Rapcabtagene autoleucel

Ad hoc announcement pursuant to Art. 53 LR Q2 net sales grew +11% (cc1, +12% USD) with core operating income1 up +21% (cc, +20% USD) Sales growth driven by continued strong performance from Kisqali (+64% cc), Entresto (+22% cc), Kesimpta (+33% cc), Scemblix (+79% cc), Leqvio (+61% cc) and Pluvicto (+30% cc)Core operating income margin1 reached 42.2%, +340 basis points (cc), mainly driven by higher net sales Q2 operating income grew +25% (cc, +21% USD); net income up +26% (cc, +24% USD)Q2 core EPS1 grew +24% (cc, +23% USD) to USD 2.42Q2 free cash flow1 of USD 6.3 billion (+37% USD) driven by higher net cash flows from operating activitiesH1 net sales up +13% (cc, +12% USD) and core operating income up +24% (cc, +21% USD)Q2 selected innovation milestones: Pluvicto Phase III PSMAddition study positive readout in PSMA+ mHSPC Vanrafia (atrasentan) FDA accelerated approval for IgANOAV101 IT US and EU submissions for SMAVotoplam Phase II PIVOT-HD study positive readout in Huntington’s diseaseRemibrutinib Phase II study positive readout in food allergy Initiating up-to USD 10 billion share buyback to be completed by year-end 2027Full-year 2025 guidance2 raised for core operating income Sales expected to grow high single digit (unchanged)Core operating income expected to grow low teens (from low double-digit) 1. Constant currencies (cc), core results and free cash flow are non-IFRS measures. An explanation of non-IFRS measures can be found on page 40 of the Condensed Interim Financial Report. Unless otherwise noted, all growth rates in this Release refer to same period in prior year. 2. Please see detailed guidance assumptions on page 7. Basel, July 17, 2025 – Commenting on Q2 2025 results, Vas Narasimhan, CEO of Novartis, said:“Novartis delivered another strong quarter, with double-digit sales and core operating income growth. We continue to drive strong performance on our ongoing launches for Kisqali, Pluvicto, and Scemblix, demonstrating the replacement power in our portfolio. We also delivered important pipeline milestones including a third positive Phase III readout for Pluvicto in hormone-sensitive prostate cancer and global filings for OAV101 IT in SMA. Our robust balance sheet and confidence in our mid and long-term growth enable us to initiate an up-to USD 10 billion share buyback as part of our commitment to balanced capital allocation." Key figures Q2 2025 Q2 2024 % change H1 2025 H1 2024 % change USD m USD m USD cc USD m USD m USD cc Net sales 14 054 12 512 12 11 27 287 24 341 12 13 Operating income 4 864 4 014 21 25 9 527 7 387 29 33 Net income 4 024 3 246 24 26 7 633 5 934 29 31 EPS (USD) 2.07 1.60 29 32 3.91 2.91 34 37 Free cash flow 6 333 4 615 37 9 724 6 653 46 Core operating income 5 925 4 953 20 21 11 500 9 490 21 24 Core net income 4 710 4 008 18 19 9 192 7 689 20 22 Core EPS (USD) 2.42 1.97 23 24 4.69 3.77 24 27 Strategy Our focus Novartis is a “pure-play” innovative medicines company. We have a clear focus on four core therapeutic areas (cardiovascular-renal-metabolic, immunology, neuroscience and oncology), with multiple significant in-market and pipeline assets in each of these areas, that address high disease burden and have substantial growth potential. In addition to two established technology platforms (chemistry and biotherapeutics), three emerging platforms (gene & cell therapy, radioligand therapy and xRNA) are being prioritized for continued investment into new R&D capabilities and manufacturing scale. Geographically, we are focused on growing in our priority geographies – the US, China, Germany and Japan. Our priorities Accelerate growth: Renewed attention to deliver high-value medicines (NMEs) and focus on launch excellence, with a rich pipeline across our core therapeutic areas.Deliver returns: Continuing to embed operational excellence and deliver improved financials. Novartis remains disciplined and shareholder-focused in our approach to capital allocation, with substantial cash generation and a strong capital structure supporting continued flexibility. Strengthen foundations: Unleashing the power of our people, scaling data science and technology and continuing to build trust with society. Financials Second quarter Net sales were USD 14.1 billion (+12%, +11% cc), with volume contributing 12 percentage points to growth. Generic competition had a negative impact of 2 percentage points, pricing had a positive impact of 1 percentage point, and currency had a positive impact of 1 percentage point. Operating income was USD 4.9 billion (+21%, +25% cc), mainly driven by higher net sales, partly offset by higher investments behind priority brands and launches and net expense from legal matters. Net income