Radhakrishnan H, Newmyer SL, Ssemadaali MA, Javitz HS, Bhatnagar P. Primary T-cell-based delivery platform for in vivo synthesis of engineered proteins. Bioeng Transl Med. 2024; 9(1):e10605. doi:10.1002/btm2.10605
Unlike the remainder of the manuscript where the data have been developed using pan CD3 T cells, Figure 4d–f used CD4 T cells. We apologize for this error. Corrections have been disclosed in the following places:
Figure 4d–f labels, edited to indicate the use of CD4 T cells
Figure 4 caption, edited to indicate the use of CD4 T cells
FIGURE 4. Functional validation of the primary T-cell-based delivery platform. (a, b) In vitro validation of target-specific delivery function proportionate to the disease burden. FRα-specific primary T cells engineered for the NFAT-RE inducible delivery function showed proportionate increase in reporter activity when cocultured with target, (a) FRα+A2780cis and (b) FRα+KPCY cells, compared to their respective nontarget (FRαneg) control cells. (c) CAR T cells manufactured using the process developed for T-cell-based delivery platform reduced tumor burden. Tumor regression was observed in intraperitoneal (i.p.) KPCY tumors in NSG mice when treated with FRα-specific CAR T cells in a dose-dependent manner (n = 5 mice per group). Bioluminescence (Luc2 activity) from the i.p. tumors was used to assess the tumor burden in vivo. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test. There was a statistically significant interaction between days and FRα-specific CAR T-cell doses on tumor burden (F[18, 96] = 4.595, p < 0.0001). (d–f) The primary T-cell-based delivery platform manufactured using the same process was functional in vivo in an antigen-specific manner (n = 5 mice per group). FRα-specific primary CD4 T cells engineered for the NFAT-RE inducible delivery function were i.p. injected in i.p. FRα+A2780cis tumor-bearing NSG mice at 24-h interval for 5 days and NFAT-RE inducible effector (Nluc) activity was measured for 6 days including the day of injection as a baseline to assess the delivery function. A control group was included to assess any background signal that may arise from using the Nluc substrate with Luc2+ tumor cells and injected with FRα-specific primary CD4 CAR T cells (engineered without the NFAT-RE inducible effector [Nluc]) to maintain equivalent tumor burden. (d) Schematic of dosing, treatment, and imaging schedules, (e) bioluminescent images, and (f) quantification. All results are represented as mean ± SEM. Statistical analysis and p values for (a), (b), and (f) were determined by multiple t test using Holm–Sidak method. *p < 0.05, **p < 0.01, and ***p < 0.001.
A result in Section 2.3, edited to indicate the use of CD4 T cells
Using primary CD4 T cells, we next manufactured FRα-CAR+ T cells with the delivery function, that is, upon engaging the target FRα antigen, the FRα-CAR activates the NFAT-RE signaling pathway to induce the expression of desired protein. The experiment schedule is detailed in Figure 4d and the results are shown in Figure 4e,f. Then, 2 × 106 FRα+Luc2+A2780cis cells were i.p. implanted in NSG mice. The 12-day-old xenograft tumors were i.p. treated with 2 × 106 FRα-CAR+ primary CD4 T cells (with NFAT-RE inducible Nluc reporter) on Days 0, 1, 2, 3, and 4. A control group was included to assess any background signal from using the Nluc substrate on Luc2+ tumor cells. This group was treated with i.p. injections of FRα-CAR+ primary CD4 T cells without NFAT-RE inducible Nluc reporter (control FRα-CAR+T cells) to maintain an equivalent tumor burden. The effector (Nluc) activity (Figure 4e) was measured and quantified (Figure 4f) at baseline (Day 0) as well as on days 1, 2, 3, 4, and 5. A significant increase in engineered effector activity (i.e., delivery function) was observed in the group treated with the FRα-CAR+ T cells with the delivery function (i.e., with NFAT-RE inducible Nluc reporter), confirming the target-inducible in situ delivery function in the engineered primary T cells.
