluding Trokendi XR® and Oxtellar XR® net product sales (non-GAAP)(1) increased 32% in the second quarter of 2024, compared to the same period in 2023. Operating income of $22.6 million and $19.4 million in the second quarter and first six months of 2024.Adjusted operating earnings (non-GAAP)(1) was $45.5 million and $67.7 million in the second quarter and first six months of 2024, respectively, compared to $10.0 million and $40.5 million in the same periods in 2023.Raising full year 2024 guidance for total revenues and operating earnings (GAAP and Non-GAAP). ROCKVILLE, Md., Aug. 06, 2024 (GLOBE NEWSWIRE) -- Supernus Pharmaceuticals, Inc. (Nasdaq: SUPN), a biopharmaceutical company focused on developing and commercializing products for the treatment of central nervous system (CNS) diseases, today announced financial results for the second quarter of 2024 and associated Company developments. Business Highlights Total IQVIA prescriptions for Qelbree were 184,342 in the second quarter 2024, an increase of 26% compared to the same period in the prior year.In August 2024, the Company resubmitted its New Drug Application (NDA) for its apomorphine infusion device (SPN-830) for the continuous treatment of motor fluctuations ("off" episodes) in Parkinson’s disease. “In the second quarter of 2024, we delivered strong net sales growth from our key growth drivers, Qelbree and GOCOVRI, as well as strong growth in adjusted operating earnings. We also advanced our product pipeline, announcing interim results from our open-label Phase 2a clinical study of SPN-817 for treatment-resistant seizures, and continued to progress SPN-820 through two Phase 2 studies in patients with depression," said Jack Khattar, President and CEO of Supernus. “Continued growth from Qelbree and GOCOVRI, together with pipeline progress, gives us confidence in delivering on our increased guidance and our operational objectives for 2024." Product Pipeline Update SPN-820 – Novel first-in-class molecule that increases mTORC1 mediated synaptic function for depression Nearly three-quarters of the planned patients have been enrolled in the ongoing Phase IIb multi-center randomized double-blind placebo-controlled parallel design study of SPN-820 in adults with treatment-resistant depression. The study will examine the efficacy and safety of SPN-820 over a course of five weeks of treatment in approximately 268 patients in up to 50 clinical sites. The primary outcome measure is the change from baseline to end of treatment period on the Montgomery-Asberg Depression Rating Scale (MADRS) Total Score. Topline data from the Phase IIb trial are expected in the first half of 2025.Enrollment is ongoing in the Phase II open-label study in patients with major depressive disorder (MDD). The primary objective of the study is to assess efficacy in MDD, as well as onset of efficacy. Topline results from the study are expected by the end of 2024. SPN-817 – Novel first-in-class highly selective AChE inhibitor for epilepsy In May 2024, the Company announced data from the planned interim analysis of its exploratory open-label Phase IIa clinical study of SPN-817 for