Raxibacumab

Third Quarter 2024 Total Revenues of $293.8 million, increase of 9% versus prior yearThird Quarter 2024 Net Income of $114.8 million, increase of 144% versus prior yearThird Quarter 2024 Adjusted EBITDA of $105.3 million, increase of 432% versus prior yearRaises FY 2024 guidance GAITHERSBURG, Md., Nov. 06, 2024 (GLOBE NEWSWIRE) -- Emergent BioSolutions Inc. (NYSE: EBS) today reported financial results for the third quarter ended September 30, 2024. "Through disciplined execution and steady, measurable progress, Emergent's financial position is the strongest it has been since 2021 as evidenced by our favorable third-quarter results," said CEO Joe Papa. "We have successfully improved efficiencies and refocused our operations related to customer demand, generated value in our core medical countermeasures and NARCAN® Nasal Spray businesses and refinanced our debt leading to increased revenue and cash flow." Papa continued, "Based on the success of our efforts since the beginning of this year, we are officially entering the turnaround phase of our multi-year transformation plan, and we will be focused on driving profitable growth, continued operational improvements and the generation of sustainable value for shareholders. We believe ongoing public health crises like the opioid overdose epidemic and mpox outbreak underscore the need for Emergent's capabilities and expertise. It is not if, but when, the next public health threat emerges, and we believe we are uniquely qualified to help respond to protect, enhance and save lives." FINANCIAL HIGHLIGHTS(1) Q3 2024 vs. Q3 2023 ($ in millions, except per share amounts)Q3 2024Q3 2023% ChangeTotal Revenues$293.8 $270.5 9%Net Income (Loss)$114.8 $(263.4)144%Net Income (Loss) per Diluted Share$2.06 $(5.08)141%Adjusted Net Income (Loss)(2)$76.2 $(56.2)236%Adjusted Net Income (Loss) per Diluted Share(2)$1.37 $(1.09)226%Adjusted EBITDA(2)$105.3 $19.8 432%Total Segment Gross Margin %(2) 57% 33% Total Segment Adjusted Gross Margin %(2) 59% 38% Year to Date (“YTD”) 2024 vs. YTD 2023 ($ in millions, except per share amounts)YTD 2024YTD 2023% ChangeTotal Revenues$848.9 $772.7 10%Net Loss$(159.3)$(711.0)78%Net Loss per Diluted Share$(3.03)$(13.97)78%Adjusted Net Loss(2)$(14.7)$(273.0)95%Adjusted Net Loss per Diluted Share(2)$(0.28)$(5.36)95%Adjusted EBITDA(2)$162.1 $(25.7)731%Total Segment Gross Margin %(2) 32% 31% Total Segment Adjusted Gross Margin %(2) 46% 33% SELECT Q3 2024 BUSINESS UPDATES Secured a new Term Loan for $250 million with OHA Agency, LLC as administrative agent.Closed on a new asset backed loan facility for $100 million with Wells Fargo Bank, National Association.Received $75 million for the sale of our RSDL® (Reactive Skin Decontamination Lotion) product to a subsidiary of SERB Pharmaceuticals ("SERB"), subject to customary adjustments based on inventory value at closingCompleted the sale of the Baltimore-Camden manufacturing site for $35 million, including customary post-closing adjustmentsSold an underutilized warehouse at our Canton, MA facility for $7 millionReceived $50 million in the third quarter related to the resolution of the contractual dispute with Janssen Pharmaceuticals, Inc.Earned $30 million development milestone payments from Bavarian Nordic as part of the sale of the Travel Health Business THIRD QUARTER 2024 FINANCIAL PERFORMANCE(1) Revenues The Company uses the following categories in discussing product/service level revenues: NARCAN® — comprises contributions from NARCAN® Nasal SprayAnthrax MCM — comprises contributions from CYFENDUS®, previously known as AV7909, BioThrax®, Anthrasil® and RaxibacumabSmallpox MCM — comprises contributions from ACAM2000®, VIGIV and TEMBEXA®Other Products — comprises contributions from BAT® and RSDL®Bioservices — comprises service and lease revenues from the Bioservices business ($ in millions)Q3 2024Q3 2023% ChangeProduct sales, net:(3) NARCAN®$95.3$142.1(33)%Anthrax MCM 11.4 32.9(65)%Smallpox MCM 132.7 24.7437%Other Products 30.1 50.1(40)%Total Product sales, net$269.5$249.88% Bioservices: Services$13.9$13.25%Leases 0.4 1.0(60)%Total Bioservices revenues$14.3$14.21% Contracts and grants$10.0$6.554% Total revenues$293.8$270.59% Products Sales, net NARCAN® For Q3 2024, revenues from NARCAN® (naloxone HCl) Nasal Spray decreased $46.8 million, or 33%, as compared with Q3 2023. The decrease was primarily driven by the discontinuation of prescription NARCAN® due to the launch of over-the-counter (“OTC”) NARCAN® in the third quarter of 2023 and lower Canadian retail sales, partially offset by higher sales of OTC NARCAN®. Anthrax MCM For Q3 2024, revenues from Anthrax MCM products decreased $21.5 million, or 65%, as compared with Q3 2023. The decrease reflects the impact of timing of sales related to CYFENDUS® and Anthrasil®, partially offset by an increase in BioThrax® sales, due to timing. Anthrax vaccine product sales are primarily made under annual purchase options exercised by the U.S. government (the “USG”). Fluctuations in revenues result from the timing of the exercise of annual purchase options, the timing of USG purchases, the availability of governmental funding and the Company’s delivery of orders that follow. Smallpox MCM For Q3 2024, revenues from Smallpox MCM products increased $108.0 million, or 437%, as compared with Q3 2023. The increase was primarily due to timing of USG purchases of ACAM2000® and VIGIV. Fluctuations in revenues from Smallpox MCM result from the timing of the exercise of annual purchase options in the existing procurement contracts, the timing of USG purchases, the availability of governmental funding and Company delivery of orders that follow. Other Products For Q3 2024, revenues from Other Product sales decreased $20.0 million, or 40%, as compared with Q3 2023. The decrease was due to lower product sales of BAT®, due to timing of deliveries, and lower product sales of RSDL®, which was sold to SERB during the third quarter of 2024. Bioservices Revenues Services For Q3 2024, revenues from Bioservices services increased $0.7 million, or 5%, as compared with Q3 2023. The increase was primarily attributable to an increase in production at the Company’s Camden facility, prior to the sale of the facility to Bora, partially offset by lower production at the Company’s Canton and Winnipeg facilities. Leases For Q3 2024, revenues from Bioservices leases decreased $0.6 million, or 60%, as compared with Q3 2023. The decrease was related to the completion of a lease for a Bioservices customer at our Canton facility, partially offset by new lease revenue associated with SERB at our Winnipeg facility. Contracts and Grants For Q3 2024, revenues from contracts and grants increased $3.5 million, or 54%, as compared with Q3 2023. The increase was primarily due to timing of funding as well as an increase related to work under the Ebanga™ program. Operating Expenses ($ in millions)Q3 2024Q3 2023% ChangeCost of Commercial product sales$47.2$60.0(21)%Cost of MCM product sales 54.0 72.5(26)%Cost of Bioservices 21.4 44.3(52)%Research and development (“R&D”) 13.8 15.3(10)%Selling, general and administrative (“SG&A”) 76.6 86.0(11)%Amortization of intangible assets 16.3 16.3—%Goodwill impairment — 218.2(100)%Total operating expenses$229.3$512.6(55)% Cost of Commercial Product Sales For Q3 2024, cost of Commercial Product sales decreased $12.8 million, or 21%, as compared with Q3 2023. The decrease was primarily due to lower prescription NARCAN® unit volume, partially offset by higher OTC NARCAN® unit volume. Cost of MCM Product Sales For Q3 2024, cost of MCM Product sales decreased $18.5 million, or 26%, as