was USD 4.0 billion (+24%, +26% cc), mainly driven by higher operating income, partly offset by higher net financial expense. EPS was USD 2.07 (+29%, +32% cc), benefiting from the lower weighted average number of shares outstanding. Core operating income was USD 5.9 billion (+20%, +21% cc), mainly driven by higher net sales, partly offset by higher investments behind priority brands and launches. Core operating income margin was 42.2% of net sales, increasing 2.6 percentage points (3.4 percentage points cc). Core net income was USD 4.7 billion (+18%, +19% cc), mainly due to higher core operating income, partly offset by higher income taxes and net financial expense. Core EPS was USD 2.42 (+23%, +24% cc), benefiting from the lower weighted average number of shares outstanding. Free cash flow amounted to USD 6.3 billion (+37% USD), compared with USD 4.6 billion in the prior-year quarter, driven by higher net cash flows from operating activities. First half Net sales were USD 27.3 billion (+12%, +13% cc), with volume contributing 14 percentage points to growth. Generic competition had a negative impact of 2 percentage points, pricing had a positive impact of 1 percentage point, benefiting from revenue deduction adjustments mainly in the US, and currency had a negative impact of 1 percentage point. Operating income was USD 9.5 billion (+29%, +33% cc), mainly driven by higher net sales and contingent consideration adjustments, partly offset by higher investments behind priority brands and launches. Net income was USD 7.6 billion (+29%, +31% cc), mainly driven by higher operating income, partly offset by higher income taxes and net financial expense. EPS was USD 3.91 (+34%, +37% cc), benefiting from the lower weighted average number of shares outstanding. Core operating income was USD 11.5 billion (+21%, +24% cc), mainly driven by higher net sales, partly offset by higher investments behind priority brands and launches. Core operating income margin was 42.1% of net sales, increasing 3.1 percentage points (3.7 percentage points cc). Core net income was USD 9.2 billion (+20%, +22% cc), mainly due to higher core operating income, partly offset by higher income taxes and net financial expense. Core EPS was USD 4.69 (+24%, +27% cc), benefiting from the lower weighted average number of shares outstanding. Free cash flow amounted to USD 9.7 billion (+46% USD), compared with USD 6.7 billion in the prior-year period, driven by higher net cash flows from operating activities. Q2 priority brands Underpinning our financial results in the quarter is a continued focus on key growth drivers (ranked in order of contribution to Q2 growth) including: Kisqali (USD 1 177 million, +64% cc) sales grew strongly across all regions, including +100% growth in the US with strong momentum from the recently launched early breast cancer indication as well as continued share gains in metastatic breast cancer Entresto (USD 2 357 million, +22% cc) sustained robust, demand-led growth globally Kesimpta (USD 1 077 million, +33% cc) sales grew across all regions driven by increased demand and strong access Scemblix (USD 298 million, +79% cc) sales grew across all regions, demonstrating the continued high unmet need in CML and continued strong momentum from the recently launched early-line indication in the US Leqvio (USD 298 million, +61% cc) continued steady growth, with a focus on increasing account and patient adoption, and continuing medical education Pluvicto (USD 454 million, +30% cc) showed encouraging demand uptake in the US following the pre-taxane metastatic castration-resistant prostate cancer (mCRPC) approval, as well as continued access expansion ex-US in the post-taxane mCRPC setting Cosentyx (USD 1 629 million, +6% cc) sales grew mainly in the US and Europe, driven by recent launches as well as volume growth in core indications Fabhalta (USD 120 million) sales grew driven by continued launch execution across all markets in PNH as well as recent launches in IgAN and C3G in the US Lutathera (USD 207 million, +17% cc) sales grew mainly in the US, Europe and Japan due to increased demand and earlier-line adoption, particularly in the US and Japan Zolgensma (USD 297 million, -17% cc) sales declined reflecting a lower incidence of SMA compared to prior year, while demand remained robust Net sales of the top 20 brands in the second quarter and first half Q2 2025 % change H1 2025 % change USD m USD cc USD m USD cc Entresto 2 357 24 22 4 618 22 22 Cosentyx 1 629 7 6 3 163 11 11 Kisqali 1 177 64 64 2 133 59 60 Kesimpta 1 077 35 33 1 976 38 38 Tafinlar + Mekinist 573 10 7 1 125 13 13 Promacta/Revolade 502 -8 -9 1 048 -2 -1 Jakavi 524 11 8 1 016 7 8 Xolair 443 4 2 899 9 10 Ilaris 477 30 