treatment-resistant seizures. The interim analysis was based on 41 enrolled subjects, of which 19 completed the maintenance period. The Company continues to expect topline results for the full study in the second half of 2024. A Phase IIb randomized, double-blind, placebo-controlled study in patients with treatment resistant focal seizures is expected to start by the end of 2024. SPN-443 – Novel stimulant for ADHD/CNS The Company plans to initiate a Phase I single dose study in healthy adults in 2024 following submission of an Investigational New Drug application. The primary objective of the study is to assess safety and tolerability. Financial Highlights This section includes information on non-GAAP financial measures. See “Non-GAAP Financial Information” section for information on non-GAAP financial measures. In addition, a reconciliation of applicable GAAP to non-GAAP financial information is included at the end of this press release. Revenues The following table provides information regarding total revenues (dollars in millions): Three Months Ended June 30, Six Months Ended June 30, 2024 2023 Change % 2024 2023 Change %Net product sales Qelbree$59.4 $31.0 92% $104.5 $56.8 84%GOCOVRI 31.7 28.8 10% 58.3 54.8 6%Oxtellar XR 29.5 23.8 24% 56.5 52.7 7%APOKYN 17.3 17.6 (2)% 33.9 34.8 (2)%Trokendi XR 17.1 19.3 (12)% 33.1 54.1 (39)%Other(2) 7.5 7.8 (4)% 14.7 15.7 (6)%Total net product sales 162.5 128.3 27% 301.0 268.9 12%Royalty, licensing and other revenues(3) 5.8 7.2 (20)% 11.0 20.4 (46)%Total revenues$168.3 $135.5 24% $312.0 $289.3 8% Total revenues excluding Trokendi XR and Oxtellar XR net product sales (non-GAAP)(1)$121.7 $92.4 32% $222.4 $182.5 22% Total revenues were $168.3 million and $312.0 million for the three and six months ended June 30, 2024, compared to $135.5 million and $289.3 million in the same period in 2023. Total net product sales were $162.5 million and $301.0 million for the three and six months ended June 30, 2024, compared to $128.3 million and $268.9 million in the same period in 2023, respectively. The increase in both periods was primarily due to the increase in net sales of Qelbree, GOCOVRI and Oxtellar XR, partially offset by the decline in net product sales of Trokendi XR due to generic erosion.Total revenues excluding Trokendi XR and Oxtellar XR net product sales (non-GAAP) increased 32% and 22% for the three and six months ended June 30, 2024, compared to the same period in 2023. Other Financial Highlights Operating earnings was $22.6 million and $19.4 million for the three and six months ended June 30, 2024, compared to operating loss of $(17.6) million and $(12.4) million for the same periods in 2023, respectively. The positive increase in both periods was primarily due to an increase in total net product sales.Adjusted operating earnings (non-GAAP) were $45.5 million and $67.7 million for the three and six months ended June 30, 2024, compared to $10.0 million and $40.5 million for the same periods in 2023, respectively.Net earnings (loss) and diluted earnings (loss) per share were $19.9 million and $0.36 for the three months and $20.0 million and $0.36 for the six months ended June 