compared with Q3 2023. The decrease was primarily due to lower sales of BAT® and CYFENDUS®, coupled with lower allocations to Cost of MCM Product sales at our Bayview facility. This decrease was partially offset by higher sales of BioThrax® and ACAM2000®. Cost of Bioservices For Q3 2024, cost of Bioservices decreased $22.9 million, or 52%, as compared with Q3 2023. The decrease was primarily due to lower overhead and remediation costs related to the sale of the Camden facility, coupled with a decrease in overhead costs at our other Maryland facilities as a result of the announced shutdowns and lower costs at our Canton facility. The decrease was partially offset by an increase in production at our Winnipeg facility. Research and Development Expenses For Q3 2024, R&D expenses decreased $1.5 million, or 10%, as compared with Q3 2023. The decrease was driven by a reduction in spend for certain funded and unfunded projects, excluding Ebanga™. The decrease was partially offset by an increase in funded R&D related to Ebanga™. Selling, General and Administrative Expenses For Q3 2024, SG&A expenses decreased $9.4 million, or 11%, as compared with Q3 2023. The decrease was primarily due to lower employee related expenses and compensation as a result of restructuring initiatives during 2023 and 2024, coupled with a decrease in legal services fees for disputes and other corporate initiatives. This decrease was partially offset by the settlement charge related to the stockholder litigation matter, net of expected insurance proceeds. Goodwill Impairment For Q3 2024, Goodwill impairment decreased $218.2 million as compared with Q3 2023. The decrease was due to the Q3 2023 non-cash impairment charge to Goodwill in the MCM Products reporting unit, which reduced the reporting unit’s goodwill balance to zero. ADDITIONAL FINANCIAL INFORMATION(1) Capital Expenditures ($ in millions)Q3 2024Q3 2023% ChangeCapital expenditures$5.8 $12.6 (54)%Capital expenditures as a % of total revenues 2% 5% For Q3 2024, capital expenditures decreased largely due to lower product development activities across the Company’s facilities. SEGMENT INFORMATION The Company manages the business with a focus on three reportable segments: (1) the Commercial Products segment consisting of our NARCAN® and other commercial products that were sold as part of our travel health business in the second quarter of 2023; (2) the MCM Products segment consisting of the Anthrax - MCM, Smallpox - MCM and Other products and (3) the services segment (“Services”) consisting of our Bioservices business. The Company evaluates the performance of these reportable segments based on revenues and segment adjusted gross margin, which is a non-GAAP financial measure. Segment revenue includes external customer sales, but does not include inter-segment services. The Company does not allocate contracts and grants revenue, R&D, SG&A, amortization of intangible assets, interest and other income (expense) or taxes to its evaluation of the performance of these segments. THIRD QUARTER 2024 SEGMENT RESULTS ($ in millions)Commercial ProductsQuarter Ended September 30, 2024 2023 $ Change% ChangeRevenues$95.3 $142.1 $(46.8)(33)%Cost of sales 47.2 60.0 (12.8)(21)%Gross margin**$48.1 $82.1 $(34.0)(41)%Gross margin %** 50% 58% Segment adjusted gross margin(2)$48.1 $82.1 $(34.0)(41)%Segment adjusted gross margin %(2) 50% 58% ** Gross margin is calculated as revenues less cost of sales. Gross margin % is calculated as gross margin divided by revenues. Commercial Products gross margin decreased $34.0 million, or 41%, to $48.1 million in the quarter, as compared with $82.1 million in the prior year quarter. Commercial Products gross margin percentage decreased seven percentage points to 50% for the quarter ended September 30, 2024. The decrease was largely due to an unfavorable price and volume mix in 2024 for NARCAN® products. Commercial Products segment adjusted gross margin is consistent with gross margin. ($ in millions)MCM ProductsQuarter Ended September 30, 2024 2023 $ Change% ChangeRevenues$174.2 $107.7 $66.5 62%Cost of sales 54.0 72.5 (18.5)(26)%Gross margin**$120.2 $35.2 $85.0 241%Gross margin %** 69% 33% Add back: Changes in fair value of financial instruments$— $(1.1)$1.1 100%Restructuring costs 4.9 5.0 (0.1)(2)%Inventory step-up provision 1.2 — 1.2 NMSegment adjusted gross margin(2)$126.3 $39.1 $87.2 223%Segment adjusted gross margin %(2) 73% 36% ** Gross margin is calculated as revenues less cost of sales. Gross margin % is calculated as gross margin divided by revenues.NM - Not Meaningful MCM Products gross margin increased $85.0 million, or 241%, to $120.2 million in the quarter, as compared with $35.2 million in the prior year quarter. MCM Products gross margin percentage increased 36 percentage points to 69% for the quarter ended September 30, 2024. The increase was largely due to overall higher sales volumes with a favorable product mix weighted more heavily to higher margin products coupled with lower allocations to cost of MCM Product sales at our Bayview facility and overall lower shutdown and overhead costs across our facilities. MCM Product segment adjusted gross margin in the current year period excludes the impact of non-cash items related to the impact of restructuring costs of $4.9 million and inventory step-up provision of $1.2 million. ($ in millions)ServicesQuarter Ended September 30, 2024 2023 $ Change% ChangeRevenues$14.3 $14.2 $0.1 1%Cost of services 21.4 44.3 (22.9)(52)%Gross margin**$(7.1)$(30.1)$23.0 76%Gross margin %**(50)%(212)% Add back: Restructuring costs 0.1 8.1 (8.0)(99)%Segment adjusted gross margin(2)$(7.0)$(22.0)$15.0 68%Segment adjusted gross margin %(2)(49)%(155)% ** Gross margin is calculated as revenues less cost of services. Gross margin % is calculated as gross margin divided by revenues. Services gross margin increased $23.0 million, or 76%, to $(7.1) million in the quarter, as compared with $(30.1) million in the prior year quarter. Services gross margin percentage increased 162 percentage points to (50)% for the quarter ended September 30, 2024. The increase was primarily due to lower overhead and remediation costs related to the sale of the Camden facility coupled with lower costs at our Bayview facility. Services segment adjusted gross margin in the current year period excludes the impact of restructuring costs of $0.1 million. YTD 2024 SEGMENT RESULTS Commercial Products Nine Months Ended September 30, 2024 2023 $ Change % Change Revenues$333.8 $386.2 $(52.4)(14)%Cost of sales 152.7 160.2 (7.5)(5)%Gross margin**$181.1 $226.0 $(44.9)(20)%Gross margin %** 54% 59% Segment adjusted gross margin(2)$181.1 $226.0 $(44.9)(20)%Segment adjusted gross margin %(2) 54% 59% ** Gross margin is calculated as revenues less cost of sales. Gross margin % is calculated as gross margin divided by revenues. Commercial Products gross margin decreased $44.9 million, or 20%, to $181.1 million for the nine months ended September 30, 2024, as compared with $226.0 million for the nine months ended September 30, 2023. Commercial Products gross margin percentage decreased five percentage points to 54% in 2024. The decrease was largely due to an unfavorable price and volume mix in 2024 for NARCAN® products, partially offset by the sale of the products associated with our travel health business to Bavarian Nordic. Commercial Products segment adjusted gross margin is consistent with gross margin. ($ in millions)MCM ProductsNine Months Ended September 30, 2024 2023 $ Change% ChangeRevenues$393.0 $309.2 $83.8 27%Cost of sales 147.3 208.4 (61.1)(29)%Gross margin**$245.7 $100.8 $144.9 144%Gross