Mice and cell lines

C57BL/6J, BALB/c, NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl)/SzJ (NSG) (female, aged 6–8 weeks) and humanized CD34+ NSG (Hu-NSG-CD34, female, aged 16 weeks) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and the Center for Comparative Medicine (CCM) of Baylor College of Medicine. The mice were housed in the animal facilities of the CCM under pathogen-free conditions. All procedures were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine.

HEK293T (Human embryonic kidney cells), RPMI-8226 (Human plasmacytoma cells with p53 gene harboring the E285K mutation), and BT-474 (Human breast ductal carcinoma cells with p53 gene harboring the E285K mutation) were purchased from the American Type Culture Collection (ATCC). The Expi293 cell line was obtained from Thermo Fisher Scientific. The mouse colon cancer MC38-p53KO/E285K cell line, characterized by stable overexpression of human p53E285K after endogenous p53 knockout (p53KO), was generated by knocking out the endogenous p53 mutant alleles (G242V & S238I) and introducing the human p53 gene with the E285K mutation. Expi293 cells, HEK293T, MC38, MC38KO, and MC38-p53KO/E285K were cultured as described previously [21]. RPMI-8226 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS and 1×anti-anti solution. BT-474 cells were cultured in Hybri-Care Medium (ATCC, 46-X) supplemented with 1.5 g/L sodium bicarbonate, 10% FBS, and 1×Antibiotic-Antimycotic solution (Gibco).

Antibody construction

Mouse antibodies against p53E285K were prepared using a previously reported method [18, 19] to obtain hybridomas. Mouse mAbs from hybridoma clones were sequenced by Syd Labs, Inc. (Hopkinton, MA) to obtain sequences coding for the heavy-chain (HL) and light-chain (LC). The HC and LC of the mouse mAbs were fused to the Fc of human IgG1 to the E285K-mAb IgG1. The expression cassettes were cloned into either pTwist (Twist Bioscience, South San Francisco, CA, USA) or gWIZ (Aldevron, Fargo, ND, USA) mammalian expression vectors. Three gWIZ constructs were made for dIgA: i.e. VH sequences, followed by mouse IgA constant region sequence, VL sequence along with mouse kappa light chain constant region sequences, and mouse J-chain (JC) sequence. The Fab region of dIgA is identical to that of IgG1 E285K-mAb. All plasmids were purified from DH5α cells, utilizing an endotoxin-free ZymoPURE™ II Plasmid Maxiprep Kit (Zymo Research, Irvine, CA, USA).

Antibody expression and purification

Antibody expression was performed using the ExpiFectamineTM 293 Transfection Kit (Gibco). For antibody purification, a NAb Protein G Spin Column Kit (Thermo Scientific, Waltham, MA, USA) was used according to the manufacturer’s protocol. The purified samples were dialyzed against PBS overnight at 4 ℃ using a Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientific). Subsequent analysis involved SDS-PAGE, and quantification was conducted on the samples using the Pierce BCA Protein Assay Kit (Thermo Scientific).

Antibody affinity detection using BLI

The interaction and specificity of His-tagged TrxA-E285K peptides and E285K-mAbs were assessed through Bio-Layer Interferometry (BLI) (Gator Bio, Palo Alto, CA, USA). Both antigens and antibodies were exchanged into Q Buffer (PBS with 0.02% Tween-20, 0.2% BSA, and 0.05% NaN3, pH 7.4). The TrxA-E285K-peptide (analyte), TrxA-R179H peptide (negative control), and purified E285K-mAb were diluted in Q buffer and loaded onto the Ni-NTA sensor chip. To initiate the experiment, the sensor was hydrated in 200 µL Q buffer for 10 min and then exposed to 250 µL Q buffer to establish an initial baseline reading. The sensor was then immersed with the E285K-mAbs for 120 s. After loading, the sensor was exposed to 200 µL antigen for 30 s to obtain another baseline measurement, followed by 120 s exposure to record an association curve. Finally, the sensor was exposed to 250 µL Q buffer to measure dissociation. Following each cycle, the sensor was regenerated using a Gly-HCl (pH 1.5) regeneration buffer. The collected data were reference-subtracted and fitted to a 1:1 binding model (Rmax global fit) using Gator Data Analysis Software (Gator Bio).