27 896 24 24 Pluvicto 454 32 30 825 26 26 Tasigna 327 -27 -27 704 -16 -15 Zolgensma 297 -15 -17 624 -3 -3 Sandostatin Group 303 -3 -3 620 -7 -6 Leqvio 298 64 61 555 67 66 Scemblix 298 82 79 536 79 78 Lutathera 207 18 17 400 16 16 Exforge Group 191 7 7 370 0 3 Lucentis 173 -37 -39 362 -39 -38 Diovan Group 154 -4 -4 304 1 3 Galvus Group 123 -18 -17 247 -17 -14 Top 20 brands total 11 584 16 14 22 421 16 17 R&D update - key developments from the second quarter New approvals Vanrafia(atrasentan) FDA granted accelerated approval for Vanrafia for the reduction of proteinuria in adults with primary IgA nephropathy (IgAN) at risk of rapid disease progression. Vanrafia can be seamlessly added to supportive care in IgAN and used as a foundational therapy. Coartem(artemether and lumefantrine) In July, Swissmedic approved Coartem Baby, the first clinically proven malaria treatment specifically designed for newborns and infants between 2-5 kg. This milestone paves the way for registration in eight African countries through the Marketing Authorization for Global Health Products (MAGHP) procedure. Regulatory updates OAV101 IT (onasemnogene abeparvovec) Regulatory submissions for OAV101 IT in patients with spinal muscular atrophy (SMA) were completed in the US and EU. Results from ongoing trials and other highlights Pluvicto(lutetium Lu177 vipivotide tetraxetan) At a prespecified interim analysis, the Phase III PSMAddition trial in PSMA+ metastatic hormone-sensitive prostate cancer (mHSPC) met its primary endpoint with a statistically significant and clinically meaningful benefit in radiographic progression-free survival (rPFS) in patients treated with Pluvicto plus standard of care (SoC) versus SoC alone. The study also showed a positive trend in overall survival in favor of the Pluvicto arm. Data will be presented at an upcoming medical meeting and, based on FDA feedback, submitted for regulatory review in H2 2025. Cosentyx(secukinumab) In the Phase III GCAptAIN study, Cosentyx did not demonstrate a statistically significant improvement in sustained remission compared to placebo in adults with newly diagnosed or relapsing giant cell arteritis (GCA). Safety in GCA was consistent with the known safety profile of Cosentyx. Kisqali(ribociclib) A new subgroup analysis of the Phase III NATALEE trial in HR+/HER2- early breast cancer (eBC) showed that patients receiving Kisqali plus endocrine therapy continued to see consistent reductions in risk of recurrence across all efficacy measures, regardless of age and menopausal status, at median follow-up of 44.2 months. Data presented at ASCO. Fabhalta(iptacopan) In the Phase IIIb APPULSE-PNH study, adult PNH patients with hemoglobin (Hb) levels ≥10g/dL who switched to Fabhalta from anti-C5 therapies experienced clinically meaningful improvements in Hb levels. The vast majority (92.7%) achieved Hb ≥12g/dL, reaching normal or near-normal levels. No patients treated with Fabhalta required transfusions, experienced breakthrough hemolysis or had any major adverse vascular events during the treatment period. Data presented at EHA. Scemblix (asciminib) In the Phase IIIb ASC4START trial evaluating the tolerability and efficacy of Scemblix versus nilotinib in adult patients with newly diagnosed Ph+ CML-CP, patients treated with Scemblix had a 55% lower risk of discontinuation due to AEs vs nilotinib, and 12.7% more patients treated with Scemblix achieved major molecular responses by week 12 vs those treated with nilotinib. Data presented at ASCO and EHA. Votoplam The Phase II PIVOT-HD study of votoplam in patients with Stage 2 and Stage 3 Huntington's disease met its primary endpoint of reduction in blood Huntingtin (HTT) protein levels at Week 12 (p<0.0001), with durable, dose-dependent lowering observed through Month 12. Across all dose levels and disease stages, votoplam showed a favorable safety and tolerability profile, with no treatment-related serious adverse events or neurofilament light chain protein (NfL) spikes. Together with our partner, PTC Therapeutics, we are evaluating the results and plan to engage with the HD community and regulatory authorities to inform next steps. Remibrutinib A Phase II study with remibrutinib in food allergy met its primary endpoint with a statistically significant and clinically meaningful benefit. These data support remibrutinib's potential as a first-in-class oral BTK inhibitor that reduces the risk of severe allergic reactions, including anaphylaxis. Phase III study planning is underway. Ianalumab Novartis will not advance investigation of ianalumab in hidradenitis suppurativa following a Phase II proof-of-concept study which did not meet our target criteria despite demonstrating efficacy