margin %** 63% 33% Add back: Changes in fair value of financial instruments$0.6 $(0.4)$1.0 250%Inventory step-up provision 1.2 1.9 (0.7)(37)%Restructuring costs 7.5 7.0 0.5 7%Segment adjusted gross margin(2)$255.0 $109.3 $145.7 133%Segment adjusted gross margin %(2) 65% 35% ** Gross margin is calculated as revenues less cost of sales. Gross margin % is calculated as gross margin divided by revenues.NM - Not Meaningful MCM Products gross margin increased $144.9 million, or 144%, to $245.7 million for the nine months ended September 30, 2024, as compared with $100.8 million for the nine months ended September 30, 2023. MCM Products gross margin percentage increased 29 percentage points to 63% for the nine months ended September 30, 2024. The increase was largely due to overall higher sales volumes with a favorable product mix weighted more heavily to higher margin products coupled with lower allocations to Cost of MCM Product sales at our Bayview facility and lower shutdown related costs, a reduction in Trobigard® related costs, due to the Trobigard® revocation, and realization of previously adjusted inventory values. MCM Product segment adjusted gross margin excludes the impact of restructuring costs of $7.5 million, inventory step-up provision of $1.2 million and changes in fair value of financial instruments of $0.6 million. ($ in millions)ServicesNine Months Ended September 30, 2024 2023 $ Change% ChangeRevenues$97.5 $57.7 $39.8 69%Cost of services 263.3 151.7 111.6 74%Gross margin**$(165.8)$(94.0)$(71.8)(76)%Gross margin %**(170)%(163)% Add back: Settlement charges, net$110.2 $— $110.2 NMRestructuring costs 0.3 8.1 (7.8)(96)%Segment adjusted gross margin(2)$(55.3)$(85.9)$30.6 36%Segment adjusted gross margin %(2)(57)%(149)% ** Gross margin is calculated as revenues less cost of sales. Gross margin % is calculated as gross margin divided by revenues.NM - Not Meaningful Services gross margin decreased $71.8 million, or 76%, to $(165.8) million for the nine months ended September 30, 2024, as compared with $(94.0) million for the nine months ended September 30, 2023. Services gross margin percentage decreased 7 percentage points to (170)% for the nine months ended September 30, 2024. The decrease was primarily due to the Settlement Agreement with Janssen and resulting revenue and write-down of related assets to net realizable value, coupled with lower production at the Company's Canton facility. This decrease was partially offset by an increase in production at the Camden facility prior to the sale of the facility to Bora and a decrease in overhead costs at our other Maryland facilities. Services segment adjusted gross margin in the current year period excludes the impact of segment settlement charge, net of $110.2 million and restructuring costs of $0.3 million. 2024 FINANCIAL FORECAST The Company provides the following updated financial forecast for full year 2024, reflecting management's expectations based on the most current information available. Full Year 2024 METRIC($ in millions)Updated Range(as of 11/6/2024)Previous Range(as of 08/06/2024)Previous Range(as of 05/01/2024)Previous Range(as of 03/06/2024)Total revenues$1,065 - $1,125$1,050 - $1,125$1,000 - $1,100$900 - $1,100Net loss$(203) - $(183)$(314) - $(274)$(148) - $(98)$(183) - $(133)Adjusted net loss(2)$(50) - $(30)$(115) - $(75)$(65) - $(15)$(130) - $(80)Adjusted EBITDA(2)$180 - $200$140 - $180$125 - $175$50 - $100Total segment adjusted gross margin %(2)43% - 45%42% - 45%44% - 47%40% - 45% Segment Level Revenue(4) Commercial Products$420 - $430$450 - $480$460 - $500$460 - $500MCM Products$510 - $550$455 - $490$440 - $490$340 - $490Services(5)$105 - $110$120 - $130$70 - $80$70 - $80 Key Assumptions($ and shares in millions)Updated Range(as of 11/6/2024) Interest expense~$75 R&D~7% of Revenue Weighted avg. fully diluted share count~53 Capex~$25 Depreciation & amortization~$109 FOOTNOTES (1) All financial information included in this release is unaudited. (2) See “Non-GAAP Financial Measures” and the "Reconciliation of Non-GAAP Financial Measures" tables for the definitions and reconciliations of these non-GAAP financial measures to the most closely related GAAP financial measures. (3) Product sales, net are reported net of variable consideration including returns, rebates, wholesaler fees and prompt pay discounts in accordance with U.S. generally accepted accounting principles. (4) Our Commercial Products forecast consists solely of NARCAN® Nasal Spray, as our Other Commercial Products, including Vivotif® and Vaxchora®, were sold to Bavarian Nordic as part of our travel health business in May 2023. (5) Our Services revenue forecast includes $50.0 million related to the Settlement Agreement with Janssen and excludes revenues related to the Baltimore-Camden Facility after August 20, 2024. CONFERENCE CALL, PRESENTATION SUPPLEMENT AND WEBCAST INFORMATION Company management will host a conference call at 5:00 pm eastern time today, November 6, 2024, to discuss these financial results. The conference call and presentation supplement can be accessed from the Company's website or through the following: By phoneTo join via telephone, please use the following dial-in details:U.S. / New York: +1-646-968-2525U.S. & Canada (Toll Free): +1-888-596-4144Conference ID: 5259189 By webcast Visit https://edge.media-server.com/mmc/p/nm3oj8g9A replay of the call can be accessed from the Emergent website. ABOUT EMERGENT BIOSOLUTIONS INC. At Emergent, our mission is to protect and enhance life. We develop, manufacture, and deliver protections against public health threats through a pipeline of innovative vaccines and therapeutics. For over 25 years, we have been at work defending people from things we hope will never happen—so that we are prepared just in case they ever do. We do what we do because we see the opportunity to create a better, more secure world. One where preparedness empowers protection from the threats we face. And peace of mind prevails. In working together, we envision protecting or enhancing 1 billion lives by 2030. For more information, visit our website and follow us on LinkedIn, Twitter, and Instagram. NON-GAAP FINANCIAL MEASURES In the accompanying analysis of financial information, we sometimes use information derived from consolidated and segment financial information that may not be presented in our financial statements or prepared in accordance with generally accepted accounting principles in the United States (“GAAP”). Certain of these financial measures are considered not in conformity with GAAP (“non-GAAP financial measures”) under the United States Securities and Exchange Commission (“SEC”) rules. Specifically, we have referred to the following non-GAAP financial measures: Adjusted Net Income (Loss)Adjusted Net Income (Loss) per Diluted ShareAdjusted EBITDATotal Segment RevenuesTotal Segment Gross MarginTotal Segment Gross Margin %Total Segment Adjusted Gross MarginTotal Segment Adjusted Gross Margin %Segment Adjusted Gross MarginSegment Adjusted Gross Margin % We define Adjusted Net Income (Loss) and Adjusted Net Income (Loss) per Diluted Share, which are non-GAAP financial measures, as net income (loss) and net income (loss) per diluted share, respectively, excluding the impact of changes in fair value of financial instruments, acquisition and divestiture-related costs, severance and restructuring costs, settlement charges, net, exit and disposal costs, impairment charges, gain (loss) on sale of business, non-cash amortization charges, contingent consideration milestones, and other income (expense) items. We use Adjusted Net Income (Loss) for the purpose of calculating Adjusted Net Income (Loss) per Diluted Share. Management uses Adjusted Net Income (Loss) per Diluted Share to assess total Company operating performance on a consistent basis. We believe that these non-GAAP financial measures, when considered together with our GAAP financial results and GAAP financial measures, provide management and investors with an additional understanding of our business operating results, including underlying trends. We define Adjusted EBITDA, which is a non-GAAP financial measure, as consolidated net income (loss) before income tax provision (benefit), interest expense, net, depreciation, amortization of intangible assets, excluding the impact of changes in fair value of financial instruments, acquisition and divestiture-related costs, severance and restructuring costs, settlement charges, net, exit and disposal costs, impairment charges, gain (loss) on sale of business, non-cash amortization charges, contingent consideration milestones and other income (expense) items. We believe that this non-GAAP financial measure, when considered together with our GAAP financial results and GAAP financial measures, provides management and investors with a more complete understanding of our operating results, including underlying trends. In addition, EBITDA is a common alternative measure of operating performance used by many of our competitors. It is used by investors, financial analysts, rating agencies and others to value and compare the financial performance of companies in our industry, although it may be defined differently by different companies. Therefore, we also believe that this non-GAAP financial measure, considered along with corresponding GAAP financial measures, provides management and investors with additional information for comparison of our operating results with the operating results of other companies. We have included the definitions of Segment Gross Margin and Segment Gross Margin %, which are GAAP financial measures, below in order to more fully define the components of certain non-GAAP financial measures presented in this press release. We define Segment Gross Margin, as a segment's revenues, less a segment's cost of sales or services. We define Segment Gross Margin %, as Segment Gross Margin as a percentage of a segments revenues. We define Segment Adjusted Gross Margin, which is a non-GAAP financial measure as Segment Gross Margin excluding the impact of restructuring costs, changes in the fair value of financial instruments, settlement charges, net and inventory step-up provision. We define Segment Adjusted Gross Margin %, which is a non-GAAP financial measure, as Segment Adjusted Gross Margin as a percentage of a segment's revenues. We define Total Segment Revenues, which is a non-GAAP financial measure, as our Total Revenues, less contracts and grants revenue, which is also equal to the sum of the revenues of our reportable operating segments. We define Total Segment Gross Margin, which is a non-GAAP financial measure, as Total Segment Revenues less our aggregate cost of sales or services. We define Total Segment Gross Margin %, which is a non-GAAP financial measure, as Total Segment Gross Margin as a percentage of Total Segment Revenues. We define Total Segment Adjusted Gross Margin, which is a non-GAAP financial measure, as Total Segment Gross Margin, excluding the impact of restructuring costs, settlement charges, net, changes in the fair value of financial instruments and inventory step-up provision. We define Total Segment Adjusted Gross Margin %, which is a non-GAAP financial measure, as Total Segment Adjusted Gross Margin as a percentage of Total Segment Revenues. Non-GAAP financial measures are not defined in the same manner by all companies and may not be comparable with other similarly titled measures of other companies. The determination of the amounts that are excluded from these non-GAAP financial measures are a matter of management judgment and depend upon, among other factors, the nature of the underlying expense or income amounts. Non-GAAP financial measures should be considered in addition to, but not as a substitute for or superior to, the information contained in our Consolidated Statements of Operations and Consolidated Statements of Cash Flows. Reconciliations of these non-GAAP financial measures to the most directly comparable GAAP financial measures are included in the financial tables accompanying this press release. SAFE HARBOR STATEMENT This press release includes forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. All statements, other than statements of historical fact, including statements regarding the future performance of the Company or any of our businesses, our business strategy, future operations, future financial position, future revenues and earnings, our ability to achieve the objectives of our restructuring initiatives and divestitures, including our future results, projected costs, prospects, plans and objectives of management, are forward-looking statements. We generally identify forward-looking statements by using words like “anticipate,” “believe,” “continue,” “could,” “estimate,” “expect,” “forecast,” “future,” “goal,” “intend,” “may,” “plan,” “position,” “possible,” “potential,” “predict,” “project,” “should,” “target,” “will,” “would,” and similar expressions or variations thereof, or the negative thereof, but these terms are not the exclusive means of identifying such statements. Forward-looking statements are based on our current intentions, beliefs, assumptions and expectations regarding future events based on information that is currently available. Readers should realize that if underlying assumptions prove inaccurate or unknown risks or uncertainties materialize, actual results could differ materially from our expectations. Readers are, therefore, cautioned not to place undue reliance on any forward-looking statement contained herein. Any such forward-looking statement speaks only as of the date of this press release, and, except as required by law, we do not undertake any obligation to update any forward-looking statement to reflect new information, events or circumstances. There are a number of important factors that could cause our actual results to differ materially from those indicated by such forward-looking statements, including, among others, the availability of USG funding for contracts related to procurement of our medical countermeasure ("MCM") products, including CYFENDUS® (Anthrax Vaccine Adsorbed (AVA) Adjuvanted), previously known as AV7909, BioThrax® (Anthrax Vaccine Adsorbed), and ACAM2000® (Smallpox (Vaccinia) Vaccine, Live) among others, as well as contracts related to development of medical countermeasures; the availability of government funding for our other commercialized products, including Ebanga™ (ansuvimab-zykl) and BAT® (Botulism Antitoxin Heptavalent (A,B,C,D,E,F,G)-(Equine)); our ability to meet our commitments to quality and compliance in all of our manufacturing operations; our ability to negotiate additional USG procurement or follow-on contracts for our MCM products that have expired or will be expiring; the commercial availability and acceptance of over-the-counter NARCAN® (naloxone HCl) Nasal Spray; the impact of a generic and competitive marketplace on NARCAN® Nasal Spray and future NARCAN® Nasal Spray sales; our ability to perform under our contracts with the USG, including the timing of and specifications relating to deliveries; our ability to provide Bioservices (as defined below) for the development and/or manufacture of product and/or product candidates of