Western blot

Western blotting was performed using the primary antibody E285K-mAb, anti-WT p53-mAb (DO-1, Santa Cruz Biotechnology, sc-126) or anti-TRIM2-mAb (Abcam, ab207728) at 4 °C overnight. This was succeeded by the secondary anti-mouse or anti-rabbit IgG HRP-conjugated antibodies (Cell Signaling Technology, 7076 V, 7074 S). The visualization was done using ECL on Chemidoc (Biorad).

ELISA

Plates were coated with p53-E285K antigen dissolved in coated buffer (R&D Systems, Minneapolis, MN, USA) overnight at 4 °C. The plates were washed with PBST (pH 7.4) containing 0.05% (v/v) Tween-20 and then blocked with 3% BSA for 1 h. The sample was then added and incubated at room temperature for 2 h. Binding was detected using an HRP-conjugated secondary antibody (Cell Signaling Technology). The reaction was developed using a TMB substrate (R&D Systems) and stopped with 2 N H2SO4. Finally, absorbance was measured at 450 nm using a plate reader (CLARIOstar, BMG Labtech, USA).

Nucleic acid encapsulation

LNPs were formulated using a NanoAssemblr Spark Formulation Device (Precision Nanosystems). Briefly, antibody-expressing plasmids (40 µg IgG1 containing HC and LC in a ratio of 1:1; 40 µg dIgA containing HC, LC, and JC in a ratio of 1:1:1) or siRNA (Trim21 siRNA, Thermo Fisher Scientific, ID# 150,993; Pigr siRNA, Thermo Fisher Scientific, ID# 151,101; Control siRNA, Thermo Fisher Scientific, AM4635) were diluted in 80 µl of 25 mM citrate buffer, pH 3.5 (bioWORLD, 40320053-1). Cationic lipid SM-102 (Cayman Chemical, 33,474), Cholesterol (Sigma-Aldrich, C3045), Phospholipid DSPC (Sigma-Aldrich Lipids, P1138) and Pegylated lipid DMG-PEG 2000 (Avanti Polar Lipids, 880,151) were diluted in 100% ethanol (Fisher Scientific, A4094) at molar ratio 51/38/8/3 in 40 µl. The lipids were dissolved in ethanol, and two volumes of nucleic acids were added to the buffer. Both phases were loaded into the NanoAssemblr Spark Cartridge (Precision Nanosystems, NIS0013) with a cap, and microfluidic mixing was performed using the recommended setting no. 9. The lipid products were subsequently purified by dialyzing against Ca2+ and Mg2+ free PBS at pH 7.4 using a Pur-A-Lyzer Maxi Dialysis Kit (0.1–3 mL, MWCO 6–8 kDa, Sigma-Aldrich, PURX60005) overnight and concentrated by 50 KDa Amicon Ultra-0.5 mL Centrifugal Filters (Merck Millipore, UFC505024) to a final pDNA concentration of 0.8 mg/mL. The mean diameter of the LNP after sonication was determined by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments Inc., Westborough, MA). The LNP-DNA or LNP-siRNA encapsulated efficiency was quantified with a Quant-iT Pico-Green dsDNA assay kit (Thermo Fisher Scientific, P11496) or Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific, R11490) according to the instructions. Before injection, the LNPs were briefly sonicated (3s for 3 times).

Cytotoxicity assay

Peripheral blood mononuclear cells (PBMCs) were prepared from human buffy coats (Gulf Coast Regional Blood Center, Houston, TX, USA) using Ficoll-Hypaque (MilliporeSigma, Chicago, IL, USA). Tumor cells (1 × 104) were incubated with E285K-mAb (10 µg/ml) for 30 min at 4°C, or cultured with LNP-pE285K-mAb (5 µg/ml) for 24 h at 37 °C. The treated tumor cells were then co-cultured with PBMCs with a ratio of 1: 50 in 96-well plates (Corning, NY, USA) at 37 °C for 72 h. Cytotoxicity was determined by measuring the amount of lactate dehydrogenase (LDH) in the supernatant using the Cytotoxicity Detection Kit PLUS (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions.

Flow cytometry assessments

Apoptosis in MC38-p53KO/E285K, RPMI-8226, and BT-474 cells was analyzed using the APC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, 640,930) according to the manufacturer’s instructions. The surface and intracellular expression of p53E285K in MC38-p53KO or MC38-p53KO/E285K cells was evaluated by surface or intracellular staining using anti-p53-E285K-mAb and Alexa Fluor 647-conjugated goat anti-mouse IgG (H + L) secondary antibody (Invitrogen, A-21,235).