30, 2024, respectively, compared to $(0.8) million and $(0.02) for the three months and $16.1 million and $0.29 for the six months ended June 30, 2023, respectively.At June 30, 2024, cash, cash equivalents, and current and long-term marketable securities were approximately $347.2 million compared to $271.5 million as of December 31, 2023. This increase was primarily due to cash generated from operations. Full Year 2024 Financial Guidance For the full year 2024, the Company is increasing prior financial guidance for total revenues, GAAP operating earnings (loss) and non-GAAP operating earnings, and maintaining guidance for combined R&D and SG&A expenses as set forth below (dollars in millions). Current Guidance(as of August 6, 2024) Previous Guidance(as of May 8, 2024)Total revenues (includes approximately $135 - $145 million of Trokendi XR and Oxtellar XR)(4)(5)$600 - $625 $580 - $620Combined R&D and SG&A expenses$430 - $460 $430 - $460Operating earnings (loss)$0 - $20 $(30) - $(0)Adjusted operating earnings (non-GAAP)(1)$100 - $125 $80 - $110 Non-GAAP Financial Information This press release contains financial measures that present financial information which do not comply with United States generally accepted accounting principles (GAAP). The non-GAAP financial measures should be considered in addition to, not as a substitute for or in isolation from, or superior to measures prepared in accordance with GAAP. Non-GAAP adjusted operating earnings on a historical and projected basis adjusts for non-cash share-based compensation expense, depreciation and amortization, intangible asset impairment charges and changes to fair value of contingent consideration, and for factors that are unusual, non-recurring or unpredictable, and excludes those costs, expenses, and other specified items presented in the reconciliation tables in this press release. In addition to non-GAAP adjusted operating earnings, we also present total revenues excluding net product sales of Trokendi XR (GAAP) and Oxtellar XR (GAAP), which is a non-GAAP measure and is calculated as total revenues (GAAP) less net product sales of Trokendi XR (GAAP) and Oxtellar XR (GAAP). Beginning in the year a product loses exclusivity due to generic entrants we generally do not expect net product sales of such products to constitute a significant part of our revenue in the future. We believe that the use of non-GAAP financial measures provides useful supplemental information to management, investors, analysts and others regarding the Company’s revenue and results of operations and assist management, investors, analysts, and others in understanding and evaluating our revenue growth and the performance of the business. There are limitations associated with the use of non-GAAP financial measures and therefore comparability may be limited. These limitations include: non-GAAP financial measures that may not be entirely comparable to similarly titled measures used by other companies; these may not reflect all items of income and expense, as applicable, that affect our operations; there may be potential differences among calculation methodologies; these may differ from the non-GAAP information used by other companies, including peer companies. We mitigate these limitations by reconciling the non-GAAP financial measure to the most comparable GAAP financial measure. Investors are encouraged to review the reconciliation. The Company’s 2024 financial guidance is also being provided on both a GAAP and a non-GAAP basis. End Notes (1) See the section titled “Non-GAAP Financial Information” for information about this non-GAAP financial measure. A reconciliation of each non-GAAP financial measure to the most directly comparable GAAP financial measure is included at the end of this press release.(2) Includes net product sales of MYOBLOC®, XADAGO® and Osmolex ER®.(3) Royalty, licensing, and other revenues include royalties on generic Trokendi XR, other licensed products and intellectual property.(4) Includes net product sales and royalty, licensing, and other revenue.(5) Reflects continued generic erosion of Trokendi XR and generic erosion of Oxtellar XR beginning in September 2024. Conference Call Details Supernus will host a conference call and webcast today, August 6, 2024, at 4:30 p.m. Eastern Time to discuss these results. A live webcast will be available in the Events & Presentations section of the Company’s Investor Relations website www.supernus.com/Investors. Participants may also pre-register any time before the call here. Once registration is completed, participants will be provided a dial-in number with a personalized conference code to access the call. Please dial in 15 minutes prior to the start time. Following the live call, a replay will be available on the Company's Investor Relations website www.supernus.com/Investors. The webcast will be available on the Company’s website for 60 days following the live call. About Supernus Pharmaceuticals, Inc. Supernus Pharmaceuticals is a biopharmaceutical company focused on developing and commercializing products for the treatment of central nervous system (CNS) diseases. Our diverse neuroscience portfolio includes approved treatments for epilepsy, migraine, ADHD, hypomobility in Parkinson's disease (PD), cervical dystonia, chronic sialorrhea, and dyskinesia in PD patients receiving levodopa-based therapy. We are developing a broad range of novel CNS product candidates including new potential treatments for hypomobility in PD, epilepsy, depression, and other CNS disorders. For more information, please visit www.supernus.com. Forward-Looking Statements This press release includes forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. These statements do not convey historical information but relate to predicted or potential future events that are based upon management's current expectations. These statements are subject to risks and uncertainties that could cause actual results to differ materially from those expressed or implied by such statements. In addition to the factors mentioned in this press release, such risks and uncertainties include, but are not limited to, the Company’s ability to sustain and increase its profitability; the Company’s ability to raise sufficient capital to fully implement its corporate