our customers at required levels and on required timelines; the ability of our contractors and suppliers to maintain compliance with current good manufacturing practices and other regulatory obligations; our ability to negotiate further commitments related to the collaboration and deployment of capacity toward future commercial manufacturing under our existing Bioservices contracts; our ability to collect reimbursement for raw materials and payment of service fees from our Bioservices customers; the results of pending government investigations and their potential impact on our business; our ability to obtain final court approval of the proposed settlement agreement relating to the stockholder litigation, including our ability to satisfy the conditions of the proposed settlement, and the source of funds to be used to resolve the litigation, and the potential impact of the settlement agreement, if approved, on our business; our ability to comply with the operating and financial covenants required by our term loan facility under a credit agreement, dated August 30, 2024, our revolving credit facility under a credit agreement, dated September 30, 2024, and our 3.875% Senior Unsecured Notes due 2028; our ability to maintain adequate internal control over financial reporting and to prepare accurate financial statements in a timely manner; our ability to successfully manage our liquidity in order to continue as a going concern; the procurement of our product candidates by USG entities under regulatory authorities that permit government procurement of certain medical products prior to FDA marketing authorization, and corresponding procurement by government entities outside the United States; our ability to realize the expected benefits of the sale of our travel health business to Bavarian Nordic, the sale of RSDL® to SERB Pharmaceuticals and the sale of our drug product facility in Baltimore-Camden to Bora Pharmaceuticals Injectables Inc.; the impact of the organizational changes we announced in January 2023, August 2023, May 2024 and August 2024; our ability to identify and acquire companies, businesses, products or product candidates that satisfy our selection criteria; the impact of cyber security incidents, including the risks from the unauthorized access, interruption, failure or compromise of our information systems or those of our business partners, collaborators or other third parties; the success of our commercialization, marketing and manufacturing capabilities and strategy; and the accuracy of our estimates regarding future revenues, expenses, capital requirements and need for additional financing. The foregoing sets forth many, but not all, of the factors that could cause actual results to differ from our expectations in any forward-looking statement. Readers should consider this cautionary statement, as well as the risks identified in our periodic reports filed with the Securities and Exchange Commission, when evaluating our forward-looking statements. Trademarks Emergent®, BioThrax®, BaciThrax®, BAT®, Trobigard®, Anthrasil®, CNJ-016®, ACAM2000®, ​NARCAN®, CYFENDUS®, TEMBEXA® and any and all Emergent BioSolutions Inc. brands, products, services and feature names, logos and slogans are trademarks or registered trademarks of Emergent BioSolutions Inc. or its subsidiaries in the United States or other countries. All other brands, products, services and feature names or trademarks are the property of their respective owners, including RSDL® (Reactive Skin Decontamination Lotion), which was acquired by SERB on July 31, 2024. Investor ContactRich LindahlExecutive Vice President, Chief Financial Officerlindahlr@ebsi.comMedia ContactAssal HellmerVice President, Communicationsmediarelations@ebsi.com Emergent BioSolutions Inc. Consolidated Balance Sheets(unaudited, in millions, except per share data) September 30, December 31, 2024 2023 ASSETS Current assets: Cash and cash equivalents$149.9 $111.7 Restricted cash 6.5 — Accounts receivable, net 121.3 191.0 Inventories, net 322.7 328.9 Prepaid expenses and other current assets 61.0 47.9 Total current assets 661.4 679.5 Property, plant and equipment, net 278.1 382.8 Intangible assets, net 517.8 566.6 Other assets 20.5 194.3 Total assets$1,477.8 $1,823.2 LIABILITIES AND STOCKHOLDERS’ EQUITY Current liabilities: Accounts payable$82.1 $112.2 Accrued expenses 16.1 18.6 Accrued compensation 63.3 74.1 Debt, current portion 0.8 413.7 Other current liabilities 67.6 32.7 Total current liabilities 229.9 651.3 Debt, net of current portion 661.8 446.5 Deferred tax liability 41.9 47.2 Other liabilities 35.8 28.9 Total liabilities$969.4 $1,173.9 Stockholders’ equity: Preferred stock, $0.001 par value per share; 15.0 shares authorized, no shares issued and outstanding — — Common stock, $0.001 par value per share; 200.0 shares authorized, 59.7 and 57.8 shares issued; 54.1 and 52.2 shares outstanding, respectively. 0.1 0.1 Treasury stock, at cost, 5.6 and 5.6 common shares, respectively (227.7) (227.7)Additional paid-in capital 924.4 904.4 Accumulated other comprehensive loss, net (7.3) (5.7)Accumulated deficit (181.1) (21.8)Total stockholders’ equity$508.4 $649.3 Total liabilities and stockholders’ equity$1,477.8 $1,823.2 Emergent BioSolutions Inc. Consolidated Statements of Operations(unaudited, in millions, except per share data) Three Months Ended September 30, Nine Months Ended September 30, 2024 2023 2024 2023 Revenues: Commercial Product sales$95.3 $142.1 $333.8 $386.2 MCM Product sales 174.2 107.7 393.0 309.2 Total Product sales, net 269.5 249.8 726.8 695.4 Bioservices: Services 13.9 13.2 96.7 52.2 Leases 0.4 1.0 0.8 5.5 Total Bioservices revenues 14.3 14.2 97.5 57.7 Contracts and grants 10.0 6.5 24.6 19.6 Total revenues 293.8 270.5 848.9 772.7 Operating expenses: Cost of Commercial Product sales 47.2 60.0 152.7 160.2 Cost of MCM Product sales 54.0 72.5 147.3 208.4 Cost of Bioservices 21.4 44.3 263.3 151.7 Research and development 13.8 15.3 61.6 82.0 Selling, general and administrative 76.6 86.0 247.2 278.7 Amortization of intangible assets 16.3 16.3 48.8 49.4 Goodwill impairment — 218.2 — 218.2 Impairment of long-lived assets — — 27.2 306.7 Total operating expenses 229.3 512.6 948.1 1,455.3 Income (loss) from operations 64.5 (242.1) (99.2) (682.6) Other income (expense): Interest expense (8.3) (19.7) (56.2) (66.2)Gain (loss) on sale of business 64.3 (0.7) 24.3 74.2 Other, net 21.9 (3.4) 15.8 (2.1)Total other income (expense), net 77.9 (23.8) (16.1) 5.9 Income (loss) before income taxes 142.4 (265.9) (115.3) (676.7)Income tax provision (benefit) 27.6 (2.5) 44.0 34.3 Net income (loss)$114.8 $(263.4) $(159.3) $(711.0) Earnings (loss) per common share Basic$2.16 $(5.08) $(3.03) $(13.97)Diluted$2.06 $(5.08) $(3.03) $(13.97) Shares used in computing earnings (loss) per common share Basic 53.1 51.8 52.6 50.9 Diluted 55.6 51.8 52.6 50.9 Nine Months Ended September 30, 2024 2023 Operating Activities Net loss$(159.3) $(711.0)Adjustments to reconcile net loss to net cash provided by (used in) operating activities: Share-based compensation expense 13.7 19.1 Depreciation and amortization 82.8 95.5 Change in fair value of contingent obligations, net 0.6 (0.4)Amortization of deferred financing costs 5.2 15.6 Deferred income taxes (5.1) (3.7)Noncash gain on sale of business (32.2) (74.2)Change in fair value of warrant and forward liabilities (1.1) — Goodwill impairment — 218.2 Impairment of long-lived assets 27.2 306.7 Loss on disposal of assets 28.9 13.9 Other 3.9 (5.0)Changes in operating assets and liabilities: Accounts receivable 52.7 (58.5)Inventories (35.5) (25.0)Prepaid expenses and other assets 