Clinical trial information and design
This study was a phase 1, open-label, single-arm clinical trial designed to evaluate the safety and efficacy of non-viral, PD1-integrated anti-CD19 CAR-T cells in treating aggressive r/r B-NHL. The clinical protocol has been registered at ClinicalTrials.gov (NCT04213469). The inclusion criteria were as follows: (1) age of 18 to 70 years; (2) diagnosis with CD19+ r/r B-NHL (stages III–IV); (3) life expectancy of >3 months; (4) Eastern Cooperative Oncology Group (ECOG) score of ≤2 and satisfactory major organ functions; and (5) a negative pregnancy test for women of reproductive potential and agreement to use birth control during the study. The exclusion criteria included (1) pregnancy or breast feeding; (2) refusal to use birth control during the next 2 years; (3) allo-haematopoietic stem cell transplantation within 6 months or previous treatment of graft-versus-host disease; (4) active autoimmune disease requiring immunosuppressive agents; (5) active infection; (6) history of other malignancies; and (7) ineligibility or lack of ability to comply with the study. To preliminarily assess the safety and effectiveness of this new CAR-T cell therapy, eight patients who had not previously been treated with CAR-T cell therapy were enrolled in the cohort with an infusion dose of 2 × 106 CAR-T cells per kg. Because of the premature manufacturing process and individual variance, the cell number for three infusion products could not meet the planned dose requirement; thus, the actual infusion doses in these patients were lower than 1 × 106 cells per kg (Extended Data Table 1 and Supplementary Table 5). This therapy included 3 d of lymphodepletion chemotherapy using combined fludarabine (25 mg m–2 from days −4 to −2) and cyclophosphamide (250 mg m–2 from days −3 to −2). CAR-T cell infusion was performed 2 d after the end of lymphodepletion chemotherapy and was followed by standard monitoring. The trial was approved by the institutional review board, and all patients provided written informed consent in accordance with the Declaration of Helsinki before enrolment. The clinical protocol was reviewed and approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (2020IIT(85)). Characteristics, clinical responses and prior therapies of the patients are shown in Extended Data Table 1 and Supplementary Table 6.
Response assessment
Treatment response was assessed according to revised criteria of the Lugano classification. PET-CT scans and bone marrow biopsy were the major methods applied to evaluate lymphoma lesions. The response assessment criteria were as follows: (1) CR (complete remission): absence of clinical symptoms and PET-CT and bone marrow evidence associated with lymphoma; (2) PR (partial remission): lymphoma volume decrease of at least 50% without new lymphoma lesions or sustained bone marrow involvement; (3) PD (progressive disease): lymphoma volume increase of at least 50% or onset of new lymphoma lesions; (4) SD (stable disease): a condition that did not meet the criteria for CR, PR or PD. Response duration was calculated from the first documentation of response until disease progression, initiation of off-study treatment or the last documentation of ongoing response.
Assessment and grading of CRS
Serum cytokines including IL-2, IL-4, IL-6, IL-10, IFNγ, TNFα and IL-17A were assessed with the Human Th1/Th2/Th17 CBA kit (BD Biosciences) within 1 month of infusion. CRS was assessed and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0 in combination with other methods45. Among the eight patients, only patient 6 was treated with an IL-6 antagonist, tocilizumab.
Assessment and grading of neurological toxicity
Neurological toxicities were assessed and graded according to NCI-CTCAE version 5.0. Once CRS symptoms such as pyrexia, hypotension and capillary leak or other types of AEs were observed, the patient would be closely monitored for signs of neurological toxicity, such as seizure, tremor, encephalopathy or dysphasia.
Assessment and grading of AEs
Patients were inpatients and were closely monitored after receiving lymphodepletion chemotherapy and CAR-T cell infusion. Physical and clinical laboratory examinations were documented during hospitalization to evaluate the toxicity of the treatment. AEs were graded using NCI-CTCAE version 5.0. All AEs are summarized in Supplementary Table 7. During hospitalization, any AEs that occurred after CAR-T cell infusion were recorded. Severe AEs, except a decrease in lymphocyte counts caused by lymphodepletion chemotherapy, were required to be reported to the Medical Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University within 24 h of occurrence. One month after infusion, patients underwent follow-up and were monitored for disease progression and toxicity once a month thereafter.