strategy; the implementation of the Company’s corporate strategy; the Company’s future financial performance and projected expenditures; the Company’s ability to increase the number of prescriptions written for each of its products and the products of its subsidiaries; the Company’s ability to increase its net revenue from its products and the products of its subsidiaries; the Company’s ability to commercialize its products and the products of its subsidiaries; the Company’s ability to enter into future collaborations with pharmaceutical companies and academic institutions or to obtain funding from government agencies; the Company’s product research and development activities, including the timing and progress of the Company’s clinical trials, and projected expenditures; the Company’s ability to receive, and the timing of any receipt of, regulatory approvals to develop and commercialize the Company’s product candidates; the Company’s ability to protect its intellectual property and the intellectual property of its subsidiaries and operate its business without infringing upon the intellectual property rights of others; the Company’s expectations regarding federal, state and foreign regulatory requirements; the therapeutic benefits, effectiveness and safety of the Company’s product candidates; the accuracy of the Company’s estimates of the size and characteristics of the markets that may be addressed by its product candidates; the Company’s ability to increase its manufacturing capabilities for its products and product candidates; the Company’s projected markets and growth in markets; the Company’s product formulations and patient needs and potential funding sources; the Company’s staffing needs; and other risk factors set forth from time to time in the Company’s filings with the Securities and Exchange Commission made pursuant to Section 13 or 15(d) of the Securities Exchange Act of 1934, as amended. The Company undertakes no obligation to update the information in this press release to reflect events or circumstances after the date hereof or to reflect the occurrence of anticipated or unanticipated events. Supernus Pharmaceuticals, Inc.Condensed Consolidated Balance Sheets(in thousands, except share data) June 30, December 31, 2024 2023 (unaudited) Assets Current assets Cash and cash equivalents$52,089 $75,054 Marketable securities 295,098 179,820 Accounts receivable, net 152,494 144,155 Inventories, net 68,155 77,408 Prepaid expenses and other current assets 23,166 16,676 Total current assets 591,002 493,113 Long-term marketable securities — 16,617 Property and equipment, net 12,274 13,530 Intangible assets, net 559,644 599,889 Goodwill 117,019 117,019 Other assets 35,890 37,505 Total assets$1,315,829 $1,277,673 Liabilities and stockholders’ equity Current liabilities Accounts payable and accrued liabilities$82,611 $79,569 Accrued product returns and rebates 175,119 154,274 Contingent consideration, current portion 47,303 52,070 Other current liabilities 3,623 4,283 Total current liabilities 308,656 290,196 Contingent consideration, long-term 697 1,380 Operating lease liabilities, long-term 30,294 33,196 