146.3 (18.3)Accounts payable (22.8) 17.7 Accrued expenses and other liabilities 32.9 (30.2)Long-term incentive plan accrual 2.5 3.7 Accrued compensation (9.9) (0.8)Income taxes receivable and payable, net 26.6 (3.5)Contract liabilities (18.8) 1.8 Net cash provided by (used in) operating activities 138.6 (238.4)Investing Activities Purchases of property, plant and equipment (21.2) (40.2)Proceeds from sale of property, plant and equipment 7.6 — Milestone payment from prior asset acquisition — (6.3)Proceeds from sale of business 110.2 270.2 Net cash provided by investing activities 96.6 223.7 Financing Activities Proceeds from the issuance of debt, net of lender fees 219.0 — Proceeds allocated to warrants issued in conjunction with debt 13.4 — Proceeds allocated to common stock issued in conjunction with debt 9.3 — Principal payments on term loan facility (198.2) (160.7)Proceeds from revolving credit facility 65.0 — Principal payments on revolving credit facility (284.2) (386.8)Debt issuance costs (14.6) — Proceeds from share-based compensation activity 0.7 1.3 Taxes paid for share-based compensation activity (0.9) (2.4)Proceeds from at-the-market sale of stock, net of commissions and expenses — 8.2 Net cash used in financing activities: (190.5) (540.4)Effect of exchange rate changes on cash, cash equivalents and restricted cash — 0.3 Net change in cash, cash equivalents and restricted cash 44.7 (554.8)Cash, cash equivalents and restricted cash, beginning of period 111.7 642.6 Cash, cash equivalents and restricted cash, end of period$156.4 $87.8 Supplemental cash flow disclosures: Cash paid for interest$55.8 $56.5 Cash paid for income taxes$35.5 $38.3 Non-cash investing and financing activities: Purchases of property, plant and equipment unpaid at period end$1.6 $9.2 Gain on extinguishments of debt$0.6 $— Issuance of common stock in conjunction with debt 7.7 — Reconciliation of cash and cash equivalents and restricted cash: Cash and cash equivalents$149.9 $87.8 Restricted cash 6.5 — Total$156.4 $87.8 Emergent BioSolutions, Inc.Reconciliation of Non-GAAP Financial MeasuresReconciliation of Net Income (Loss) and Net Income (Loss) per Diluted Share to Adjusted Net Income (Loss) and Adjusted Net Income (Loss) per Diluted Share(1) ($ in millions, except per share data)Three Months Ended September 30, Nine Months Ended September 30, 2024 2023 2024 2023 SourceNet income (loss)$114.8 $(263.4) $(159.3)$(711.0) Adjustments: Non-cash amortization charges$9.7 $21.9 $54.0 $65.0 Amortization of intangible assets (IA), Other IncomeImpairments — 218.2 27.2 524.9 Impairment of long-lived assets and goodwillSeverance and restructuring costs 6.3 20.6 22.9 34.5 Cost of MCM Products, Cost of Services, SG&A and R&DInventory step-up provision 1.2 — 1.2 1.9 Cost of MCM ProductsAcquisition and divestiture costs — — — 2.8 SG&AExit and disposal costs — — 13.3 6.1 R&DLoss (gain) on sale of business (64.3) 0.7 (24.3) (74.2)Other Income (Expense)Settlement charges, net 10.0 — 120.2 — Cost of Services and SG&AContingent consideration milestones (30.0) — (30.0) — Other Income (Expense)Changes in fair value of financial instruments (1.1) (1.1) (0.5) (0.4)Cost of MCM Products and Other Income (Expense)Other expense (income), net items 6.7 — 9.8 — Other Income (Expense)Tax effect 22.9 (53.1) (49.2) (122.6) Total adjustments:$(38.6)$207.2 $144.6 $438.0 Adjusted net income (loss)$76.2 $(56.2) $(14.7)$(273.0) Net income (loss) per diluted share$2.06 $(5.08) $(3.03)$(13.97) Adjustments: Non-cash amortization charges$0.17 $0.42 $1.03 $1.28 Amortization of IA, Other Income (Expense)Impairments — 4.21 0.52 10.31 Impairment of long-lived assetsSeverance and restructuring costs 0.12 0.40 0.44 0.68 Cost of MCM Products, Cost of Services, SG&A and R&DInventory step-up provision 0.02 — 0.02 0.04 Cost of MCM ProductsAcquisition and divestiture costs — — — 0.06 SG&AExit and disposal costs — — 0.25 0.12 R&DLoss (gain) on sale of business (1.16) 0.01 (0.46) (1.46)Other Income (Expense)Settlement charges, net 0.18 — 2.29 — Cost of Services and SG&AContingent consideration milestones (0.54) — (0.57) — Other Income (Expense)Changes in fair value of financial instruments (0.02) (0.02) (0.01) (0.01)Cost of MCM Products and Other Income (Expense)Other expense (income), net items 0.12 — 0.19 — Other Income (Expense)Tax effect 0.42 (1.03) (0.95) (2.41) Total adjustments:$(0.69)$3.99 $2.75 $8.61 Adjusted net income (loss) per diluted share$1.37 $(1.09) $(0.28)$(5.36) Diluted shares used in computing Adjusted net income (loss) per diluted share 55.6 51.8 52.6 50.9 Emergent BioSolutions, Inc.Reconciliation of Net Income (Loss) to Adjusted EBITDA(1) ($ in millions)Three Months Ended September 30, Nine Months Ended September 30, 2024 2023 2024 2023 Net income (loss)$114.8 $(263.4) $(159.3)$(711.0)Adjustments: Depreciation & amortization$26.4 $27.9 $82.8 $95.5 Income taxes 27.6 (2.5) 44.0 34.3 Total interest expense, net 7.7 19.4 54.8 59.9 Impairments — 218.2 27.2 524.9 Inventory step-up provision 1.2 — 1.2 1.9 Changes in fair value of financial instruments (1.1) (1.1) (0.5) (0.4)Severance and restructuring costs 6.3 20.6 22.9 34.5 Exit and disposal costs — — 13.3 6.1 Acquisition and divestiture costs — — — 2.8 Loss (gain) on sale of business (64.3) 0.7 (24.3) (74.2)Settlement charges, net 10.0 — 120.2 — Contingent consideration milestones (30.0) — (30.0) — Other expense (income), net items 6.7 — 9.8 — Total adjustments$(9.5)$283.2 $321.4 $685.3 Adjusted EBITDA$105.3 $19.8 $162.1 $(25.7) Emergent BioSolutions, Inc.Reconciliations of Total Revenues to Total Segment Revenues and of Segment and Total Segment Gross Margin and Gross Margin % to Segment and Total Segment Adjusted Gross Margin and Adjusted Gross Margin %(1) Three Months Ended September 30, 2024(unaudited, in millions)Commercial ProductsMCM ProductsServices Total Segment Contracts & Grants Total RevenuesRevenues$95.3 $174.2 $14.3 $283.8 $10.0$293.8 Cost of sales or services 47.2 54.0 21.4 122.6 Gross margin$48.1 $120.2 $(7.1) $161.2 Gross margin % 50% 69%(50)% 57% Add back: Inventory step-up provision$— $1.2 $— $1.2 Restructuring costs — 4.9 0.1 5.0 Adjusted gross margin$48.1 $126.3 $(7.0) $167.4 Adjusted gross margin % 50% 73%(49)% 59% Three Months Ended September 30, 2023(unaudited, in millions)Commercial ProductsMCM ProductsServices Total SegmentContracts & Grants Total RevenuesRevenues$142.1 $107.7 $14.2 $264.0 $6.5$270.5 Cost of sales or services 60.0 72.5 44.3 176.8 Gross margin$82.1 $35.2 $(30.1) $87.2 Gross margin % 58% 33%(212)% 33% Add back: Changes in fair value of financial instruments$— $(1.1)$— $(1.1) Restructuring costs — 5.0 8.1 13.1 Adjusted gross margin$82.1 $39.1 $(22.0) $99.2 Adjusted gross margin % 58% 36%(155)% 38% Emergent BioSolutions, Inc.Reconciliations of Total Revenues to Total Segment Revenues and of Segment and Total Segment Gross Margin and Gross Margin % to Segment and Total Segment Adjusted Gross Margin and Adjusted Gross Margin %(1) Nine Months Ended September 30, 2024(unaudited, in millions)Commercial ProductsMCM ProductsServices1 Total SegmentContracts & Grants Total RevenuesRevenues$333.8 $393.0 $97.5 $824.3 $24.6$848.9 Cost of sales or services 152.7 147.3 263.3 563.3 Gross margin$181.1 $245.7 $(165.8) $261.0 Gross margin % 54% 63%(170)% 32% Add back: Changes in fair value of financial instruments$— $0.6 $— $0.6 Inventory step-up provision — 1.2 — 1.2 Settlement charges, net — — 110.2 110.2 Restructuring costs — 7.5 0.3 7.8 Adjusted gross margin$181.1 $255.0 $(55.3) $380.8 Adjusted gross margin %(1) 54% 65%(57)% 46% (1) Total Segment results for the nine months ended September 30, 2024 