Immunohistochemistry
Immunohistochemistry (IHC) analysis was undertaken on formalin-fixed, paraffin-embedded tissue sections. In brief, after sections were deparaffinized in xylene and rehydrated in a graded alcohol series, endogenous peroxidase was blocked with 3% hydrogen peroxide. Antigen retrieval was performed using EDTA buffer (pH 9.0). After rinsing sections in PBS, antibodies against human CD19 (Biolynx) and PD-L1 (Agilent) were used for IHC staining. Staining was carried out on an automated immunostainer (Leica Bond-III, Dako Autostainer Link 48) using a Bond Polymer Refine Detection system.
Cell lines
293T and Nalm-6 cells were purchased from the American Type Culture Collection, and Raji cells were purchased from the Cell Bank of the Chinese Academy of Sciences. All cell lines were authenticated by short-tandem-repeat profiling. 293T cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Thermo Fisher). Nalm-6 and Raji cells were maintained in RPMI-1640 (Thermo Fisher) supplemented with 10% FBS (Thermo Fisher). A Raji cell line stably expressing firefly luciferase (ffLuc) was established by lentiviral infection. Raji cells stably expressing PD-L1 were generated using a lentiviral vector containing a co-expression cassette for PD-L1 and ffLuc. All stable cell lines underwent selection with puromycin. All cell lines were regularly tested to ensure they were free of mycoplasma contamination.
Isolation and expansion of human primary T cells
Fresh peripheral blood mononuclear cells (PBMCs) from healthy donors were provided by the First Affiliated Hospital, College of Medicine, Zhejiang University and Shanghai SAILY Biological Technology Co., Ltd. Recruitments of healthy human blood donors was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University and by Shanghai Zhaxin Traditional Chinese Ethics Committee and Western Medicine Hospital. All donors signed an informed consent form. Fresh PBMCs from patients were collected by apheresis. PBMCs were isolated by density gradient centrifugation using Ficoll (Sigma-Aldrich). T cells were enriched through magnetic separation using anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec) and activated with T Cell TransAct (Miltenyi Biotec). T cells were cultured in X-VIVO 15 medium (Lonza) supplemented with 2% human AB serum or CTS Immune Cell Serum Replacement (Thermo Fisher) and recombinant human IL-2 (100 U ml–1), IL-7 (5 ng ml–1) and IL-15 (5 ng ml–1). Cells were collected once cell number reached the requirement for administration and then washed, formulated and cryopreserved.
Construction of CAR cassette
The anti-CD19 CAR cassette was composed of the single-chain variable fragment derived from clone FMC63, the extracellular domain and transmembrane regions of CD8α, the intracellular domain of 4-1BB (CD137) and the intracellular domain of CD3ζ. Transcription of the CAR element was driven by an EF1α promoter and terminated by an SV40 poly(A) signal sequence. In this study, the same anti-CD19 CAR cassette was used in different constructs.
Ribonucleoprotein and linear dsDNA production
A two-component sgRNA was chemically synthesized (GenScript) and resuspended with TE buffer. Ribonucleoproteins (RNPs) were produced by complexing one sgRNA and recombinant spCas9 (Thermo Fisher) for 10 min at room temperature. RNPs were subjected to electroporation immediately after complex formation. For linear dsDNA production in preclinical experiments, plasmids containing an mTurquoise2 or anti-CD19 CAR sequence flanked by homology arms were first constructed. Linear dsDNA was then obtained by restriction endonuclease digestion and purified by TIANgel DNA Purification kit (Tiangen Biotech). The sgRNAs used were as follows: AAVS1 sgRNA (5′-AGAGCUAGCACAGACUAGAG-3′; chr19:55115996, intron 1 of PPP1R12C), PD1 sgRNA (site 1) used for the preparation of PD1-19bbz and LV-19bbz_PD1-KO cells (5′- CGACUGGCCAGGGCGCCUGU-3′; chr2:241858824, exon 1 of PD1), PD1 sgRNA (site 2) (5′-GGGCGGUGCUACAACUGGGC-3′; chr2:241858788, exon 1 of PD1), TRAC sgRNA (5′-AGAGCAACAGTGCTGTGGCC-3′; chr14:22547693, exon 1 of TRAC), B2M sgRNA (5′-GAGTAGCGCGAGCACAGCTA-3′; chr15:44711569, exon 1 of B2M).