Deferred income tax liabilities, net 11,440 24,963 Other liabilities 7,288 6,422 Total liabilities 358,375 356,157 Stockholders’ equity Common stock, $0.001 par value; 130,000,000 shares authorized; 55,046,049 and 54,723,356 shares issued and outstanding as of June 30, 2024 and December 31, 2023, respectively 55 55 Additional paid-in capital 455,170 439,493 Accumulated other comprehensive loss, net of tax (372) (593)Retained earnings 502,601 482,561 Total stockholders’ equity 957,454 921,516 Total liabilities and stockholders’ equity$1,315,829 $1,277,673 Supernus Pharmaceuticals, Inc.Condensed Consolidated Statements of Earnings (Loss)(in thousands, except share and per share data) Three Months EndedJune 30, Six Months EndedJune 30, 2024 2023 2024 2023 (unaudited) (unaudited)Revenues Net product sales$162,538 $128,336 $300,999 $268,911 Royalty, licensing and other revenues 5,787 7,227 10,970 20,416 Total revenues 168,325 135,563 311,969 289,327 Costs and expenses Cost of goods sold(a) 17,916 21,091 34,225 44,551 Research and development 26,183 24,379 51,113 45,591 Selling, general and administrative 85,904 86,782 172,420 172,379 Amortization of intangible assets 20,108 20,108 40,245 40,074 Contingent consideration expense (gain) (4,355) 790 (5,450) (857)Total costs and expenses 145,756 153,150 292,553 301,738 Operating earnings (loss) 22,569 (17,587) 19,416 (12,411) Other income (expense) Interest and other income, net 3,733 1,370 7,129 6,716 Interest expense — (910) — (2,415)Total other income (expense) 3,733 460 7,129 4,301 Earnings (loss) before income taxes 26,302 (17,127) 26,545 (8,110) Income tax expense (benefit) 6,386 (16,296) 6,505 (24,227)Net earnings (loss)$19,916 $(831) $20,040 $16,117 Earnings (loss) per share Basic$0.36 $(0.02) $0.37 $0.30 Diluted$0.36 $(0.02) $0.36 $0.29 Weighted average shares outstanding Basic 54,978,781 54,502,993 54,890,265 54,442,463 Diluted 55,724,283 54,502,993 55,675,474 59,035,154 (a) Excludes amortization of intangible assets. Supernus Pharmaceuticals, Inc.Reconciliations of GAAP to Non-GAAP Financial Information(Unaudited) Reconciliation of GAAP Total revenues to Non-GAAP Total revenues excluding Trokendi XR and Oxtellar XR net product sales An itemized reconciliation between total revenues on a GAAP basis and Total revenues excluding Trokendi XR and Oxtellar XR net product sales, a non-GAAP measure, is as follows (dollars in millions): Three Months Ended June 30, Six Months Ended June 30, 2024 2023 Change % 2024 2023 Change %Total revenues (GAAP)(1)$168.3 $135.5 24% $312.0 $289.3 8%Adjustments: Trokendi XR net product sales (17.1) (19.3) (12)% (33.1) (54.1) (39)%Oxtellar XR net product sales (29.5) (23.8) 24% (56.5) (52.7) 7%Total revenues excluding Trokendi XR and Oxtellar XR net product sales (non-GAAP)(1)$121.7 $92.4 32% $222.4 $182.5 22% (1) Includes net product sales and royalty, licensing, and other revenues. Reconciliation of GAAP Operating earnings (loss) to Non-GAAP Adjusted Operating earnings An itemized reconciliation between operating earnings (loss) on a GAAP basis and adjusted operating earnings on a non-GAAP basis is as follows (dollars in