includes $50.0 million attributable to the Settlement Agreement with Janssen. The revenue and cost of services is related to raw materials purchased for the Janssen Agreement which Janssen had not reimbursed. Excluding the impacts of the Settlement Agreement, Total Segment Adjusted Gross Margin % would have been 3% higher for the nine months ended September 30, 2024. Nine Months Ended September 30, 2023(in millions)Commercial ProductsMCM ProductsServices Total SegmentContracts & Grants Total RevenuesRevenues$386.2 $309.2 $57.7 $753.1 $19.6$772.7 Cost of sales or services 160.2 208.4 151.7 520.3 Gross margin$226.0 $100.8 $(94.0) $232.8 Gross margin % 59% 33%(163)% 31% Add back: Changes in fair value of financial instruments$— $(0.4)$— $(0.4) Inventory step-up provision — 1.9 — 1.9 Restructuring costs — 7.0 8.1 15.1 Adjusted gross margin$226.0 $109.3 $(85.9) $249.4 Adjusted gross margin % 59% 35%(149)% 33% Emergent BioSolutions, Inc.Reconciliation of Net Loss Forecast to Adjusted Net Income (Loss) Forecast ($ in millions)2024 Full Year ForecastSourceNet loss$(203) - $(183) Adjustments: Non-cash amortization charges$65Amortization of intangible assets and Other Income (Expense)Changes in fair value of financial instruments(1)Other Income (Expense)Impairments27Impairment of long-lived assetsSeverance and restructuring costs23Cost of MCM Products, Cost of Services, SG&A and R&DInventory step-up provision1Cost of MCM ProductsExit and disposal costs13R&DLoss (gain) on sale of business(24)Other Income (Expense)Settlement charges, net120Cost of Services and SG&AContingent consideration milestones(30)Other Income (Expense)Other expense (income), net items10Other Income (Expense)Tax effect(51) Total adjustments:$153 Adjusted net loss$(50) - $(30) Reconciliation of Net Loss Forecast to Adjusted EBITDA Forecast ($ in millions)2024 Full Year ForecastNet loss$(203) - $(183)Adjustments: Depreciation & amortization$109Income taxes60Total interest expense, net75Impairments27Inventory step-up provision1Changes in fair value of financial instruments(1)Severance and restructuring costs23Exit and disposal costs13Loss (gain) on sale of business(24)Settlement charges, net120Contingent consideration milestones(30)Other expense (income), net items10Total adjustments$383Adjusted EBITDA$180 - $200 Reconciliations of Forecasted Total Revenues to Forecasted Total Segment Revenues and of Forecasted Segment and Total Segment Gross Margin and Gross Margin % to Forecasted Segment and Total Segment Adjusted Gross Margin and Adjusted Gross Margin %(1) (in millions)2024 Full Year Forecast Total revenues$1,065 - $1,125Contracts & Grants(30) - (35)Total segment revenues$1,035 - $1,090 Cost of sales or services$710 - $719Total segment gross margin$325 - $371Total segment gross margin %31% - 34%Add back: Changes in fair value of financial instruments$1Inventory step-up provision1Settlement charges, net110Restructuring costs8Total segment adjusted gross margin$445 - $491Total segment adjusted gross margin %43% - 45%

摘要：单克隆抗体（mAbs）及其衍生物已成为近几十年来最重要的生物治疗药物类别之一。mAb的成功归因于其高度多功能性、高目标特异性、卓越的临床安全性和有效性。抗体发现，作为抗体开发流程的最上游阶段，在决定mAb产品的临床结果中起着关键作用。噬菌体展示技术最初是为肽定向进化而开发的，由于其前所未有的优势，已被广泛应用于全人源抗体的发现。噬菌体展示技术的价值已通过多项获批的mAbs得到证明，包括一些来自该技术的顶级畅销mAb药物。自从30多年前首次建立抗体噬菌体展示以来，噬菌体展示平台已经被开发出来，用于生成针对难以靶向的抗原的mAbs，并解决体内抗体发现方法中存在的问题。最近，新一代噬菌体展示库已经针对发现具有“药物样”属性的mAbs进行了优化。本综述将总结抗体噬菌体展示的原理和三代抗体噬菌体展示库的设计。 1.引言 单克隆抗体（mAbs）及其衍生物1代表了一类主要的治疗药物，并且近年来已成为美国最畅销的药物。全球mAb市场规模在2020年达到了约1500亿美元，并预计在未来五年内翻一番。迄今为止，获批的全人源治疗性抗体是通过体内动物免疫（包括人源化小鼠和恢复期人类供体）或体外噬菌体展示技术发现的。对于体内动物免疫，通过重复免疫目标兴趣物来产生抗体。在免疫反应的发展过程中，选择具有高亲和力和可开发性的抗体。然而，这一过程耗时且需要抗原具有免疫原性和无毒性。此外，由于选择完全在体内完成，对特异性和表位等属性的控制有限。作为另一种方法，噬菌体展示技术通过提供高度多功能的方法，克服了体内抗体生成技术中存在的许多缺点，极大推进了治疗性mAb的发现过程。由于完全体外选择系统，噬菌体展示通过使抗体能够针对几乎任何目标或表位进行发现，包括那些对动物免疫有毒或无免疫原性的目标，从而超越了体内发现方法。此外，由于完全控制的选择条件，噬菌体展示可以量身定制，以选择无法通过体内方法实现的所需属性，例如，选择特定的表位识别，pH依赖性结合，抗体内化， 甚至催化活性。此外，噬菌体展示库已成功地用于针对最具挑战性的目标或表位的抗体发现，例如流感血凝素的茎区，G蛋白偶联受体（GPCRs），以及离子通道的特定构象。最近开发的具有蛋白质质量控制步骤的新一代噬菌体库将进一步完善体内和体外衍生抗体的可开发性差距。此外，人们越来越考虑到，基于抗体的亲和试剂和治疗剂的开发应该从基于动物的方法转向体外方法，以保护动物。 尽管迄今为止大多数获批的治疗性mAbs是通过动物免疫发现的，但近年来噬菌体衍生抗体的数量有所增加，包括一些畅销药物，如Humira®、Lucentis®和Tecentriq®（表1）。截至2022年11月，已有17种噬菌体衍生的mAbs获得批准，并且许多正在临床试验中积极评估（表1）。由于技术的不断发展以及相关关键专利的到期，预计未来将有越来越多的噬菌体衍生mAbs进入临床试验和市场。 噬菌体展示的成功主要依赖于库的质量和选择（也称为“淘选”）策略。两种最广泛使用的淘选策略是固相和液相淘选。在固相淘选中，目标被固定在微孔板上，特定的噬菌体结合物被捕获。通过在大肠杆菌宿主细胞中的洗脱和扩增，特异性噬菌体命中被放大。经过迭代淘选循环后，目标特异性噬菌体被富集（图1）。在液相淘选中，生物素标记的目标与抗体噬菌体库一起孵育，并被短暂地通过链霉亲和素包被的磁性珠拉下来捕获结合的噬菌体。可以通过替代淘选策略或整合其他技术实现表位定向抗体选择，例如竞争淘选和位点特异性光交联。一些其他的淘选策略，例如基于细胞的和基于纳米盘的淘选，已经被开发用于具有挑战性的目标，例如多跨膜蛋白。此外，功能性选择可以直接集成在选择过程中。尽管已经开发了各种淘选策略以满足不同的抗体发现目标，但选择程序通常是确立和标准化的。然而，库设计高度可变，在基于噬菌体展示的抗体发现中起着核心作用。在这篇综述中，我们将简要总结抗体噬菌体展示和噬菌体工程的原理。这篇综述将重点讨论三代噬菌体展示库的设计原理，以及几个特别设计的库。 图1. 抗体噬菌体展示库的类型（左上角面板）、库构建（左下角面板）和抗体选择（右侧面板）。根据多样性的来源，抗体噬菌体展示库可以分为三种类型：天然库、合成库和半合成库。在一个典型的噬菌体载体系统中，从天然免疫球蛋白储备库中克隆或在体外设计并合成的轻链和重链的可变区被克隆到噬菌体展示载体（噬菌体载体）中，其中一个链与噬菌体的pIII基因融合以展示。然后将库池转化到大肠杆菌宿主细胞中。通过辅助噬菌体感染，提供噬菌体生产的所有组分，生成噬菌体库。以固相淘选为例，噬菌体库与固定化的抗原一起孵育。洗涤后，非特异性噬菌体被去除，抗原特异性噬菌体留在抗原上。然后通过多种方法将结合的噬菌体从抗原上解离，例如，低pH洗脱、酶解。最后，洗脱的噬菌体在大肠杆菌宿主细胞中进行扩增。经过几轮迭代淘选后，富集了抗原特异性克隆。通常，来自淘选中后期的输出噬菌体进行测序和结合特性分析，以分别获得序列多样性和高亲和力。图注：三种类型的抗体噬菌体展示库，包括天然、合成和半合成库。构建抗体噬菌体库包括生成抗体可变区片段、克隆、转化和噬菌体产生。抗体选择包括抗原固定、噬菌体结合、洗涤、洗脱和噬菌体扩增。 2.噬菌体展示 在1985年，George P. Smith首次描述了在不影响噬菌体感染的情况下在噬菌体表面展示外来肽。从那时起，噬菌体展示技术已被广泛应用于各种应用，例如蛋白质进化（也称为定向进化）、表位确定、酶底物的识别和药物发现。由于在噬菌体展示系统中表型和基因型在物理上是链接的，因此可以通过淘选快速选择具有所需属性的蛋白质变体，并且可以轻松提取遗传信息。在噬菌体展示系统中，构建了一个在噬菌体表面展示多样化肽或蛋白质变体的库（通常为1010 − 1012的多样性）。在迭代选择循环期间，具有有利属性的变体被富集。然后可以筛选出独特的序列以供进一步应用。虽然噬菌体展示的成功已在广泛领域得到证明，但这篇综述将重点讨论其在治疗性抗体发现中的应用。 抗体噬菌体展示最初在三个研究所开发：德国癌症研究中心的细胞与肿瘤生物学研究所（德国），英国医学研究委员会分子生物学实验室和美国斯克里普斯研究所。从那时起，各种抗体片段格式已常用于噬菌体展示，如单链可变片段（scFv），抗原结合片段（Fab），单域抗体（VHH），和双特异性抗体片段。尽管也有报道在噬菌体上展示全长IgG，但这个平台并未广泛使用，可能由于展示不稳定、展示水平低和噬菌体传播偏差，导致库的功能多样性有限。然而，已报道了在噬菌体淘选后对全长IgG格式进行功能性筛选的策略，例如双宿主载体，供体-受体系统，高通量重格式化，和哺乳动物表达噬菌体衍生抗体。尽管如此，全长抗体的展示可以通过真核展示平台轻松实现，如酵母和哺乳动物细胞展示。展示格式的选择取决于最终将在应用中使用的格式。由于重新格式化有时会导致抗体属性的实质性变化，例如亲和力和稳定性，因此从库到最终产品保持格式一致被认为是“经验法则”。因此，噬菌体展示提供了一个高度多功能的平台，关于抗体格式的选择。 在几种被充分研究的丝状细菌噬菌体（Ff）中，包括f1、M13和fd，M13是最广泛用于噬菌体展示的。Ff噬菌体通过宿主F菌毛和噬菌体次要衣壳蛋白pIII之间的特定相互作用感染大肠杆菌宿主细胞。Ff噬菌体基因组编码11种蛋白质，其中5种是衣壳蛋白（pIII、pVI、pVII、pVIII和pIX）。尽管所有五种衣壳蛋白都被证明可以用于蛋白质展示，pIII作为噬菌体展示平台上的抗体融合伙伴最广泛使用，因为它可以容纳大蛋白，对噬菌体功能干扰最小，并且与单价噬菌体展示兼容。抗体-pIII融合通常通过遗传融合实现，但也可以通过其他结合方法实现，如亮氨酸拉链二聚体化，合成Fc结合ZZ结构域，等。 在早期展示系统中，感兴趣的肽与噬菌体基因组中的pIII框架内融合。因此，肽在噬菌体表面所有pIII副本上展示，这可能导致噬菌体感染力下降。此外，可以高效展示的肽的大小限于12个氨基酸。在基于phagemid的系统中，抗体-pIII融合在单独的质粒中编码（指定为phagemid），并且需要与辅助噬菌体共感染以提供噬菌体增殖的所有蛋白质。结果是，每个噬菌体颗粒包含野生型和抗体融合pIII蛋白质。这个系统的关键优势是可以实现单克隆展示，这有助于通过避免淘选过程中的亲和力效应，选择高亲和力克隆。同时，由于野生型pIII的存在，噬菌体感染力得到最大程度的保留。系统的一个缺点是野生型pIII在噬菌体颗粒中更有效地组装，导致群体中只有一小部分（<10%）的抗体展示噬菌体颗粒。因此，由于过多的野生型噬菌体背景，淘选效率或任何噬菌体介导的结合测定的灵敏度可能会受到严重影响。一种策略是增加展示的多重性，这可以通过不同的方法实现，如pVIII/pVII展示系统，适配器导向展示，并使用抗体-pIII融合基因的诱导性启动子。为了最大化展示价，通过生成一个在pIII供应大肠杆菌宿主中具有pIII基因缺陷型和野生型感染力表型的辅助噬菌体，开发了hyperphage。因此，在常规phagemid系统中使用hyperphage，产生了只展示抗体-pIII融合蛋白的噬菌体颗粒。这不仅大大提高了噬菌体ELISA的灵敏度，而且显著提高了淘选效率。 pIII蛋白由N1和N2结构域组成，这些结构域对噬菌体感染是必需的，以及一个C末端结构域（CT）用于pIII组装。在另一项努力中，通过在基因组中删除N1和N2结构域，产生了一种突变辅助噬菌体，称为CT辅助噬菌体。与hyperphage系统类似，CT辅助噬菌体的感染力通过在异质表达完整pIII基因的大肠杆菌宿主中传播来恢复。因此，使用CT辅助噬菌体可以显著提高淘选效率，因为只有结合了抗体-pIII融合的噬菌体才具有感染力。还报告了其他一些pIII缺陷辅助噬菌体系统，例如ex-phage，phaberge，和用低N1表达工程化的辅助噬菌体。值得注意的是，由于这些辅助噬菌体变体采用多价展示，它们导致噬菌体产量减少或噬菌体感染力下降。因此，它们可能不是噬菌体库构建的理想选择。为了平衡展示水平和噬菌体功能，开发了XP5辅助噬菌体，通过在pIII基因中引入多个稀有密码子和改变核糖体结合位点间距，减少WT pIII的产生。 3.三代抗体噬菌体展示库 通用抗体噬菌体展示库，通常包含超过一百亿个独特序列，已成为发现针对任何类型目标的mAbs的宝贵来源。根据多样性的来源，抗体噬菌体库可以分为天然、全合成和半合成库（图1）。在天然库中，多样性完全来自自然储备库，可以是健康、自身免疫或免疫接种的供体（分别称为天然、非免疫和免疫库）。从理论上讲，一个大型天然库可以用来分离针对任何目标的抗体，但是，如果库是从免疫接种的供体构建的，多样性将高度偏向于特定目标。对于从人类储备库构建的免疫库，它可以是从接种疫苗的供体或从感染或疾病中恢复的供体构建的。在全合成库中，抗体框架通常从表现良好且具有优越可开发性的人类抗体种系中选择。互补决定区（CDRs）基于抗体结构和应用目的设计。用于创建CDR多样性的合成DNA的质量在决定合成库的功能大小时起着关键作用。通过使用单核苷酸或三核苷酸膦酰胺（TRIM技术）作为构建块，可以在高通量方式下合成多样化的CDRs，如基于阵列的寡核苷酸合成和Slonomics。