Human primary T cell electroporation
Electroporation was performed 2–3 d after T cell stimulation. For preparation of non-viral, gene-specific targeted CAR-T cells from healthy donors and some patients, the procedure was conducted following the manufacturer’s instructions using a Lonza 4D electroporation system. In brief, 2.5 × 106 to 1.5 × 107 prewashed T cells were resuspended in 100 μl electroporation buffer P3. Meanwhile, RNPs were prepared, followed by mixture with the DNA template (1.5–20 μg). Cells in electroporation buffer were then added and moved into electroporation cuvettes. Programme EO115 was chosen for electroporation. After electroporation, cells were immediately supplemented with prewarmed medium and transferred out of the electroporation cuvettes.
To prepare PD1-19bbz cells for some patients, we followed the manufacturer’s instructions using the GT Flow Transfection System (MaxCyte). In brief, prewashed T cells were resuspended in MaxCyte electroporation buffer. Meanwhile, RNPs were prepared, followed by mixture with the DNA template. Cells in electroporation buffer were then added and moved into a static processing assembly (CL-1.1). After electroporation, cells were transferred for recovery and then added to culture medium. The reaction conditions were scaled up according to those used in the Lonza 4D electroporation system.
CAR-T cell generation by lentivirus
The CAR sequence was cloned into the pCDH lentiviral vector backbone containing an EF1α promoter. Lentivirus was produced by transfecting 293T cells with CAR plasmid, pMD2.G and psPAX2 using polyethylenimine. Virus-containing supernatants were collected after 3 d to infect primary human T cells stimulated for 2–3 d. LV-19bbz_PD1-KO cells were prepared by lentiviral infection followed by electroporation using recombinant spCas9 (Thermo Fisher) and PD1 sgRNA after 2 d.
Indel percentage analysis
Genomic DNA was obtained using a Genomic DNA Purification kit (Thermo Fisher). Fragments containing indel sites were amplified by PCR using specific primers and purified by TIANgel DNA Purification kit (Tiangen Biotech). DNA sequencing was carried out, and the indel percentage was measured by ICE analysis (Synthego). The primers used were as follows: AAVS1-Forward, 5′-CACCACGTGATGTCCTCTGA-3′; AAVS1-Reverse, 5′-CCGGCCCTGGGAATATAAGG-3′; PD1-Forward, 5′-CCACGTGGATGTGGAGGAAG-3′; PD1-Reverse, 5′-CCACACAGCTCAGGGTAAGG-3′.
Genotyping of PD1-19bbz cells
PD1-19bbz cells were prepared using T cells from two independent healthy donors. Some CAR+ cells were sorted by fluorescence-activated cell sorting (FACS). Genomic DNA was isolated using the Genomic DNA Purification kit (Thermo Fisher). Semiquantitative PCR using three primers in one reaction was carried out. One forward primer (5′-CCCTGCAACTGATGGTGACT-3′) specifically binds to the anti-CD19 CAR cassette. A reverse primer (5′-TCACAGTGTACACAGAGGGC-3′) recognizes a genomic sequence outside the right homology arm. Another forward primer (5′-GACAGTTTCCCTTCCGCTCA-3′) recognizes a genomic sequence within the left homology arm. The intensity ratio of wild-type/indel and CAR-specific bands in unsorted and sorted cells was calculated by densitometry quantification to genotype PD1-19bbz cells, where A was the wild-type/indel percentage in sorted cells, B was the CAR percentage in sorted cells, C was the wild-type/indel percentage in unsorted cells and D was the CAR percentage in unsorted cells. The genotype percentages in samples were calculated with the following equations: wild-type/indel (%) = (B × C – A × D)/B × 100%, heterozygous (%) = 2 × A × D/B × 100% and homozygous (%) = (B – A) × D/B × 100%.
Deep sequencing
Deep sequencing (10,000× coverage) was carried out to detect indels at the PD1 on-target site and 29 top-ranked off-target sites predicted by the Benchling CRISPR tool or to validate possible indels preliminarily indicated by whole-genome sequencing or off-target events at 24 top-ranked potential sites identified by iGUIDE in one representative infusion product (patient 2). Deep sequencing (50,000× coverage) was also performed to validate off-target events at the PHACTR1 site in different infusion products. Genomic DNA from untreated T cells and infusion products was isolated using the Genomic DNA Purification kit (Thermo Fisher). Fragments containing indel sites were amplified by PCR using specific primers and subjected to sequencing on a Hi-TOM platform as described previously46. Deep sequencing was carried out by the XI’AN CyanSnow Gene Company.