millions): Three Months EndedJune 30, Six Months EndedJune 30, 2024 2023 2024 2023 Operating earnings (loss) - As Reported (GAAP)$22.6 $(17.6) $19.4 $(12.4)Adjustments: Amortization of intangible assets 20.1 20.1 40.2 40.1 Share-based compensation 6.6 6.1 12.4 12.4 Contingent consideration expense (gain) (4.4) 0.8 (5.5) (0.9)Depreciation 0.6 0.6 1.2 1.3 Operating earnings - As Adjusted (non-GAAP)$45.5 $10.0 $67.7 $40.5 Non-GAAP adjusted operating earnings adjusts for non-cash items including amortization of intangible assets, share-based compensation expense, change in fair value of contingent consideration, and depreciation. Reconciliation of Full Year 2024 Financial Guidance - GAAP Operating earnings (loss) to Non-GAAP Adjusted Operating earnings An itemized reconciliation between projected operating earnings (loss) on a GAAP basis for the full year 2024 and projected adjusted operating earnings on a non-GAAP basis for the full year 2024 is as follows (dollars in millions): Current Guidance(as of August 6, 2024) Previous Guidance(as of May 8, 2024)Operating earnings (loss) - GAAP$0 - $20 $(30) - $0Adjustments: Amortization of intangible assets$78 - $80 $80 - $81Share-based compensation$27 - $29 $27 - $29Contingent consideration expense (gain)$(7) - $(7) $1 - $2Depreciation$2 - $3 $2 - $3Operating earnings - As Adjusted (non-GAAP)$100 - $125 $80 - $110 CONTACTS: Jack A. Khattar, President and CEOTimothy C. Dec, Senior Vice President and CFOSupernus Pharmaceuticals, Inc.(301) 838-2591 or INVESTOR CONTACT:Peter VozzoICR Westwicke(443) 213-0505peter.vozzo@westwicke.com

导读：多聚体纳米抗体指将不同的纳米抗体（也称VHH）连接在一起，从而产生能够同时靶向超过一种抗原的抗体分子。多聚体纳米抗体共同靶向相同抗原分子上的非重叠表位有助于提升抗体分子对靶标的亲和力，变体特异性和中和效力。有研究基于最新的自复制RNA(saRNA)技术，设计了一款六聚体纳米抗体saRNA药物，有效维持纳米抗体在体内的表达水平和治疗疗效。 2022年7月8号，塔夫茨大学Jean Mukherjee等人在Scientific Reports发布文章：Intramuscular delivery of formulated RNA encoding six linked nanobodies is highly protective for exposures to three Botulinum neurotoxin serotypes。他们设计了一款编码 VHH 异六聚体的repRNA-LNP，并以VHH 异源六聚体蛋白为对照，在BoNT 肉毒杆菌中毒小鼠模型中，验证了repRNA-LNP能够赋予小鼠对超致死剂量的肉毒毒素 A、B、E 型产生广谱的免疫保护作用。这项工作证明将纳米抗体与mRNA技术结合起来，有望成为一种非常有前景的广谱型免疫治疗手段。 01  纳米抗体与自复制RNA（saRNA）技术的联合 单克隆抗体免疫疗法具有非常广泛的临床应用。为提升单克隆抗体的效力，扩大其特异性，最近有些研究设计出能够识别1个或者更多表位的多功能抗体融合蛋白，这种类型的抗体能够提升靶向单个靶标的结合能力或者通过结合多个靶标增加抗体的新功能。 下一代免疫疗法非常有前景的方法是使用连接的单域抗体（sdAbs），即来自于骆驼或者鲨鱼体内，天然缺失轻链，只含有重链可变区的纳米抗体，也称为VHHs。与传统抗体相比，纳米抗体具有很多不同的优势，包括分子量小（14KDa），对于PH和温度具有更高的稳定性，可通过微生物表达体系进行批量生产。VHHs还可以作为多聚体蛋白表达，提升中和效力，实现多种治疗性靶标的识别。 最后，VHHs异多聚体非常适合利用复制 repRNA实现快速表达。与非复制 mRNA 相比，使用repRNA可将免疫球蛋白在肌肉细胞中的表达量提高 32 倍。repRNA技术的一个优势在于剂量节省优势，这对于体内有限数量的转染repRNA的肌肉细胞实现抗体的治疗性表达水平是至关重要的。 02  VHH异源六聚体的序列设计 研究者设计了两种类型的异源六聚体VAN蛋白序列，分别编码6个相连的VHH组分，包含靶向 BoNT 血清型（A、B 和 E）的 VHH 对，以不同的方式组装产生的异源六聚体称为：VNA1-ABE与VAN2-ABE。每一种VHH靶向BoNT的精确位点和分子机制已经通过结构与功能方面的研究得以确认。本研究中使用的 VHHs 包括中和 BoNT/A 的 ciA-H7 (VA1) 和 ciA-B5 (VA2) ；中和 BoNT/B的JLI-G10 (VB1) 和 JLK-G12 (VB2)；中和 BoNT/E的JLE-G6 (VE1) 和 JLE-E9 (VE2)。在异六聚体VNA1-ABE中，识别每个BoNT血清型的VHH按照顺序连接在一起，即NH 2 - VA1/VA2/VB1/VB2/VE1/VE2。VNA1-ABE 使用柔性间隔序列进行组装，以允许VHH对结合同一靶蛋白上的两个非重叠表位。在 VNA2-ABE 中，VHH对相互串联在一起，即NH 2 - VA1/VB1/VE1/VA2/VB2/VE2。在VNA2-ABE序列中，每个 VHH 之间包含短 SGGGG 间隔序列，以减小总蛋白的尺寸。VNA1-ABE 和 VNA2-ABE 编码区两侧均存贮于检测的 E-tags，并且在C 端含有小鼠白蛋白结合肽以促进更长的血清半衰期。 