The tumor tissues were excised and minced into approximately 1 mm3 cubic pieces. They were then digested using a mouse Tumor Dissociation Kit (Miltenyi Biotec) and incubated on a rocker (Gentle MACS Octo 8, Miltenyi Biotec) at 37 °C for 25–40 min. The resulting digested cells were filtered through 70-µm cell strainers (BD Pharmingen) and washed twice with cold PBS containing 2% FBS. The total isolated cells were counted using a cell counter, and some cells were stained and detected by flow cytometry (FACS) to obtain the percentage of immune subsets after blocking Fc receptors and removing dead cells with a Zombie Aqua Fixable Viability Kit (BioLegend). Cell surface staining was performed by incubating with the following antibodies for 30 min at 4 °C, followed by intracellular staining. All data were obtained on a Cytek® NL-3000 FACS system (Cytek Biosciences) and analyzed using FlowJo V10 (BD Biosciences). The antibodies used in this study are as follows: anti-mouse CD16/32 (clone 93, BioLegend), Human TruStain FcX™ (BioLegend, 422,302), APC anti-human IgG Fc (clone M1310G05, BioLegend), Brilliant Violet 421™ anti-human CD45 (clone HI30, BioLegend), PE anti-human IFN-γ (clone W19227A, BioLegend), APC/Cy7 anti-mouse CD45 (clone 30-F11, BioLegend), Brilliant Violet 750™-conjugated anti-mouse CD45 (clone 30-F11, BioLegend), Percp/cy5.5 anti-mouse CD19 (clone 1D3/CD19, BioLegend), PE anti-mouse CD3 (clone 17A2, BioLegend), Pacific Blue™ anti-mouse CD4 (clone RM4-5, BioLegend), PerCP/Cyanine5.5 anti-mouse CD8a (clone 53 − 6.7, BioLegend), APC anti-mouse NK-1.1 (clone S17016D, BioLegend), APC-conjugated anti-mouse IFN-γ (clone XMG1.2, 505,810), FITC-conjugated anti-mouse TNF-α (clone MP6-XT22, Biolegend), PE-conjugated anti-mouse IL-2 (JES6-5H4, Biolegend), APC anti-mouse CD11c (clone N418, BioLegend), FITC anti-mouse/human CD11b (clone M1/70, BioLegend), PE anti-mouse CD103 (clone 2E7, BioLegend), PerCP anti-mouse F4/80 (clone BM8, BioLegend), Alexa Fluor® 647 anti-mouse FOXP3 (clone MF-14, BioLegend), FITC anti-mouse CD107a (clone 1D4B, BioLegend), PE anti-mouse/human CD44 (clone IM7, BioLegend), FITC anti-mouse CD62L (clone MEL-14, BioLegend), PE anti-mouse CD80 (clone 16-10A1, BioLegend), FITC anti-mouse CD86 (clone GL-1, BioLegend), Pacific Blue™ anti-mouse I-A/I-E (clone M5/114.15.2, BioLegend), PerCP/Cyanine5.5 anti-mouse H-2Kd/H-2Dd (clone 34-1-2 S, BioLegend).

Flow PLA

The Proximity Ligation Assay (PLA) was conducted following the Duolink PLA FACS protocol with modification using the Duolink flowPLA Detection Kit–Voilet (Sigma-Aldrich, DUO94005). MC38-p53KO/E285K cells were washed, fixed, and permeabilized. Following three PBS washes, samples were blocked with Duolink Blocking Solution (Sigma-Aldrich, DUO82007) for 1 h at 37 °C. Primary antibodies, including the purified E285K-mAb and TRIM21-mAb (Abcam, ab207728), were added at 5 µg/mL in antibody dilution solution at 37 °C for 1 h. After washing twice with PBS, samples were incubated with the Duolink In Situ PLA Probe Anti-Mouse MINUS (Sigma-Aldrich, DUO92001) and Duolink In Situ PLA Probe Anti-Rabbit PLUS (Sigma-Aldrich, DUO92005) in PLA antibody diluent (MilliporeSigma, DUO82008) for 1 h at 37 °C. Following another PBS wash, ligase (1:40 in Duolink Ligation buffer) was added to the cells and incubated for 30 min at 37 °C to facilitate ligation. After washing twice with Duolink Wash Buffer A, the cells were incubated with DNA polymerase (1:80 in amplification buffer) overnight at 37 °C. Following two washes with Duolink Wash Buffer, the cells were incubated for 30 min at 37 °C with 1× flowPLA Detection Solution. After two additional washes with Duolink Wash Buffer B, the cells were subjected to measurement using the Cytek® Northern Lights cytometer (Cytek Biosciences).