Whole-genome sequencing
Genomic DNA from untreated T cells and the infusion product of patient 2 was extracted using the Blood & Cell Culture DNA kit (Qiagen) according to the manufacturer’s instructions and subjected to library construction. Sequencing libraries were generated using the TruSeq Nano DNA HT sample preparation kit (Illumina) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. These libraries including untreated and edited T cells were sequenced on the HiSeq platform (Illumina) with 100× coverage. BWA (Burrows–Wheeler aligner)47 was used to align clean reads for each sample against the reference genome (settings: mem -t 5 -M -R). Alignment files were converted to BAM files using SAMtools48 (settings: -bS -t). In addition, potential PCR duplications were removed using the sambamba command ‘markdup’. If multiple read pairs had identical external coordinates, only the pair with the highest mapping quality was retained. Indels (<50 bp) were calculated and identified with MPILEUP in SAMtools48 and were processed using picard-tools. To reduce the indel detection error rate, we filtered out indels for which the number of supporting reads was less than 4, the quality value (MQ) was less than 30 and QUAL was less than 20. Indels were filtered, with those near other variants and within the pseudoautosomal region (PAR) removed. Whole-genome sequencing was carried out by Novogene Co., Ltd.
We used Cas-OFFinder (http://www.rgenome.net/cas-offinder/) to predict potential off-target sites. Any sequence, followed by an NRG protospacer adjacent motif, having no more than five mismatches (a bulge penalty equals two base mismatches) with the PD1 sgRNA was screened with a total of 2,219 sites (not including those around the on-target site) identified. Indels exclusively detected in the edited sample and located around potential off-target sites were searched. No indel events were found within 15 bp upstream and downstream (±15 bp) of the sites. Indel events were detected within 200 bp upstream and downstream (±200 bp) of eight sites. Deep sequencing with 10,000× coverage was performed to validate these indel events.
iGUIDE
The locations of off-target cleavage sites were mapped using iGUIDE31, an improvement of the GUIDE-seq method32. In brief, stimulated T cells were mixed with RNP (spCas9–PD1 sgRNA) and a protected double-stranded oligodeoxynucleotide (dsODN) and were then subjected to electroporation. Genomic DNA was isolated as described previously after 6 d of cell culture. Next, DNA was cleaved by sonication, adaptors were ligated to free DNA ends and PCR was performed using primers that annealed to the adaptor and dsODN. PCR products were analysed by Illumina sequencing, and reads were mapped to the human genome. Given the presence of a flanking reporter dsODN sequence in correct priming, reads resulting from mispriming could be identified and filtered out. The sequencing data were analysed using the iGUIDE pipeline, available at https://github.com/cnobles/iGUIDE. The gene lists used are available at http://bushmanlab.org/assets/doc/allOnco_Feb2017.tsv and http://bushmanlab.org/assets/doc/humanLymph.tsv.
scRNA-seq
Various CAR-T cells (LV-19bbz, LV-19bbz_PD1-KO, AAVS1-19bbz, PD1-19bbz) were prepared in parallel using T cells from two patients (patients 1 and 2) by different methods and then collected after 9 d. Several CAR-T cell types (LV-19bbz, AAVS1-19bbz, PD1-19bbz) were prepared in parallel using T cells from two healthy donors (D1 and D2) by different methods and immediately collected after 4 h. Fresh PBMCs from patients were collected by apheresis at the peak (day 7 or 12) and stable (day 28 or 29) stages of CAR-T cell expansion after infusion and then isolated by density gradient centrifugation using Ficoll (Sigma-Aldrich). Differently prepared CAR-T cells, infusion products and PBMCs from three patients (patients 1–3) were subjected to scRNA-seq.
scRNA-seq libraries were generated using a 10x Genomics Chromium Controller instrument and Chromium Single-Cell 3′ V3.1 reagent kits (10x Genomics). In brief, cells were concentrated to 1,000 cells per μl and approximately 7,000 cells were loaded into each channel to generate single-cell Gel Bead-In-Emulsions (GEMs), resulting in mRNA barcoding of an expected 5,000 single cells for each sample. After the reverse transcription step, GEMs were broken and barcoded cDNA was purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed, ligated with adaptors and amplified by index PCR. The final libraries were quantified using the Qubit High-Sensitivity DNA assay (Thermo Fisher), and the size distribution of the libraries was determined using a High-Sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). All libraries were sequenced on an Illumina sequencer using a 150-bp paired-end run.