vs placebo. No new safety signals were observed and all other studies for ianalumab in B-cell driven diseases continue as planned. Rapcabtagene autoleucel (YTB323) A Phase I/II study of rapcabtagene autoleucel, a rapidly manufactured CD19 CAR-T therapy using the T-Charge platform, demonstrated the expansion of CAR-T cells, deep B cell depletion, early and sustained improvement in overall disease activity, and a favorable benefit/risk profile in 21 patients with severe refractory SLE up to 12 months after treatment. Data presented at EULAR. Zigakibart Updated results from the Phase I/II study for zigakibart in IgAN showed a robust and clinically meaningful reduction in proteinuria of 60.4% from baseline and eGFR stabilization over 100 weeks of treatment. To date, this is the longest duration of treatment reported for an anti-APRIL agent, demonstrating long-term safety and efficacy. Data presented at ERA. The Phase III BEYOND trial is ongoing with anticipated readout in 2026. Selected transactions Novartis has completed the acquisition of Regulus Therapeutics, a clinical-stage biopharmaceutical company focused on developing microRNA therapeutics. Regulus’ lead asset, farabursen, is a potential first-in-class oligonucleotide targeting miR-17 for the treatment of autosomal dominant polycystic kidney disease (ADPKD) that recently completed Phase Ib. The acquisition is aligned with the therapeutic area focus of Novartis and leverages its strength and expertise in renal disease. In July, Novartis entered into an agreement with Sironax, granting Novartis an exclusive option to acquire its Brain Delivery Module (BDM) platform, a differentiated blood-brain-barrier crossing technology designed to enhance the brain delivery of therapeutics of various modalities. Capital structure and net debt Retaining a good balance between investment in the business, a strong capital structure, and attractive shareholder returns remains a priority. During the first half of 2025, Novartis repurchased a total of 48.8 million shares for USD 5.3 billion on the SIX Swiss Exchange second trading line under the USD 15 billion share buyback (announced in July 2023 and completed on July 1, 2025, with a total of 140.9 million shares repurchased over this period). In addition, 1.6 million shares (equity value of USD 0.2 billion) were repurchased from employees. In the same period, 11.2 million shares (equity value of USD 0.6 billion) were delivered to employees related to equity-based compensation plans. Novartis aims to offset the dilutive impact from equity-based compensation plans of employees over the remainder of the year. Consequently, the total number of shares outstanding decreased by 39.2 million versus December 31, 2024. These treasury share transactions resulted in an equity decrease of USD 4.9 billion and a net cash outflow of USD 5.4 billion. Net debt increased to USD 23.8 billion at June 30, 2025, compared to USD 16.1 billion at December 31, 2024. The increase was mainly due to the free cash flow of USD 9.7 billion being more than offset by the USD 7.8 billion annual dividend payment, cash outflows for treasury share transactions of USD 5.4 billion and net cash outflow for M&A, intangible assets transactions and other acquisitions of USD 3.1 billion. As of Q2 2025, the long-term credit rating for the company is Aa3 with Moody’s Ratings and AA- with S&P Global Ratings. 2025 outlook Barring unforeseen events; growth vs. prior year in cc Net sales Expected to grow high single-digit Core operating income Expected to grow low-teens Key assumption: We continue to assume Entresto US generic entry in mid-2025 for forecasting purposes, though timing of generic entry is subject to ongoing IP and regulatory litigation Foreign exchange impact If mid-July exchange rates prevail for the remainder of 2025, the foreign exchange impact for the year would be positive 1 percentage point on net sales and negative 1 percentage point on core operating income. The estimated impact of exchange rates on our results is provided monthly on our website. Key figures1 Q2 2025 Q2 2024 % change H1 2025 H1 2024 % change USD m USD m USD cc USD m USD m USD cc Net sales 14 054 12 512 12 11 27 287 24 341 12 13 Operating income 4 864 4 014 21 25 9 527 7 387 29 33 As a % of sales 34.6 32.1 34.9 30.3 Net income 4 024 3 246 24 26 7 633 5 934 29 31 EPS (USD) 2.07 1.60 29 32 3.91 2.91 34 37 Net cash flows from operating activities 6 664 4 875 37 10 309 7 140 44 Non-IFRS measures Free cash flow 6 333 4 615 37 9 724 6 653 46 Core operating income 5 925 4 953 20 21 11 500 9 490 21 24 As a % of sales 42.2 39.6 42.1 39.0 Core net income 4 710 4 008 18 19 9 192 7 689 20 22 Core EPS (USD) 2.42 1.97 23 24 4.69 3.77 24 27 