在TRIM技术中，每个氨基酸由一个定义的密码子编码。因此，可以在所需位置精确定义氨基酸分布。此外，由于每个氨基酸的密码子被选择以获得最佳抗体表达，功能库的大小增加了。在自然抗体储备库中，除了CDRH3外，CDRs的多样性是展示典型结构的确定序列集合。与传统的基于列的DNA合成方法（每列产生一个单一基因）相比，高通量DNA合成技术允许在基于硅的芯片上的同一足迹上并行合成大量预定序列。这不仅去除了影响可开发性的不良基序，而且精确模仿了自然抗体多样性。因此，半合成库结合了合成CDRs（通常是三个轻链CDRs和重链CDR1和CDR2）和来自自然储备库的重链CDR3。噬菌体库可以为抗体发现或工程而设计。后者的设计基于一个亲本抗体，并针对一个特定目标量身定制。这篇综述将重点讨论为抗体发现而设计的三代通用抗体噬菌体展示库（表2）。 3.1.第一代库 1989年首次描述了通用抗体噬菌体库的生成及其在淘选中的成功应用。由小鼠抗体储备库的可变区放大构建的Fab库包含2.5×107个菌落形成单位（PFUs）。不久之后，使用类似程序创建了一个免疫scFv噬菌体库。尽管库大小很小（2×105），但命中率显著提高（两轮淘选后>90%）。天然库在体外抗体发现方面的成功已在不同机构得到证明。这些天然库的一个共同特征是轻链和重链是随机结合的，因此这些被称为组合库。由于重链和轻链的重组，库的多样性显著增加。例如，如果一个天然抗体储备库包含106的多样性（即106个独特的轻链和重链对），经过完全的轻链和重链重组后，多样性将达到1012。一个显著的优势是这种方法可以产生自然储备库中不存在的特异性，这使得能够发现针对任何给定抗原的抗体，包括自身抗原。相比之下，由于免疫耐受（一种免疫系统不对自身抗原反应的保护机制），对于天然配对的抗体来说，针对自身抗原的发现可能具有挑战性。此外，一个大的天然库包含了大多数种系基因，这进一步由于多样的框架结构增加了库的结构复杂性。尽管天然库中的种系基因分布通常与自然储备库中的一致，但观察到选择后某些家族的种系基因使用是有偏差的，例如，重链的VH1–69、VH1–46（Kabat命名法）和/或IGHV3–30（IMGT命名法）。对于轻链，Vλ1-c、Vλ2-2a（Kabat命名法）和/或IGLV1–47、IGKV1–12、IGKV1-D33（IMGT命名法）在选择后被富集。与此同时，在1990年代初生成了几个合成和半合成抗体库。尽管由于库大小小（107-108），只分离出了低亲和力（微摩尔范围）的抗体，但这些库证明了可以体外生成人工抗体。后来的研究表明，通过生成更大尺寸的天然库（1010-1012），可以直接分离出低至亚纳摩尔亲和力的抗体。值得注意的是，由Cambridge Antibody Technology开发的大型天然库及相关噬菌体展示技术已导致六种mAbs获批：adalimumab, belimumab,moxetumomab pasudotox,raxibacumab, emapalumab,和 tralokinumab.另一个例子是n-CoDeR库，其中重链和轻链CDR片段分别使用针对VH3–23和VL1–47种系的引物放大。然后六个CDR在单个轻链和重链框架中重新组装，该框架在人类储备库中表达良好，在细菌宿主细胞中表达良好。这种策略克服了传统天然抗体噬菌体库的缺点，其中由于某些种系基因在原核系统中表达和/或展示水平低，可能会丢失部分多样性。n-CoDeR库被用于发现五个mAbs及其衍生物，这些进入临床试验。根据我们的经验，可以从内部构建的大型天然库（库大小=1012）中轻松实现高命中率（>90%），并且可以常规分离出高亲和力（低纳摩尔）的抗体。根据目标，筛选的总序列中有10-20%是独特的。 综上所述，第一代噬菌体库证明了抗体发现可以完全在体外进行，并且可以直接从噬菌体库中分离出高亲和力和序列多样性的抗体。 3.2.第二代库 在第二代库的设计中，以几个半合成和全合成库为代表，基于对自然抗体结构和抗体-抗原相互作用的了解，仔细选择了氨基酸分布和多样化的位置。通过利用TRIM技术，使用预合成的三核苷酸作为寡核苷酸合成的构建块，可以精确定制CDR氨基酸组成以满足特定的设计标准。此外，库设计中还考虑了可开发性。 在几个合成库的设计中，例如HelL-11、HelL-13和Library F，经常在抗体-抗原相互作用中使用的氨基酸在CDRL3和CDRH3中占主导地位。更具体地说，在ETH2和ETH-2-Gold库的设计中，预测在silico中与抗原直接接触的选定残基被随机化，而结构支持的CDR残基保持不变。基于ETH-2-Gold库，PHILO和PHILODiamond库被设计为通过在抗原接触位点的特定位置纳入亲水残基，覆盖更广泛的表位景观。ETH系列库导致两种mAbs，Teleukin和Dekavil，在临床试验中被研究。尽管CDRL3和CDRH3在种系多样性中发挥关键作用，但已观察到CDR1和CDR2在体细胞高频突变过程中优先选择用于亲和力成熟。在Dyax库的设计中，一个半合成库，多样性的轻链和CDRH3来自自身免疫供体的抗体储备库，以增加发现针对自身抗原的抗体的可能性。然而，CDRH1和CDRH2是完全合成的，并且使用所有氨基酸（除半胱氨酸外）随机化了预测为表面暴露的残基。事实上，通过对四个选定抗原进行淘选，平均抗体亲和力（由KD指示）有三个目标低于20 nM。值得注意的是，另一个目标的中等平均亲和力（131 nM）可能是由于库构建中使用了单一的VH/VL框架对。Dyax库被用于发现四种获批的mAbs：ramucirumab,necitumumab,avelumab,和 lanadelumab.在对抗体-蛋白质和抗体-肽相互作用倾向进行系统分析时，机器学习被用来预测CDR热点残基分布。通过在所有CDRs中密集富集热点残基，GH库在高亲和力和特异性方面增强了抗体-抗原识别。实际上，通过使用最小库，已经证明只有一小部分氨基酸类型（四氨基酸代码（Tyr、Asp、Ser和Ala）或二进制代码（Tyr和Ser））足以介导与蛋白质的相互作用。 在HuCAL系列库中，包含了覆盖主要CDR规范结构类型的框架，以最大化结构多样性。通过利用TRIM技术，设计了六个CDR，精确模仿自然储备库中出现的氨基酸分布。因此，HuCAL库更有可能产生具有类似自然属性的抗体。由于TRIM技术允许在寡核苷酸合成中精确定义核苷酸组成，它还通过优化密码子使用和避免终止密码子，实现了在大肠杆菌宿主细胞中稳定和高表达抗体，这些是使用简并密码子的缺点。因此，合成库的功能多样性可以大大改善。值得注意的是，重链H137上丙氨酸的百分比可以调整，以有利于结合蛋白质或半抗原/肽。HuCAL库的另一个特点是在所有六个CDR环周围引入了限制位点。因此，通过快速CDR重组，可以促进抗体工程。在HuCAL-PLATINUM库的设计中，为了避免影响抗体的可开发性，CDR设计中避免了翻译后修饰热点，例如N-糖基化。此外，根据环长度调整了CDRH3中的氨基酸分布，以进一步模仿自然储备库。在另一项努力中，为了进一步提高从噬菌体库中选择的抗体的整体可开发性，确定了400对VH/VL对中的36对具有最佳生物物理属性，并用于构建Ylanthia库。结果，直接从库中选择的抗体显示出优越的亲和力、蛋白质表达水平、热稳定性和聚集倾向。值得注意的是，HuCAL-GOLD库产生了2017年获批的guselkumab。 总结来说，在第二代库的设计中，通过去除序列责任基序、包含CDR中的自然氨基酸分布，并选择具有优越生物物理属性的重链和轻链框架，考虑了抗体结构和可开发性属性。 3.3.第三代库 通常，第二代库中可开发性属性的改进是通过基于序列的优化实现的。虽然我们对基于序列的蛋白质责任预测的知识仍然有限，但许多生物物理属性，如稳定性、溶解性和表达，与蛋白质的高阶结构密切相关。尽管可以为库构建选择具有高稳定性和表达的支架，但噬菌体库中抗体的整体生物物理属性也高度取决于CDR序列。因此，在第三代库的构建中，通过实验改进了这些可开发性属性。 为了增强库的溶解性和热稳定性，在半合成ALTHEA Gold库的构建中，引入了一个热休克步骤，随后是蛋白A回收。这是基于观察到热变性选择了稳定且折叠良好的抗体。库的验证表明，在热休克和蛋白A选择后，多样化CDR位置上疏水性残基的总体频率降低了。相比之下，带电残基被过滤过程正向选择。通过对多样化的抗原组进行淘选，选择的scFvs显示出高亲和力（KD范围从个位数nM到亚nM）、溶解性（>50 mg/L）和热稳定性（Tm > 70°C），这与治疗性抗体的生物物理参数一致。 在另一项努力中，为了创建具有“药物样”属性的半合成噬菌体库，应用了酵母展示过滤来选择具有临床开发所需的最佳可开发性属性的序列，包括亲和力、聚集性、热稳定性、多特异性和表达水平。这一步利用了真核蛋白质质量控制系统，选择分泌的正确行为的蛋白质。具体来说，创建了五个单CDR酵母展示库，其中每个库中一个CDR（除CDRH3外）是从复制的自然多样性中合成的，分别创建。正确显示并且在高水平显示的抗体序列被选择并进行了下一代测序（NGS）分析。然而，CDRH3序列来源于人类自然抗体储备库。这样做有两个原因：1）根据长度和氨基酸使用，合成CDRH3多样性通常远远超过实际库大小；2）自然CDRH3来源可以提供足够的高多样性，并且这些序列也已经在体内筛选，以获得最佳生物物理属性，例如高稳定性和表达，低免疫原性。为了验证库，对四种抗原进行了淘选。值得注意的是，在总共分离的81个抗体中，约80%显示出个位数到亚nM的亲和力。更引人注目的是，通过确定可开发性参数，包括热稳定性、多特异性和自相互作用，97%的抗体测量值表现与相应的获批亲本抗体相似或更好。这项研究强调了真核质量控制系统在选择高质量抗体中的重要性。 总结来说，第三代库利用体外或体内实验方法进一步提高整体库质量，从而产生属性与治疗性抗体药物相当的抗体。 4.为特定应用设计的库 尽管上述通用噬菌体库为抗体发现提供了宝贵的资源，但它们在针对具有挑战性的目标和表位（例如，GPCR、凹形表位）以及特定应用（例如，pH依赖性抗体）时的性能可能会受到影响。这是由于传统抗体库的固有特性，无论是天然库还是模仿人类抗体储备的合成库。噬菌体展示技术的优势之一是库可以定制以适应不同的应用。实际上，已经设计并创建了几种专门的噬菌体库。 4.1.GPCR库 GPCR代表一类七次跨膜受体。由于大约三分之一的美国食品药品监督管理局（FDA）批准的药物针对GPCR，GPCR已被公认为成功的药物靶点。然而，由于高疏水性、构象柔性和细胞外部分表位的有限可及性，GPCR对抗体来说是具有挑战性的目标。迄今为止，只有两种针对GPCR的FDA批准抗体药物：mogamulizumab和erenumab，分别针对CC趋化因子受体4和降钙素基因相关肽受体。噬菌体展示为针对具有挑战性的目标提供了宝贵的抗体发现平台，包括离子通道、转运体和GPCR。例如，通过挖掘所有已知GPCR配体相互作用的序列，并在CDRH3中纳入已识别的结合基序，设计了一种合成抗体噬菌体展示库。结果，这个专注于GPCR的库成功地发现了一组针对胰高血糖素样肽-1受体的高亲和力拮抗性抗体。 4.2.选择pH依赖性抗体的库 通常，通过传统的高亲和力抗体消除可溶性目标通常需要大剂量。这是因为抗体通常在中性pH和轻微酸性pH（pH 5.5–6.0）下与抗原结合的强度相似；因此，与抗体结合在细胞外环境中的抗原不会在内体内的复合物中解离。结果，抗原可以从新生儿Fc受体（FcRn）介导的循环中逃避溶酶体降解并返回。因此，能够在生理pH中和靶标，并在酸性内体pH中释放它的抗体预计将提高治疗指数。过去，通过抗体工程实现了pH依赖性结合，例如组氨酸扫描。另外，开发了扫荡抗体技术，以在中性pH下增强与FcRn的结合，以快速摄取抗体-抗原复合物进行目标清除。虽然这些技术证明了成功，但它们都是劳动密集型的，并且都基于现有的抗体。在另一次尝试中，从合成抗体噬菌体展示库中去新分离了pH依赖性抗体。在库设计中，CDRH3中富含组氨酸残基有两个原因。首先，组氨酸在生理pH下是中性带电，但在pH 6.0时变为正电荷。因此，蛋白质-蛋白质相互作用区域内的组氨酸残基可以施加pH依赖性结合。其次，组氨酸在自然储备库中较少见。结合修改的选择策略，分离了几种在pH 7.4下具有高结合亲和力和强大的中和活性，但在pH 6.0下结合力弱的抗CXCL10抗体。 4.3.带有延长CDRH3的库 人类或小鼠抗体的一个限制是针对目标的凹形表位，例如离子通道的孔和酶的口袋，可能具有挑战性。这主要是因为相对较短的CDRH3长度，通常小鼠和人类分别为7-12和8-20个氨基酸，它们倾向于形成洞穴或平坦的副位。因此，目标的凹形构象通常对传统抗体是不可达的。尽管已经分离出具有大约30个氨基酸的扩展CDRH3的抗人类免疫缺陷病毒（HIV）广泛中和抗体，但它们只在外周感染人群中的少数人中发现，并且需要多年的发展。有趣的是，一些物种的抗体，如牛和驼类，具有自然延长的CDRH3（例如，牛可达70个氨基酸），这提供了额外的多样性和副位复杂性。牛超长CDRH3通常采用“柄和旋钮”结构，其中β链“柄”支撑具有多个二硫键稳定的结构复杂的“旋钮”域。“旋钮”域从抗体表面突出，使其可以接触到凹形表位。在一项概念验证研究中，构建了一个携带延长CDRH3（23-27个氨基酸）的合成Fab噬菌体展示库。通过对库进行淘选，鉴定出了一系列有效抑制基质金属蛋白酶-14的抗体。值得注意的是，其中一个抗体被指示与酶活性口袋附近结合。展示具有延长CDRH3的非典型抗体的库进一步扩展了噬菌体展示技术的应用。 5.结论与展望 噬菌体展示在抗体发现方面已被证明是无可争议的成功，这一点由17种获批的mAbs和越来越多的噬菌体衍生抗体正在进行临床研究所证实。作为一种完全体外的技术，噬菌体展示不仅弥补了体内抗体发现方法所固有的许多限制，而且还提供了一个高度多功能和可定制的平台，该平台不断发展以满足不同的开发目标。 亲和力是评估噬菌体库质量的关键因素。观察到可实现的亲和力与库大小相关。我们研究了已发布通用噬菌体库中获得的最高亲和力值与库大小之间的相关性（数据未显示）。与先前的观察一致，这两个参数之间存在正相关性。这是因为通用库旨在针对任何给定目标进行抗体发现。因此，更大的库（更高的多样性）提供了更大的机会来识别高亲和力抗体。在合成库的情况下，尽管使用的框架数量有限，但设计的CDR多样性通常远远超过自然CDR多样性。因此，获得高亲和力抗体的机会主要取决于CDR序列多样性。在天然库的情况下，库多样性不仅来自序列多样性，还来自轻链和重链的重组。值得注意的是，随机的重链和轻链重组过程非常类似于链重组，这是体外抗体亲和力成熟的一种常规策略。这可能解释了为什么即使从没有或非常有限的体细胞高频突变的种系序列构建的天然噬菌体库中，也能分离出具有非常高亲和力的抗体。虽然预期确定库大小是决定亲和力的关键因素，但与库类型或库代的相关性并未观察到（数据未显示）。一个原因是，除了一些早期库外，三代噬菌体库的库大小差异很小。此外，三代噬菌体库的进步主要反映了整体可开发性属性的改进。 已报道，不显示抗体片段的库克隆（秃头噬菌体）的存在部分是由于抗体基因中存在的终止密码子或移码。这些克隆经常因为减少了抗体融合蛋白的生产负担和由于噬菌体颗粒上所有野生型pIII分子导致的更高感染力而过度生长，导致库多样性丧失。为了增加功能性库大小，已经开发了几种策略。例如，使用抗标签抗体进行校验淘选，以选择内框序列，因为标签与抗体序列内框。在HuCAL GOLD库的设计中，使用β-内酰胺酶基因来消除移码序列。 关于噬菌体展示库中的框架使用，人们普遍认为使用多个框架的库比使用单一框架对的库表现更好，因为多个支架提供了更多的结构多样性。在选择框架时，考虑了诸如在自然储备库中的频率、稳定性、表达和展示水平等参数。此外，应该注意到几个重链种系基因，例如某些IGVH4家族基因，在噬菌体淘选中被发现被去选择。此外，VH4–34被发现与B细胞毒性相关。因此，这些种系应该从库设计中排除。对于大多数噬菌体展示库，VH和VL是随机重组的，这与自然储备库中没有明显的VH/VL配对偏好的观察一致。然而，在药物开发方面，由于不同的VH/VL配对确实表现出非常不同的生物物理特性，应该关注框架配对的选择。 与体内抗体发现方法相比，噬菌体展示的另一个优势是序列信息可以快速且容易地检索。然而，这种优势部分被传统的单克隆表征筛选方法所削弱，其中只有一小部分序列信息，即最丰富的序列，从淘选输出中被评估。这部分是由于在大肠杆菌宿主细胞中抗体展示噬菌体的固有扩增偏差，导致一些序列在几轮淘选后变得稀少。为了克服这一限制，NGS，允许深入挖掘样本中的序列空间，最近已被应用于抗体噬菌体展示技术，特别是用于识别那些具有潜在有趣特性的稀有序列。最近，结合NGS的机器学习已被应用于预测噬菌体淘选中序列的结合特性（例如，亲和力、表位、可开发性），甚至生成具有改进属性的新序列。 尽管噬菌体展示有许多优点，但由于缺乏体内蛋白质质量控制过程，体外衍生的mAbs的可开发性一直是一个关注点。如前所述，体内或真核质量控制步骤已集成到第三代噬菌体展示库的构建中。因此，只有具有有利可开发性的库成员被选择。事实上，已报道从第三代库中选择的mAbs显示出整体增强的可开发性属性，包括高亲和力、改善的稳定性和溶解性，以及较少的自相互作用。或者，可以使用哺乳动物展示来进一步筛选或优化mAbs的可开发性属性，基于最佳生物物理属性和展示水平之间的强相关性。值得一提的是，核酸编码生物制剂的体内递送的出现，即DNA和mRNA技术，能够在体内直接生产治疗性mAbs。这些递送技术绕过了复杂的蛋白质制造、储存和运输过程，这些过程需要具有出色生物物理属性的蛋白质。因此，可以预见，随着新药物递送技术的进步，许多对可开发性的要求，特别是制造能力，将在未来得到缓解。 随着噬菌体展示技术的不断发展，以及与其他尖端技术（如NGS和机器学习）的结合，噬菌体展示技术将继续为未来的创新药物发现做出巨大贡献。 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。