We applied fastp49 with default parameter to filter out the adaptor sequence and remove low-quality reads to achieve clean data. Feature-barcode matrices were then obtained by aligning reads to the human genome (GRCh38 version 91, Ensembl) using CellRanger v3.1.0. We applied downsampled analysis among the samples sequenced according to the mapped barcoded reads for each cell of each sample to achieve the aggregated matrix. Cells with over 200 expressed genes and a mitochondrial unique molecular identifier (UMI) rate below 20% passed the cell quality filtering, and mitochondrial genes were removed from the expression table.
The Seurat package (v.3.1.4; https://satijalab.org/seurat/) was used for cell normalization and regression based on the expression table according to the UMI counts of each sample and the mitochondrial rate to obtain scaled data. Principal-component analysis (PCA) was performed on the basis of the scaled data with the top 2,000 most highly variable genes and the top ten principal components used for t-SNE construction and UMAP construction. Using the graph-based cluster method, we acquired the unsupervised cell cluster result on the basis of the top ten principal components from PCA and calculated the marker genes by the FindAllMarkers function with the Wilcoxon rank-sum test algorithm under the following criteria: (1) ln(fold change) > 0.25; (2) P < 0.05; (3) min.pct > 0.1. To characterize the relative activation of a given gene set such as the KEGG pathway ‘memory, dysfunction and cytotoxicity’ as described previously, we used QuSAGE (v.2.16.1)50 to calculate the score for each cluster/sample and GSVA (v.1.32.0)51 to calculate the score for each cell. GSEA (http://broadinstitute.org/gsea) was used to analyse differentially enriched gene sets between samples. scRNA-seq and data analysis were performed by NovelBio Bio-Pharm Technology Co., Ltd.
Flow cytometry
CAR and membrane protein expression was determined by flow cytometry. Cells were prewashed and incubated with antibodies for 30 min on ice. After washing twice, samples were run on an LSRFortessa (BD Biosciences) or DxFLEX Flow Cytometer (Beckman Coulter) and analysed with FlowJo software. The following antibodies were used: FITC anti-human CD3, APC anti-human CD69, APC anti-human CD137, APC anti-human CD25, APC anti-human PD1, APC anti-human LAG3, APC anti-human TIM3, BV421 anti-human CD45RO, APC anti-human CD62L, APC anti-human CD3, FITC anti-human CD19, FITC anti-human CD4, APC anti-human CD4, APC anti-human CD8, BV421 anti-human CD45 (all from BioLegend), PerCP-Cy5.5 anti-human CD4, BV421 anti-human CD8, PerCP-Cy5.5 anti-human CD45 (all from BD Biosciences) and APC anti-human PD-L1 (Thermo Fisher). For detection of CAR expression, biotinylated human CD19 (amino acids 20–291; ACRO Biosystems) and PE streptavidin (BioLegend) were added sequentially or PE-labelled human CD19 (amino acids 20–291; ACRO Biosystems) was used. For some experiments, CAR-T cells were co-cultured with target cells at an effector/target ratio of 1:1 (Raji cells) or 1:2 (PD-L1-expressing Raji cells) for 24 h before collection. For detection in clinical samples, peripheral blood cells were stained with antibodies, followed by addition of Lysis Buffer (BD Biosciences) before being run. The percentage of CAR+ cells was analysed in CD45+CD3+ gated cells.
CAR copy number analysis by qPCR
Blood samples were collected before and after CAR-T cell infusion. Lysis Buffer (BD Biosciences) was added, and genomic DNA was acquired using the Genomic DNA Purification kit (Thermo Fisher). A seven-point standard curve was generated by using 5 × 100 to 5 × 106 copies per μl of lentiviral vector DNA containing the 19bbz sequence. TaqMan qPCR assays were performed to measure CAR copy number in peripheral blood cells. qPCR was run on a QuantStudio 3 Real-Time PCR System (Thermo Fisher). Each sample was analysed in triplicate. Primers specifically targeting the 19bbz sequence were as follows: forward, 5′-GCTGTAGCTGCCGATTTCCA-3′; reverse, 5′-GGTTCTGGCCCTGCTTGTAC-3′; probe, 5′-AGTGAAGTTCAGCAGGAGCGCAGACG-3′.