1. Constant currencies (cc), core results and free cash flow are non-IFRS measures. An explanation of non-IFRS measures can be found on page 40 of the Condensed Interim Financial Report. Unless otherwise noted, all growth rates in this Release refer to same period in prior year. Detailed financial results accompanying this press release are included in the Condensed Interim Financial Report at the link below:https://ml-eu.globenewswire.com/resource/download/1403b1dc-887b-4bec-8b02-080540c4ebf4/ DisclaimerThis press release contains forward-looking statements within the meaning of the United States Private Securities Litigation Reform Act of 1995, that can generally be identified by words such as “anticipate,” “can,” “will,” “continue,” “ongoing,” “growth,” “launch,” “expect,” “expand,” “deliver,” “accelerate,” “guidance,” “outlook,” “priority,” “potential,” “momentum,” “commitment,” or similar expressions, or by express or implied discussions regarding potential new products, potential new indications for existing products, potential product launches, or regarding potential future revenues from any such products; or regarding results of ongoing clinical trials; or regarding potential future, pending or announced transactions; regarding potential future sales or earnings; or by discussions of strategy, plans, expectations or intentions, including discussions regarding our continued investment into new R&D capabilities and manufacturing; or regarding our capital structure. Such forward-looking statements are based on the current beliefs and expectations of management regarding future events and are subject to significant known and unknown risks and uncertainties. Should one or more of these risks or uncertainties materialize, or should underlying assumptions prove incorrect, actual results may vary materially from those set forth in the forward-looking statements. You should not place undue reliance on these statements. There can be no guarantee that the investigational or approved products described in this press release will be submitted or approved for sale or for any additional indications or labeling in any market, or at any particular time. Nor can there be any guarantee that such products will be commercially successful in the future. Neither can there be any guarantee that the expected benefits or synergies from the transactions described in this press release will be achieved in the expected timeframe, or at all. In particular, our expectations could be affected by, among other things: uncertainties concerning global healthcare cost containment, including ongoing government, payer and general public pricing and reimbursement pressures and requirements for increased pricing transparency; uncertainties regarding the success of key products, commercial priorities and strategy; uncertainties in the research and development of new products, including clinical trial results and additional analysis of existing clinical data; our ability to obtain or maintain proprietary intellectual property protection, including the ultimate extent of the impact on Novartis of the loss of patent protection and exclusivity on key products; uncertainties regarding our ability to realize the strategic benefits, operational efficiencies or opportunities expected from our external business opportunities; uncertainties in the development or adoption of potentially transformational digital technologies, including artificial intelligence, and business models; uncertainties surrounding the implementation of our new IT projects and systems; uncertainties regarding potential significant breaches of information security or disruptions of our information technology systems; uncertainties regarding actual or potential legal proceedings, including regulatory actions or delays or government regulation related to the products and pipeline products described in this press release; safety, quality, data integrity, or manufacturing issues; our performance on and ability to comply with environmental, social and governance measures and requirements; major macroeconomic and geo- and socio-political developments, including the impact of any potential tariffs on our products or the impact of war in certain parts of the world; uncertainties regarding future global exchange rates; uncertainties regarding future demand for our products; and other risks and factors referred to in Novartis AG’s most recently filed Form 20-F and in subsequent reports filed with, or furnished to, the US Securities and Exchange Commission. Novartis is providing the information in this press release as of this date and does not undertake any obligation to update any forward-looking statements as a result of new