图1：异源六聚体VNA 抗毒素的序列设计 03  VHH异源六聚体的体内表达 为了确认异源六聚体VNA的中和能力，首先将编码 VNA1-ABE 和 VNA2-ABE 的DNA序列与人 Igκ 前导序列连接到哺乳动物细胞表达载体 pSecB 上面。同时，还制备类似的表达载体 pSecB，分别携带能够中和BoNT/A 的 VHHs DNA序列【即 VA1/VA2 (VNA-BoNTA) 异源二聚体】和能够中和BoNT/B 的VHHs DNA序列【即VB1/VB2 (VNA-BoNTB) 异源二聚体】。将上述质粒转染CHO 细胞，并在 3-4 天后收集培养基，通过 SDS-PAGE 电泳进行总蛋白染色或免疫印迹分析，以确认全长 VNA 异六聚体的表达（图 2A/B）。从胶图中可以看到，分泌的携带E标签的全长异源六聚体VNA可正常表达，具有预期的大小，并且未检测到截断的异常表达产物。与两种编码异二聚体 VNA 的表达质粒相比，编码全长异源六聚体VNA的质粒转染细胞后与其具有相似的蛋白表达水平，每个异六聚体 VNA 的表达水平约为 10 µg/ml。 图2：利用SDS-PAGE、蛋白质印迹和ELISA分析不同VNA表达载体转染的CHO细胞后的蛋白表达情况 04  VHH异源六聚体识别BoNT抗原靶标 为了确定与异二聚体 VNA 相比，六个 VHHs组装成的异六聚体VNA 的靶标识别能力是否会受到影响，利用ELISA分析异六聚体VNA 对 BoNT/A/B /E毒素的中和能力。结果发现，与异二聚体 VNA相比， VNA1-ABE 和 VNA2-ABE具有相似的靶向BoNT/A/B /E毒素的中和能力（图2/C）。因此，与异二聚体VHHs相比，将六个VHH表达为一个单一蛋白并不会对其靶向毒素的中和能力造成影响，两种异源六聚体构型之间也没有明显差异。 05  VHH异源六聚体重组蛋白的体内抗毒素效果 研究者在小鼠体内评估通过CHO 表达系统生产出来的异六聚体蛋白的抗毒素功效。他们首先在小鼠体内注射空白对照，VNA1-ABE重组蛋白或者VNA2-ABE重组蛋白，然后以小鼠腹膜内中位致死剂量对小鼠进行3种血清型肉毒杆菌毒素的攻毒试验。 结果发现，空白对照组小鼠在注射毒素3h后死亡，而所有注射 VNA1-ABE重组蛋白 或 VNA2-ABE 重组蛋白的小鼠均存活了7 天。虽然经过治疗的小鼠会出现短暂的轻微肉毒中毒迹象，但所有症状在一周内完全消失。这些研究表明，与单体VHHs相比，异源六聚体VNA 的协同中和活性赋予机体针对不同毒素的免疫保护效果，这说明构成异源六聚体VNA 的每种VHHs成分均保留了全部或者大部分的靶向不同毒素的中和活性。 图3：注射异六聚体VNA重组蛋白保护小鼠免受100MIPLD50剂量的BoNT/A/B/E肉毒杆菌毒素攻毒后的致死效应 接下来，研究者想知道异六聚体VNA的构象是否影响治疗效果，因此他们比较注射异六聚体 VNA1-ABE 重组蛋白与VNA2-ABE 重组蛋白的治疗效果。首先用 500 MIPLD 50剂量的 BoNT/B 对小鼠进行攻毒试验，然后注射两种不同类型的异六聚体VNA。结果发现，对照组小鼠在进行攻毒后的两小时内死亡，而用使用 VNA 治疗的小鼠显示100% 的存活率(图4/A)。接着，他们用 10,000 MIPLD 50 BoNT/A 对小鼠进行攻毒试验，在注射如此高的毒素剂量后，对照组小鼠攻毒 1 小时内死亡，但所有先前注射过 VNA 重组蛋白的小鼠增加了存活时间。此外，与注射 VNA1-ABE 重组蛋白相比，注射VNA2-ABE 重组蛋白的小鼠能够增加存活时间，这表明 在VNA2-ABE 重组蛋白序列中同源 VHH 之间间距的增加提升了其靶向毒素的中和能力（图4/B）。 图4：异六聚体VNA重组蛋白保护小鼠免受500MIPLD50剂量的BoNT/B的致死效应，并增加小鼠 在10,000MIPLD50高剂量BoNT/A下的存活时间 06  编码VHH异源六聚体的 repRNA体内抗毒素效果 研究者想知道肌肉内注射编码VNA 异六聚体的repRNA 是否同样具有抗肉毒杆菌毒素的治疗效果。他们首先设计出编码新六聚体VNA的repRNA序列，在 VNA2-ABE的序列基础上做出以下优化，以便降低异六聚体的总大小：（1）移除非核心 的VHH 残基；（2）保留 C 端用于检测的 E-tag 序列；（3）用五个甘氨酸残基间替换VHH 之间的间隔序列，以缩短间隔序列。通过体外转录合成编码VNA3-ABE 的repRNA，并且用LION™ (HDT Bio) 阳离子纳米载体进行包封，以方便进行肌肉注射。 首先，研究者测定注射VNA3-ABE -repRNA后的药代动力学特征，以评估给药后VNA3-ABE的在血清中的表达水平。在小鼠双侧肌肉注射LION包封的10 µg VNA3-ABE -repRNA。在注射后 4 小时和 5 天之间进行定点取血，并通过 ELISA 评估血清的中的VNA3-ABE 蛋白水平。结果显示，血清VNA3-ABE 滴度在注射后8h便可检测到，在注射后2天达到峰值，在 5 天时血清VNA3-ABE 滴度下降到检测限以下（图5/A）。 接下来，研究者在小鼠 BoNT 攻毒模型中，测定VNA3-ABE -repRNA的治疗效果。将LION 包封的VNA3-ABE -repRNA（每只小鼠 10 ug）或单独的 LION 通过小鼠双侧大腿注射给药。在注射VNA3-ABE -repRNA后24小时，在小鼠体内，通过静脉内注射含有各100 MIPLD 50的BoNT/A/B/E混合毒素对小鼠进行攻毒。所有对照小鼠在 BoNT 中毒后 3 小时内死亡，这与高剂量毒素攻击后小鼠的存活时间一致。相比之下，所有接受VNA3-ABE -repRNA的小鼠均在攻击中存活。尽管在高剂量毒素攻击的情况下，注射VNA3-ABE -repRNA的小鼠表现出肉毒杆菌中毒的轻微迹象，但是这种中毒迹象会迅速消退（图5）。总的来说，这些数据证明LION 包封的VNA3-ABE -repRNA在体内具有 抵抗多种 BoNT 血清型毒素攻击的治疗效果。 图5：在小鼠 BoNT肉毒杆菌毒素模型中，注射LION 包封的VNA3-ABE -repRNA具有抵御多种毒素攻击的治疗效果 07  总结 在这篇文章中，研究者利用靶向BoNT 的单域抗体 VHH 来设计构建出3个异源六聚体 VNA，每个 VNA 都包含六个相同的靶向BoNT的中和抗体 VHH。在每个VNA结构序列中，六个 VHH 的顺序或者间距有所不同。研究者证实在小鼠模型中，注射每种 VNA 均可保护小鼠免受三种高剂量肉毒毒素的攻击。 无论是静脉注射基于重组蛋白的异源六聚体 VNA（VNA1-ABE、VNA2-ABE）还是肌肉注射阳离子脂质包封的VNA3-ABE-repRNA（VNA3-ABE），异六聚体 VNA 都具有有效的抗毒素治疗效果。经过测试，分泌的最小异六聚体的分子质量仅为 84 kDa。与单体VHHs相比，异源六聚体VNA 的协同中和活性赋予机体针对不同毒素的免疫保护效果，这说明构成异源六聚体VNA 的每种VHHs成分均保留了全部或者大部分的靶向不同毒素的中和活性。 总之，研究者证明将纳米抗体形成高效异源多聚体的巨大优势与mRNA技术结合，借助高效的递送技术，能够为基于抗体的免疫治疗方法开拓新的思路。 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。