Antigen stimulation and proliferation of CAR-T cells
As antigen for stimulation, Raji or PD-L1-expressing Raji cells were pretreated with mitomycin C (50 μg ml–1) for 90 min at 37 °C. CAR-T cells were co-cultured with target cells at an effector/target ratio of 1:1 (Raji cells) or 1:2 (PD-L1-expressing Raji cells) for 3–4 d per stimulation. The number of CAR+ cells was determined by multiplying the total viable cell number and the percentage of CAR+ cells. Cell viability was measured by Trypan blue staining.
CellTrace Violet proliferation assays
AAVS1-19bbz cells were labelled with CellTrace Violet (Thermo Fisher) according to the manufacturer’s instructions. Raji cells were pretreated with mitomycin C (50 μg ml–1) for 90 min at 37 °C. CAR-T cells and target cells were mixed at an effector/target ratio of 1:1. After 5 d, cells were collected and run on an LSRFortessa (BD Biosciences).
Bead-based immunoassays
In preclinical experiments, CAR-T cells were co-cultured with Raji or PD-L1-expressing Raji cells at an effector/target ratio of 1:1 (Raji cells) or 1:2 (PD-L1-expressing Raji cells) in medium without exogenous cytokines. The supernatant was collected after 24 h, and cytokines were measured using LEGENDplex bead-based immunoassays (BioLegend) according to the manufacturer’s instructions.
ELISAs
For in vitro evaluation of infusion products, CAR-T cells were co-cultured with Nalm-6 cells at an effector/target ratio of 1:1 in medium without exogenous cytokines. The supernatant was collected after 18–24 h, and IFNγ secretion was measured using the Human IFN-γ ELISA kit (StemCell) according to the manufacturer’s instructions.
Flow cytometry-based cytotoxicity assays
AAVS1-19bbz cells were co-cultured with Raji cells at an effector/target ratio of 1:1 for 18 h. Flow cytometry was used to detect residual tumour cells by staining with APC anti-human CD3 and FITC anti-human CD19 antibodies. Cells were enumerated using CountBright Absolute Counting Beads (Thermo Fisher) following the manufacturer’s instructions.
LDH cytotoxicity assays
CAR-T cells were co-cultured with Nalm-6, Raji or PD-L1-expressing Raji cells at the indicated effector/target ratios. Cytotoxicity was measured by release of LDH using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer’s instructions.
In vivo mouse experiments
All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (2011) issued by the National Research Council (USA), Laboratory Animal Administration Regulation (2017) issued by the National Science and Technology Committee (China) and the laboratory animal administration regulations (Shanghai, Jiangsu). The care and use of animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the East China Normal University Center for Animal Research or InnoStar Bio-tech Nantong Co., Ltd. For the experiment comparing the LV-19bbz and AAVS1-19bbz groups at a high infusion dose, 6- to 8-week-old B-NDG (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) male mice (Biocytogen) were injected intravenously with 2 × 105 ffLuc-transduced Raji cells. Subsequently, 2 × 106 CAR-T cells were administered intravenously after 5 d. For the experiment comparing the LV-19bbz, AAVS1-19bbz and PD1-19bbz groups at a low infusion dose, 6- to 9-week-old NCG (NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt) female mice (GemPharmatech) were injected intravenously with 2 × 105 ffLuc-transduced Raji cells. Subsequently, 1 × 106 CAR-T cells were administered intravenously after 5 d. For other experiments, 6- to 9-week-old NCG female mice (GemPharmatech) were inoculated intravenously with 5 × 105 ffLuc-transduced PD-L1-expressing Raji cells. Subsequently, 1 × 106 or 2 × 106 CAR-T cells were injected intravenously after 5 d. Bioluminescence images were acquired and analysed using the IVIS Imaging System and software (PerkinElmer). Mice were randomized on the basis of tumour radiance before CAR-T cell injection. Mice were killed according to the experimental protocols or when they met prespecified endpoints defined by the IACUC. Animal technicians were blinded to expected outcomes. The experiments were performed in the East China Normal University Center for Animal Research or InnoStar Bio-tech Nantong Co., Ltd.
Statistics
Experimental data are presented as the mean ± s.d. or the mean ± s.e.m. as described in the figure legends. Data were analysed by one-way ANOVA, two-way ANOVA or log-rank Mantel–Cox test as indicated using GraphPad software. P < 0.05 was considered statistically significant. Asterisks are used to indicate significance: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. NS, not significant.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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