information, future events or otherwise. All product names appearing in italics are trademarks owned by or licensed to Novartis. About Novartis Novartis is an innovative medicines company. Every day, we work to reimagine medicine to improve and extend people’s lives so that patients, healthcare professionals and societies are empowered in the face of serious disease. Our medicines reach nearly 300 million people worldwide. Reimagine medicine with us: Visit us at https://www.novartis.com and connect with us on LinkedIn, Facebook, X and Instagram. Novartis will conduct a conference call with investors to discuss this news release today at 14:00 Central European time and 8:00 Eastern Time. A simultaneous webcast of the call for investors and other interested parties may be accessed by visiting the Novartis website. A replay will be available after the live webcast by visiting https://www.novartis.com/investors/event-calendar. Detailed financial results accompanying this press release are included in the Condensed Interim Financial Report at the link below. Additional information is provided on our business and pipeline of selected compounds in late-stage development. A copy of today's earnings call presentation can be found at https://www.novartis.com/investors/event-calendar. Important dates October 28, 2025Third quarter & nine months 2025 resultsNovember 19-20, 2025 Meet Novartis Management 2025 (London, UK)December 1, 2025 Social Impact & Sustainability annual investor event (virtual)February 4, 2026 Fourth quarter & full year 2025 results # # # Novartis Media RelationsE-mail: media.relations@novartis.com Novartis Investor RelationsCentral investor relations line: +41 61 324 7944E-mail: investor.relations@novartis.com Please find full media release in English attached and on the following link: Media release (PDF) Further language versions are available through the following links: German version is available through the following link:Medienmitteilung (PDF)

摘要：嵌合抗原受体（CAR）-T细胞疗法已显示出强大的治愈潜力，并成为一系列B细胞恶性肿瘤治疗的关键组成部分。CAR-T细胞疗法成功用于治疗血液癌和实体癌以及自身免疫性疾病等其他适应症，取决于有效的CAR-T细胞生产，这不仅会影响产品的安全性和有效性，还会影响有需要的患者的整体可及性。这篇综述讨论了自体CAR-T细胞生产的主要工艺参数，以及监管考虑因素和正在进行的发展，这些因素将使下一代CAR-T细胞疗法成为可能。【NO.1】介绍文中描述了几种FDA批准的CAR-T细胞产品的生产工艺，具有广泛的相似性和独特性（表1）。整个过程包括从白细胞分离法产物中分离起始细胞群、T细胞活化、基因修饰、离体扩增、最终产品配方和产品放行测试（图1）。本综述旨在概述每个关键生产步骤中的工艺参数如何影响所得细胞产品，并讨论有可能在治疗效果和患者可及性方面显着改变CAR-T细胞疗法格局的下一代生产策略。表1.FDA批准的CAR-T细胞产品生产总结【NO.2】选择起始细胞群选择CAR-T细胞生产的起始细胞群是一个早期决策点，对生产过程和最终产品有重大影响。自体CAR-T细胞生产过程通常从纯化形式的成熟T细胞开始，或作为外周血单核细胞（PBMC）群体的一部分。成熟T细胞从初始（TN）表型分化为干细胞记忆（TSCM）、中枢记忆（TCM）、效应记忆（TEM）、效应（TE）和耗竭（TEXH）细胞。此外，T细胞可分为CD8+细胞毒性T细胞、CD4+辅助性T细胞和CD4+调节性T细胞。在辅助性T细胞类别中，可以进一步识别亚群，例如Th1、Th2、Th17和滤泡辅助细胞。T细胞表型的多样性使起始细胞群的选择成为CAR-T细胞生产中的主要工艺参数。然而，生物学并不是影响起始材料选择的唯一因素，因为患者的临床病史和健康状况也对可以获得的细胞生产材料施加了实际限制。图1.自体CAR-T细胞生产过程示意图，自体CAR-T细胞生产通常涉及初始细胞分离、T细胞活化、CAR转基因（或mRNA）的引入、细胞扩增和最终制剂。大多数产品在输注到患者体内之前在床边冷冻保存和解冻。传统的制造工艺通常涉及1-2周的离体细胞作和扩增，而简化的制造工艺可以将离体时间缩短到24-72小时。然而，由于运输、产品放行测试和患者临床护理考虑所需的时间，实际的静脉到静脉时间可能会长得多。【NO.3】CD4+与CD8+T细胞CAR-T细胞通过CAR抗原结合直接激活，无需辅助受体与MHC分子结合。因此，CAR-T细胞功能原则上不需要CD4和CD8分子作为辅助受体。CAR-T细胞的早期开发工作考虑了选择性富集CD8+T细胞以最大限度地提高细胞毒性的优势。然而，CD4+辅助性T细胞在促进CD8+T细胞的效应功能、扩增和持久性方面也起着关键作用，并且已经表明CD4+T细胞在肿瘤微环境中的存在导致CD8+细胞毒性T细胞的募集、增殖和功能增加。因此，目前批准的CAR-T细胞产品都含有CD4+和CD8+T细胞，尽管采用了不同的生产方法。细胞分离通常通过基于磁珠的细胞分选来实现。CD4+和CD8+细胞的混合物可以通过去除非T细胞或使用CD4和CD8结合微珠的组合进行阳性选择来分离。分离混合T细胞群的过程相对简化，与平行生产单独的CD4+和CD8+细胞培养物相比，可以以更低的成本完成，但它排除了在最终产品中精确控制CD4：CD8比率的可能性。或者，CD4+和CD8+T细胞可以单独分离和培养，从而可以配制具有精确CD4：CD8比率的最终产品。但是，这种方法的代价是增加劳动力、时间和生产成本。此外，最佳的CD8+T细胞扩增需要CD4+T细胞的存在，这使得分离的CD8+T细胞的生产容易出现生产失败。应对这一挑战的一个潜在解决方案是分别分离CD4+和CD8+T细胞，但以规定的起始比例共培养，目的是产生具有大致所需比例的CD4：CD8T细胞的最终细胞产品。这种策略已被证明可以克服CD8+T细胞扩增的困难，导致与单独培养的CD4+和CD8+T细胞相比，小鼠的产物具有更高的细胞毒功能。然而，这种“定义”的生产方法并不能完全防止生产失败。重要的是，在产品中产生所需的最终比例所需的起始CD4：CD8比率可能因供体而异，因此这种方法仍然难以实现精确的产品特征。【NO.4】T细胞分化状态除了CD4：CD8比率之外，T细胞产物的分化状态已被证明与抗肿瘤疗效特征相关。在抗原刺激后，T细胞经历克隆扩增，一个亚群分化成TE细胞，TE细胞具有强大的细胞毒性和细胞因子产生能力，但寿命相对较短，相比之下，活化的T细胞亚群变成记忆T细胞，这些细胞寿命长，响应继生抗原攻击而迅速激活。与终末分化的TE细胞相比，TN、TSCM和TCM细胞具有更大的长期持续潜力并产生较低水平的炎性细胞因子，它们可以共同实现持久的抗肿瘤反应，同时避免严重的急性毒性，如细胞因子释放综合征（CRS）。这些发现支持了对从富集幼稚和记忆T细胞开始的生产过程的探索，早期证据表明此类产品可以实现强大的疗效和良好的安全性。CD45RA、CD45RO、CD62L和CCR7的表面表达通常用于识别T细胞亚型，并且可以通过选择CD62L+细胞从白细胞去除术产物中分离出幼稚和记忆T细胞。然而，CD62L+选择可同时导致同样表达CD62L的CD14+髓系细胞和CD25+Treg的意外富集。髓系细胞已被证明会显着降低T细胞活化和随后的病毒转导的效率，这可能是由于用于激活T细胞的抗CD3/CD28磁珠的吞噬作用。此外，据报道，CAR-T产品中存在Tregs与抗肿瘤疗效差相关。这些发现表明，CAR-T产品可能受益于CD14+和CD25+细胞类型在CD62L+细胞富集之前耗尽，并且在多项临床试验（例如，NCT02208362和NCT04007029）中评估了使用CD14-/CD25-/CD62L+细胞作为CAR-T细胞生产的起始材料。出乎意料的是，一项试验（NCT04007029）的数据显示，在CD62L富集之前未耗尽CD14+和CD25+细胞的起始群体制成的CAR-T细胞产物与由CD14/CD25耗尽的起始群体制成的产品相比，具有相似的活化、转导、扩增和最终CD3+纯度水平。由非CD14/CD25耗尽细胞制成的产品在细胞生产结束时显示CD4+T细胞中明显富集，但基于该1期试验中的少量患者，由CD14/CD25耗尽或未耗尽起始细胞群制成的细胞产品之间未观察到治疗结果的显着差异。值得注意的是，该特定临床试验中使用的细胞生产过程使用聚合物纳米基质T细胞兴奋剂而不是基于磁珠的激活试剂，并且髓系细胞对不同大小和刚度的激活试剂表现出的差异吞噬反应可能在所得细胞产物的行为中发挥了关键作用。