Animal experiments

A syngeneic mouse colon cancer model was established by subcutaneous inoculation of MC38-p53KO/E285K cells (3 × 105 cells/mouse). The LNP-pE285K-mAb (IgG1 or IgA) or control plasmid (40 µg/mouse) was administered intratumorally at specified time points. Meanwhile, combination therapy with 250 µg/mouse αPD-1 (BioXcell, BE0146) or 500 µg/mouse αCD4 (BE0119), αCD8 (BP0117), αNK1.1(BE0036), and αCD19 (BE0150) was intraperitoneally administered after intratumor injection. For the lung metastasis model, 1 × 106 MC38-p53KO/E285K cells were injected into the tail vein on day 0. The mice were given an intravenous injection of the same amount of LNP-pE285K-mAb (40 µg/mouse) on day 7 and repeated treatment on day 12 post tumor inoculation. On day 22 post tumor inoculation, mice were sacrificed, and the lungs were surgically excised. Metastatic nodules in the lung tissue were quantified by observing histological sections of the entire transverse plane of lung tumors in each mouse and calculating the number of tumor metastatic foci. To establish intestinal tumors, the mice were injected intraperitoneally with 2 × 106 MC38-p53KO/E285K cells suspended in 100 µl of PBS. The mice were then intraperitoneally treated with LNP-pE285K-mAb on days 7 and 14. Tumor growth was monitored, and animal survival was evaluated. To calculate the tumor inhibition rate, the following formula was used: (C-T)/C×100%; T is the tumor weight from each animal, and C is the weight of the largest tumor from the control group.

NSG mice were subcutaneously inoculated with RPMI-8226 cells or BT-474 (5 × 106 cells/mouse) resuspended in a mixture of serum-free medium and Matrigel (Corning, 354,230) at a 1:1 volume ratio into the right flank. On day 10 post-tumor inoculation, 1 × 107 PBMCs were administered intravenously to each mouse. Fourteen days post tumor inoculation, 5 mice were randomly grouped and treated with either an intratumor injection of LNP-pE285K-mAb or LNP-Ctrl (40 µg/mouse) on days 14, 19 and 24. Similarly, Hu-NSG-CD34 mice were subcutaneously inoculated with BT-474 (5 × 106 cells/mouse) and intratumorally treated on days 14 and 21 post tumor inoculation. Humane endpoints were defined as tumor length reaching 1.5 cm, tumor burden equal to or greater than 10% of the normal body weight, or severe tumor necrosis. The tumor burden and mouse weight were measured, and tumor volume was calculated using the following equation: V = (length × width2)/2. All procedures were performed with the approval of the Institutional Animal Care and Use Committee of the Baylor College of Medicine.

scRNA-seq sequencing

Subcutaneous tumors were harvested 14 days after a single-dose treatment, excised, and minced in RPMI-1640 medium supplemented with 10% FBS. The dissected tumor samples were dissociated into single-cell suspension using a mouse Tumor Dissociation Kit (Miltenyi Biotech, 130-096-730) with a gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech). Cells were collected by using a 40 μm cell strainer (Corning, 431,750), centrifuged at 300 g for 10 min, and lysis red cells using ACK buffer, and resuspended in RPMI1640with 5% FBS. Single-cell suspensions were refined by excluding dead cells using Zombie Green viability dye, and Fc receptors were blocked before the addition of APC-Cy7-conjugated anti-mouse CD45 antibody. Subsequently, CD45+ cells were sorted from each tumor sample using a FACSAria II instrument. Equivalent numbers of sorted CD45+ cells from five mice per group were combined, concentrated by centrifugation, and resuspended at a density of 1 × 103 cells/µl in RPMI1640 medium containing 10% FBS. Approximately 10,000 cells were allocated for the 10 × Genomics 5′ v2 single-cell assay for both experimental groups. TCR/BCR libraries were prepared using the Chromium™ single cell V(D)J enrichment kit (10× Genomics). Libraries were prepared according to the manufacturer’s protocol at the Single Cell Genomics Core at Baylor College of Medicine (BCM). BCM Genomic and RNA Profiling (GARP) Core sequenced the libraries on NovaSeq 6000.