Addition to the Key Resource Table in Section 4.1, to indicate the source of CD4 T cells
Human primary CD4 T cells
Stanford Blood Center
A1015
A method in Section 4.10, edited to indicate the use of CD4 T cells
4.10 In vivo validation of delivery function of the engineered T cells (engineered for delivery function with NFAT-RE delivery system). The in vivo validation of our T-cell based delivery system was performed in mice at SRI International in accordance with the guidelines from the Institutional Animal Care and Use Committee (Approval # 22001). Six- to 8-week-old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from The Jackson Laboratory. After mandatory quarantine, the NSG mice were anesthetized and 2 × 106 FRα+Luc2-2A-E2Crimson+A2780cis cells in 100 μL 1× PBS were i.p. implanted. The tumor growth was monitored every 3–4 days for the next 12 days using i.p. injected 150 mg d-Luciferin per kg of mouse dissolved in 1× PBS. At 11 days after implantation, the mice were randomized into two groups (n = 5 each). The two groups were then treated with 2 × 106 primary CD4 T cells engineered for delivery function (i.e., FRα-CAR with NFAT-RE inducible Nluc reporter) or the control primary CD4 T cells (FRα-CAR only, i.e., without NFAT-RE inducible Nluc reporter) every day for 5 days. The bioluminescent reporter (Nluc) activity was determined by i.p. injection of the Nano-Glo® substrate (1:20 dilution of the substrate in 1× PBS, equivalent to 0.5 mg per kg of mouse) on Days 0, 1, 2, 3, 4, and 5 after treatment. Imaging was performed in a IVIS Lumina X5 imaging system. The data were quantified by analysis of the ROI using Living Image software. The tumor luminescence is plotted as the mean ± SEM of total flux (photons/s) against days after treatment.
中文翻译：
对“用于工程蛋白质体内合成的初级 T 细胞递送平台”的更正
Radhakrishnan H、Newmyer SL、Ssemadaali MA、Javitz HS、Bhatnagar P。用于工程蛋白体内合成的主要 T 细胞递送平台。生物工程翻译医学。2024；9（1）：e10605。doi：10.1002/btm2.10605
与手稿的其余部分使用泛 CD3 T 细胞开发数据不同，图 4d-f 使用 CD4 T 细胞。对于这个错误，我们深表歉意。更正已在以下地方披露：
图4d-f标签，经过编辑以指示CD4 T细胞的使用
图 4 标题，经过编辑以表明 CD4 T 细胞的使用
图 4.基于 T 细胞的主要递送平台的功能验证。（a，b）与疾病负担成比例的靶标特异性递送功能的体外验证。与各自的非靶细胞 (FRα neg )相比，针对 NFAT-RE 诱导递送功能而设计的 FRα 特异性原代 T 细胞与靶细胞 (a) FRα + A2780cis 和 (b) FRα + KPCY 细胞共培养时，报告基因活性成比例增加控制细胞。(c) 使用为基于 T 细胞的递送平台开发的工艺制造的 CAR T 细胞减少了肿瘤负担。当以剂量依赖性方式使用 FRα 特异性 CAR T 细胞治疗时，在 NSG 小鼠的腹膜内 (ip) KPCY 肿瘤中观察到肿瘤消退（ 每组n = 5 只小鼠）。腹膜内肿瘤的生物发光（Luc2 活性）用于评估体内肿瘤负荷。使用双向方差分析和Tukey多重比较检验进行统计分析。天数和 FRα 特异性 CAR T 细胞剂量对肿瘤负荷的影响存在统计学上显着的交互作用（F [18, 96] = 4.595，p < 0.0001）。(d–f) 使用相同工艺制造的主要 T 细胞递送平台在体内以抗原特异性方式发挥作用（ 每组n = 5 只小鼠）。将针对 NFAT-RE 诱导递送功能设计的 FRα 特异性原代 CD4 T 细胞以 24 小时间隔腹腔注射到 FRα + A2780cis 荷瘤 NSG 小鼠中，持续 5 天，并测量 NFAT-RE 诱导效应子 (Nluc) 活性包括注射当天在内的 6 天作为评估递送功能的基线。纳入对照组以评估使用 Nluc 底物与 Luc2 +肿瘤细胞并注射 FRα 特异性原代 CD4 CAR T 细胞（不含 NFAT-RE 诱导效应子 [Nluc] 的工程设计）可能产生的任何背景信号，以维持同等水平肿瘤负荷。(d) 给药、治疗和成像方案示意图，(e) 生物发光图像，以及 (f) 量化。所有结果均表示为平均值±SEM。(a)、(b) 和 (f) 的统计分析和p值通过使用 Holm-Sidak 方法的多重t检验确定。* p < 0.05，** p < 0.01，*** p < 0.001。
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