【NO.5】T细胞活化方法T细胞活化对于细胞生产过程的成功至关重要，因为它直接影响CAR转基因整合的效率以及离体培养期间的T细胞扩增。T细胞活化通常是通过刺激CD3和CD28结合细胞因子支持来实现的。CD3信号传导（信号1）触发T细胞活化，而CD28信号传导提供必要的共刺激（信号2）以避免无反应。此外，细胞因子混合物（信号3）——最常见的是IL-2、IL-7和/或IL-15——用于支持T细胞扩增。目前，生产方案通常使用涂有抗CD3和抗CD28抗体的磁珠或胶体聚合物纳米基质来提供信号1和2。磁珠提供固体支持，模拟呈递肽-MHC复合物和共刺激配体的靶细胞以进行T细胞结合，抗CD3/CD28磁珠可同时用作基于磁性的细胞分选的T细胞分离剂。然而，磁珠容易被髓细胞吞噬，因此使用抗CD3/CD28珠子进行细胞分选存在CD14+细胞富集的风险，同时减少了可用于刺激T细胞的激活试剂的数量。此外，在回输之前，需要通过磁分离进行去珠步骤以生成纯CAR-T细胞产物。相比之下，胶体聚合物纳米基质似乎不太容易被髓系细胞消除，并且可以通过简单的离心去除。然而，尚未报道这些试剂的头对头比较，无法对所得细胞产物进行严格比较。无论形式如何，市售活化剂都是以固定磁珠：细胞比例或每体积稀释比例应用的标准化产品。最近的发展探索了根据患者的疾病类型为每种产品量身定制的个性化T细胞活化试剂的可能性。例如，利用涂有脂质双层的介孔二氧化硅微棒，Mooney及其同事开发了APC模拟支架，该支架不仅可以提供抗CD3和-CD28信号，还可以实现IL-2的持续释放。可以精确调整抗CD3和-CD28抗体的密度，以提供最佳刺激强度，从而最大限度地提高T细胞适应性。实施经过微调、高度个性化的试剂必须与生产通量和工艺稳健性的实际考虑相平衡。尽管如此，可以适应不同产品要求的“智能”材料的出现可以显着提高生产过程的灵活性和质量。应该注意的是，尽管T细胞活化是当今CAR-T细胞生产中不可或缺的步骤，但下一代生产工艺正在考虑消除T细胞活化的可能性。例如，最近的一份报告描述了慢病毒载体成功转导未活化的T细胞。【NO.6】CAR转基因CAR转基因递送方法可以显着影响CAR转基因表达水平以及遗传毒性，进而影响所得CAR-T细胞产品的安全性和有效性。在本节中，我们广泛评估了CAR-T细胞生产的病毒和非病毒基因递送方法。6.1病毒基因递送目前，所有FDA批准的CAR-T细胞产品都使用慢病毒或逆转录病毒转导来实现CAR转基因整合。病毒转导受益于相对较高的整合效率，表达稳定整合的CAR构建体的T细胞已被证明在T细胞输注后持续>10年。然而，病毒整合的转基因缺乏插入位点特异性，存在插入诱变的理论风险。一项全基因组分析研究比较了用γ逆转录病毒载体与慢病毒载体制成的CAR-T细胞产品中的转基因整合位点，结果表明，与逆转录病毒相比，慢病毒载体更有可能整合到内含子和基因间区域;相比之下，逆转录病毒整合到启动子、非翻译区和外显子区的频率更高，导致对mRNA转录水平的影响更大。值得注意的是，迄今为止还没有报道病毒整合导致CAR-T细胞致癌转化的实例。相反，一项案例研究报告了一名患者，其对CD19CAR-T细胞疗法的反应似乎与单个CAR-T细胞克隆的扩增相关，该克隆的CAR转基因随机插入TET2基因座，从而敲除唯一功能性的TET2拷贝，因为该患者在另一个TET2等位基因中具有先天性错义突变。同样，在B细胞急性淋巴细胞白血病患者（B-ALL）。尽管这些病例表明随机插入可能会偶然导致积极的治疗结果，但插入到不需要的基因座的风险仍然是探索可以确保位点特异性的替代转基因整合策略的动机。除了整合位点的考虑外，病毒载体还以具有复制能力的逆转录病毒（RCR）和慢病毒（RCL）的形式存在另一种潜在风险。因此，FDA要求对病毒载体以及病毒转导的细胞产品进行RCR/RCL检测测定。然而，迄今为止，尚未在临床CAR-T细胞产品中检测到RCR和RCL的报道，并且平衡RCR/RCL测试造成的成本和潜在延迟与此类分析的实际好处仍然是一个积极科学探索的话题。尽管病毒转导原则上可以实现高转导效率，但在患者细胞产品中实现一致水平的转基因整合是一项技术挑战。事实上，10%或更低的CAR阳性率——基于转导细胞在抗原暴露后将在体内扩增的前提设定的水平——作为临床试验中使用的细胞产品中转导效率的最低阈值并不少见。为了最大限度地降低由于转基因表达不良而导致生产失败的风险，生产方案可以结合转导增强剂的使用，例如硫酸鱼精蛋白、retronectin和泊洛沙姆。病毒转导中的限速步骤是病毒颗粒最初附着在细胞膜上，这一过程可以通过使用retronectin进行逆转录病毒转导和聚阳离子（如硫酸鱼精蛋白）进行慢病毒转导来促进。聚阳离子充当将病毒连接到细胞表面的静电桥。然而，硫酸鱼精蛋白对T细胞的毒性与提高转导效率的好处相抵消。相比之下，retronectin不会导致T细胞毒性，但其使用仅限于逆转录病毒载体。已经开发了具有更有利毒性特征的专有转导增强剂，尽管它们的高成本和专有访问可能会阻碍广泛使用。除了病毒转导的生物学特性外，实际考虑也会影响病毒在CAR-T细胞生产中的使用。能够生产临床级病毒的认证设施数量仍然有限。因此，病毒生产所需的时间和财务成本可能成为研究药物开发和商业CAR-T细胞生产的瓶颈，突出了可能更灵活且更具成本效益的替代非病毒递送系统的吸引力。6.2非病毒基因递送在CAR-T细胞生产的背景下，已经探索了几种不同的非病毒基因递送方法，包括CRISPR/Cas9、转座子和mRNA转染。CRISPR/Cas9作为一种有效的基因组修饰方法已被广泛采用，其增强CAR-T细胞疗法的潜力并未被忽视。为了实现位点特异性基因修饰，Cas9核酸酶与单向导RNA（sgRNA）复合，通过与sgRNA的序列互补性识别基因组DNA上的靶位点，并引入双链DNA断裂。接下来，通过编码所需转基因（例如CAR）和基因组切割位点的DNA模板之间的同源重组来促进转基因插入。指定转基因整合位点的能力为工程化具有所需功能的T细胞开辟了新的可能性。已经通过计算确定了许多基因外“安全港”位点并进行了实证测试，以支持T细胞的精确基因改造以实现治疗功能。特别是，已经表明CAR转基因可以使用腺相关病毒载体作为同源定向修复模板有效地整合到编码内源性TCRα链的TRAC基因座中。这种策略征用了响应T细胞激活状态动态控制TCR表达水平的内源性基因调控机制，数据表明所得的CAR-T细胞可能比病毒整合的CAR-T细胞更有效。此外，基于非病毒CRISPR/Cas9的基因编辑策略也被用于用肿瘤特异性TCR取代内源性TCR，从而能够开发高度个性化的T细胞疗法。作为另一个例子，上述涉及TET2突变的案例研究激发了随后对有意将CAR转基因整合到TET2位点作为增强CAR-T细胞增殖和持久性的一种手段的评估。有趣的是，结果表明，TET2破坏对体内抗肿瘤疗效的益处因表达的特定CAR构建体而异，这与CAR整合到TRAC基因座中的类似发现相呼应，并强调了确定CAR转基因最佳整合位点的非平凡性质。除了促进位点特异性转基因整合外，CRISPR/Cas9还与病毒整合结合使用，以敲除不需要的内源性基因，同时非位点特异性整合编码肿瘤靶向受体的转基因。例如，第一个涉及CRISPR/Cas9编辑的T细胞的FDA批准的临床试验利用了一种生产工艺，该工艺用CRISPR/Cas9敲除PD-1和内源性TCR，同时慢病毒整合了靶向NY-ESO-1的转基因TCR。值得注意的是，据观察，输注后PDCD1基因座突变的NY-ESOTCR表达细胞的频率随着时间的推移而降低，这表明PD-1编辑的T细胞可能缺乏形成长期记忆的能力。在另一项1期临床研究中，CRISPR/Cas9用于敲除PD-1和TCRα链，同时慢病毒整合靶向间皮素的CAR。尽管两项研究都显示出可接受的安全性，但都没有导致疗效的显着改善，突出了持续改进的必要性。此外，CRISPR/Cas9介导的双链DNA断裂带来了意外基因组变化的重要风险，需要仔细分析和质量控制，最近的一项研究揭示了Cas9工程CAR-T细胞中染色体丢失的可能性。有趣的是，同一项研究观察到，特定的作顺序会影响染色体丢失的频率，如果在T细胞被激活之前进行Cas9介导的双链断裂，则可以减少染色体丢失的频率。作为CRISPR/Cas9的替代品，转座子也被用于实现CAR转基因稳定整合到T细胞产品中。特别是，piggyBac和睡美人（SB）转座子系统已在临床环境中进行了评估。转座子系统执行“剪切和粘贴”过程，其中转座酶与靶基因元件两侧的末端反向重复序列（即转座子）结合，通过双链DNA断裂切除转座子，并将转座子重新整合到合适的基因组位点（例如，在SB转座子的情况下，包含AT二核苷酸的回文序列）。通过这种方式进行的转基因整合不是位点特异性的，据报道，piggyBac转座酶表现出类似于逆转录病毒的优先插入转录起始位点，再次增加了插入诱变的潜在风险。在一项1期临床试验中，两名接受piggyBac转座子系统产生的同种异体CD19CAR-T细胞产品治疗的患者患上了CAR-T细胞衍生的淋巴瘤，导致一人死亡。事后分析表明，恶性CAR-T细胞不包含转基因插入已知的致癌位点，但显示出多条染色体的显着拷贝数增加和丢失以及来自转基因启动子的转录通读。重要的是，导致恶性转化的产物具有异常高的载体拷贝数（VCN），其中一种产物的VCN为25。相比之下，在没有产品特定理由的情况下，FDA推荐的病毒转导细胞产品的最大拷贝数为每个转导细胞5个拷贝。消除致癌插入风险的一种策略是避免编码CAR的转基因的稳定整合，而是从mRNA模板瞬时表达CAR。除了消除遗传毒性的风险外，mRNA的瞬时CAR表达也被探索为降低潜在毒性的一种手段，特别是当CAR靶向也存在于健康组织中的抗原时。编码转基因的mRNA模板通常通过电穿孔递送到T细胞中，转基因表达持续约1周，电穿孔后表达水平每天下降。因此，瞬时基因表达的潜在安全优势必须与治疗的短暂性质相平衡。临床评估表明，mRNA编码的CAR-T细胞是安全的，并且可以表现出抗肿瘤功效，但实现完全和持久的肿瘤控制的能力仍然是一个研究领域。【NO.7】离体细胞扩增一旦T细胞被修饰以表达CAR转基因，产物必须扩增到足够大的数量，以满足给药所需的剂量。此扩展期的持续时间因不同的方案而异，但典型的过程从T细胞活化到细胞收获持续1-2周。扩增条件针对T细胞生长进行了优化，具有细胞因子支持，如IL-2、IL-7和/或IL-15。虽然扩增期的主要目的是增加T细胞数量，但它也通过将非T细胞暴露于不如其他细胞类型的培养条件来消除非T细胞，因此即使生产过程从混合PBMC开始，也能产生高度富集的T细胞产品。除了细胞因子外，FBS等培养补充剂在支持T细胞扩增方面也起着关键作用，尽管能够实现更精确配方和降低人畜共患病原体风险的确定培养基成分正在迅速取代FBS等成分。最后，最近的研究探索了使用各种药物补充剂来促进理想的T细胞表型并增强所得CAR-T细胞产品的治疗潜力。例如，酪氨酸激酶抑制剂达沙替尼已用于CAR-T细胞生产，以抑制CAR强直信号传导（即在没有抗原刺激的情况下的受体信号传导），从而防止T细胞过早耗竭并增加CAR-T细胞功能。该策略用于生产靶向GD2的CAR-T细胞——已知这些细胞具有强烈的强直信号并表现出耗竭倾向——用于1期试验，早期结果显示多名H3K27M突变弥漫性中线神经胶质瘤患者有显着改善。