Processing of scRNA seq data

Raw sequence reads in the FASTQ formats were aligned to the mouse reference genome using CellRanger Count v7.1.0 pipeline (https://cloud.10xgenomics.com) with the default settings for alignment, barcode assignment, and UMI counting of the raw sequencing data with the genome reference Mouse (mm10) 2020-A. BCR and TCR raw gene expression matrices were generated using the CellRanger count pipeline with default parameters and mouse GRCm38/mm10 as the reference genome.

Doublet cells were eliminated using the R package scDblFinder(1.14.0) [29], and additional processing was performed using the R package Seurat (v4.4.0) [30]. Filtered cells had > 10% mitochondrial, nFeature RNA > 7500, or < 200 identified genes. After log-normalization, the highly variable genes were determined using the “vst” selection method in Seurat and the FindVariableFeatures tool. The ScaleData function in Seurat was used to scale the data. The RunHarmony function from the R package harmony (1.01) [31] combined multiple data after PCA analysis. Neighbors and clusters were defined using Seurat’s FindNeighbors and FindClusters function and a resolution of 0.5. RunUMAP was used to calculate the UMAP dimensionality reduction with default parameters utilizing the top 30 principal components (PCs) and harmony reduction. For the analysis focusing on T cells, B cells, NK cells, and NK-like cells, which are subsets of the annotation cell types, data were re-run from the NormalizeData function following the Seurat pipeline. To assist with annotating the T cell subcluster, we utilize the R packages STACAS (2.1.3) [32] and ProjecTILs (3.2.0) [33].

Differentially expressed genes (DEGs) and enrichment analysis

DEGs between clusters were obtained with a Wilcoxon followed by Bonferroni correction using the FindAllMarkers function in Seurat. For GO and KEGG enrichment analysis, Clusterprofiler (4.8.3) [34] was used to examine DEGs in cell clusters. The AddModuleScore function was used to evaluate the gene set score of T cell transcription factors, memory markers, cytotoxic markers, and checkpoint markers. To plot specific genes or gene set scores of interest, the FeaturePlot function in Seurat and the plot_density function in Nebulosa (1.10.0) [35] were used to show the genes or gene set score distribution on cells. UMAP and heatmap plot modification was done using scRNAtoolVis (0.0.7).

Processing of scVDJ seq data

scVDJ and scRNA seq analysis were performed using the Seurat and scRepertoire (1.11.0) [36] packages. In brief, VDJ sequences were extracted using the combine TCR or combine BCR function in scRepertoire. We used the Python module CoNGA to calculate and show the TCR motifs within TRAV and TRAB from T cells subclusters.

Pseudotime analysis

Pseudotime analysis was performed via two different methods. R package FitDevo (1.2.1) [37] was used to analyze the developmental trajectories of T, B, NK, and NK-like cells and to infer the CD8+ and CD4+ T cell starting cell populations through global cell differentiation. R package Slingshot (2.8.0) [38] was used to infer the differentiation paths of CD8+ T cells and CD4+ T cells. Combining the UMAP dimensionality reduction results in Seurat and the starting trajectory of FitDevo analysis, we used the slingPseudotime function to predict the cell differentiation path and the potential developmental pathways in each cell group.

Receptor–ligand interaction analysis

The CellChatR toolkit (2.2) [39] was used to explore cellular interactions different between control and treatment groups. Communication probabilities were calculated based on the CellChatDB database of literature and PPI-supported ligand–receptor interactions in mouse datasets. First, we filtered the incoming and outgoing parameters for each sample using the selectK function before aggregating the data from the two cell groups using the mergeCellChat function. The Cellchat protocol “Comparison analysis of multiple datasets using CellChat” was used to analyze the merged data items.

Statistical analyses

Unless otherwise stated, data were expressed as the mean ± standard deviation (SD). Two groups were compared using a two-tailed Student’s t-test. GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Values of p < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).