由于对参加该试验的每位患者进行了多剂量的CAR-T细胞给药，因此关于达沙替尼是否能防止耗竭和/或实现持续T细胞功能的明确结论仍然难以捉摸。尽管如此，这个例子证明了药理学调节CAR-T细胞生产过程以产生用于临床转化的功能性CAR-T细胞产品的可行性。【NO.8】产品放行测试利用CAR-T细胞在2017年治疗血液系统恶性肿瘤方面的突破性成功，全球正在进行1000多项活跃的CAR-T细胞临床试验，集中在北美和欧亚大陆。临床研究的热潮加深了对成功CAR-T细胞产品相关参数的理解。与任何拟议的疗法一样，寻求临床评估的CAR-T细胞必须满足与安全性、纯度、效力、特性和稳定性相关的既定产品特性。所有FDA批准的CAR-T细胞产品使用以下指标的组合来衡量商品特征：•安全性：支原体、无菌、内毒素、残留病毒试剂定量、活力和CAR转基因定量•纯度：T细胞纯度、活力、残留试剂定量、转导效率和是否存在污染肿瘤细胞•效价：抗原刺激后转导效果、活力、CAR表达、细胞毒性或细胞因子（如IFN-γ）的产生•鉴定：CAR表达、观察外观、清晰度、剂量和活力•稳定性：配方、运输和储存。FDA最近起草了CAR-T细胞产品的标准化期望。特别是，早期研究性新药申请提交的批次放行标准不需要经过验证的效价测定和规格，但在生成支持生物制品许可申请的数据时必须包括此类测定。根据正在进行的临床试验和实际经验的积累数据，还提出了额外的参数，以推动未来开发更具可重复性、更有效和安全的治疗方法。例如，鉴于证据表明分化较少、记忆样表型较多的CAR-T产品更有可能实现持久的肿瘤清除，未来的商业产品可能会受益于设置一个功效特征指标，该指标指定产品中有利T细胞亚群的比例。然而，更改和收紧产品规格必须以严格的科学证据和基本的生物学理解为指导，以避免引发不必要的生产失败，从而可能减少患者获得治疗的机会。在某些涉及非危及生命的批次放行标准（例如剂量、转导功效、细胞因子产生水平或CAR表达）的产品失败的情况下，在获得必要的监管批准后，可以向患者施用不合格产品。在扩大访问方案中，患者收到了不合格的商业和临床试验产品，并经历了与接受标准产品治疗的患者相当的临床结果。在现实世界环境中积累CAR-T细胞产品的临床经验对于了解哪些产品特性真正影响患者安全性和治疗效果非常宝贵，并可能作为进一步完善CAR-T细胞产品放行测试监管指南的指南。【NO.9】CAR-T细胞生产的下一代策略迄今为止讨论的生产策略支持了迄今为止多种CAR-T细胞产品的开发，并展示了CAR-T细胞疗法克服晚期恶性肿瘤的潜力。然而，FDA批准后CAR-T细胞疗法的实际经验也凸显了传统细胞生产工艺的局限性，这些工艺通量低且资源密集，导致患者获得可能挽救生命的疗法的机会有限。截至2023年初，90%的MM患者出现疾病进展，25%的患者在等待BCMA定向的CAR-T细胞产品插槽时死于疾病，在接受单采术之前等待时间从1到10个月不等。单采术后，细胞必须经过往返生产地点的运输、离体修饰和扩增以及严格的质量控制测试。大多数患者需要桥接疗法来对抗细胞生产过程中的进一步临床恶化，这可能导致出现与桥接相关不良反应的患者进一步延迟输注。即使在静脉到静脉期间没有医疗并发症的情况下，4-7%的患者也因生产失败而无法接受他们的CAR-T细胞产品，风险因素包括多线治疗后患者T细胞的健康状况降低和漫长的生产过程本身。产品生产失败后的治疗选择包括重复单采术或过渡到替代疗法，尽管这两种情况下的生存结果都很差。总而言之，导致生命损失的多种延误来源强调了及时获得CAR-T细胞产品的重要性。在这里，我们讨论了正在开发的下一代策略，以应对CAR-T细胞生产中的关键挑战（表2）。表2.下一代CAR-T细胞生产策略9.1加速细胞生产为了应对CAR-T细胞生产中的这些挑战，已经开发了新策略来显着缩短细胞生产的持续时间，从而降低生产成本，生产造失败的可能性和静脉到静脉的时间。这些下一代生产工艺可以在短短24小时内形成最终产品配方（图1），从而有可能显著加快患者获得治疗的机会。大部分时间节省是由于离体扩增期的显着缩短，从而限制了可以实现的细胞计数的倍数增加。然而，离体细胞分裂次数的减少也可能导致T细胞群分化程度降低，输注后具有更大的长期增殖潜力。事实上，早期结果表明，在早期时间点收获的产物表现出更大比例的TCM细胞（CD45RO+CD62L+）和TN/TSCM细胞（CD45RO-/CCR7+），据报道，它们表现出治疗上有利的表型和功能。使用加速生产的CAR-T细胞产品的一个例子是使用“T-Charge”平台生产的CD19定向YTB323细胞，该平台需要<2天的离体培养。据报道，与使用传统CAR-T细胞生产工艺生产的等效产品（tisa-cel）。值得注意的是，2期试验的初步结果表明，YTB323在复发/难治性弥漫性大B细胞淋巴瘤（DLBCL）患者中可以产生与tisa-cel相当的疗效，但剂量低25倍，这表明减少的离体培养时间可能产生了功能优越的产品。另一个例子是，在复发/难治性MM患者的1期试验中，使用相同的T-Charge平台生产的BCMA定向PHE885细胞实现了98%的总体缓解率。值得注意的是，93%（13/14）的患者在输注后6个月可检测到CAR-T细胞，在71%（5/7）的患者在12个月时可检测到CAR-T细胞，表明体内T细胞具有强大的持久性。另一个名为FasTCAR-T的加速生产平台已被用于为B-ALL患者生成“CD19F-CAR-T”细胞，其中92%（23/25）的患者实现了最小残留病阴性完全缓解。该试验中的大多数（20/25）患者继续接受同种异体造血干细胞移植，因此无法评估对CAR-T细胞治疗反应的长期持久性。随着来自多项试验的临床数据不断成熟，通过不同生产平台产生的CAR-T细胞产品诱导的反应的相对持久性将是一个重要的关注点。与加速细胞生产过程相关的理论问题是，在离体激活后不久冷冻保存的CAR-T细胞可能表现出过度刺激的表型。此外，特别是在血液系统恶性肿瘤的情况下，来自高度简化的生产工艺的产品增加了包含肿瘤细胞的可能性，否则这些细胞在针对T细胞生长优化的条件下，在延长的离体培养期间会被耗尽。然而，现有的临床数据表明，通过这些缩短方案生产的CAR-T细胞产品的安全性在临床上仍然是可控的。YTB323治疗DLBCL的1期研究结果显示，33%（15/45）的患者经历过CRS，伴有1例4级事件，11%（5/45）出现免疫效应细胞相关神经毒性综合征（ICANS），伴有2例3级事件。在B-ALL患者中，CD19F-CAR-T细胞表现出更大的毒性，CRS的发生率增加了两倍（总体为96%，≥级为24%），ICANS的发生率增加了一倍（28%，均为≥3级）。25名患者中有21名在CRS发作期间需要介入性皮质类固醇，尽管治疗并未消融CAR-T细胞扩增峰值。来自正在进行的临床试验的额外数据将有助于建立我们对通过不同平台生产的CAR-T细胞产品之间生物学差异的理解。最后，需要注意的是，关于产品放行测试的监管要求仍然适用于采用加速工艺生产的产品，并且在估计静脉到静脉的时间时，仍需要考虑此类放行测试所需的时间。9.2流程自动化改进商业CAR-T细胞生产的另一种途径涉及生产过程的自动化，这有可能降低生产成本、人为错误导致生产失败的可能性以及接触点的污染。在某些司法管辖区（例如美国），一些自动化系统被视为全封闭系统，允许在符合良好生产规范标准的设施外部运行。此外，自动化可以显着减少生产人员所需的数量和经验水平。反过来，这些因素支持了现场生产的可能性，从而为患者提供新鲜、非冷冻保存的产品。在一项针对非霍奇金淋巴瘤患者的1期试验中，患者接受了CD19/CD20双特异性CAR-T细胞，这些细胞要么新鲜给药，要么从冻存的等分试样中解冻，早期数据显示新鲜CAR-T细胞产品具有更高的活力（93%新鲜对63%冻存）。该试验的后续报告证实，与使用冷冻保存的CAR-T细胞治疗的患者相比，接受新鲜CAR-T细胞产品治疗的患者的完全缓解率要高得多（80%新鲜对29%冷冻保存）以及更大的CAR-T细胞扩增和持久性。尽管细胞冷冻保存程序的改进仍有可能减少新鲜和冷冻保存的CAR-T细胞产品之间观察到的功效差异，但这些结果突出了现场细胞生产的潜在优势。然而，在确保产品质量始终如一的同时，将这种做法扩展到大量医疗中心仍然是一个关键挑战。重要的是，需要进行几项监管改革才能允许即时CAR-T细胞生产，而全球司法管辖区之间的差异代表了现场细胞生产实际实施的重大障碍。此外，使用新鲜细胞产品会妨碍完成需要较长潜伏期的放行测试，因此需要依赖过程中的样本测试以及应急临床管理计划，以应对在细胞产品输注后收到无菌或其他测试结果失败的情况。此外，新鲜产品的管理带来了重大的物流挑战，因为它需要细胞生产和患者接受细胞输注的准备之间密切的时间协调。最后，当需要根据患者调整最佳过程参数设置时，自动化系统的高度标准化性质可能会带来挑战。例如，来自不同患者的细胞可能具有截然不同的扩增速率，因此每天在培养物中维持适当细胞密度所需的培养基量可能因生产活动而异。为了实现最佳结果，需要配备仪器来感应关键参数并做出相应的响应。9.3体内细胞生产展望未来，下一代生产工艺可能会完全消除离体细胞作和扩增，而是在体内产生治疗性细胞群。几项临床前研究表明，能够使用慢病毒或腺相关病毒在体内转导T细胞。此外，脂质纳米颗粒（LNP）和聚合物纳米载体已被证明可以在体内将核酸有效载荷递送到T细胞。为了实现T细胞靶向，病毒颗粒、LNP和纳米载体通常用单链可变片段或其他靶向CD3、CD4或CD8的结合结构域修饰。然而，T细胞靶向的特异性可能不需要是绝对的，人们可以考虑同时生成CAR-T细胞、CAR-自然杀伤细胞和CAR-巨噬细胞的潜在优势。然而，不将转基因递送到恶性细胞中仍然至关重要，因为这样的整合事件可能导致CAR蛋白掩盖靶抗原并保护肿瘤细胞免受表达CAR的效应细胞的检测。此外，在可以实现稳定转基因整合的病毒载体的情况下，脱靶基因递送增加了遗传毒性风险。在实践中，专门针对T细胞的转基因递送载体和促进高效转基因整合的策略，而无需外部T细胞激活，对于产生能够实现持久抗肿瘤疗效的有效剂量的CAR-T细胞可能是必要的。最后，在体内CAR-T细胞生产平台的临床转化之前，需要进行深入的药代动力学和安全性验证。尽管仍然存在许多障碍，但绕过离体细胞生产的能力有可能显著降低成本，并增加需要这种治疗选择的患者获得CAR-T细胞疗法的机会。【NO.10】总结CAR-T细胞疗法已成为血液系统恶性肿瘤越来越重要的治疗选择，临床试验不断将CAR-T细胞疗法的应用扩展到实体瘤和自身免疫性疾病的治疗。因此，能够高效可靠地生产高质量细胞产品的CAR-T细胞生产工艺对于支持及时、安全和有效的患者护理至关重要。积累的临床经验、真实世界的生产数据收集、自动化系统工程的进步以及对T细胞生物学的基本理解都在自体CAR-T细胞生产工艺的持续改进中发挥着关键作用。此外，同种异体细胞疗法的快速发展前景涉及额外的免疫效应细胞类型以及干细胞群产生的T细胞，在未来几年将继续成为有趣的科学问题和实际工程挑战的来源。识别微信二维码，添加抗体圈小编，符合条件者即可加入抗体圈微信群！请注明：姓名+研究方向！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。