Targeting of intracellular oncoproteins with peptide-centric CARs – Nature

Neuroblastoma samples and cell lines

Five neuroblastoma CDX and three PDX that show a range of HLA expression by RNA sequencing and immunohistochemistry were selected for the initial immunopeptidomics experiment (Extended Data Table 1). All had whole exome sequencing and single nucleotide polymorphism array data available in addition to RNA sequencing¹⁸. Eight high-risk tumours with a mean mass of 0.56 g, ranging from 0.17 to 1.7 g, were obtained from the Children’s Oncology Group (COG; https://childrensoncologygroup.org/) with matched sequencing from Therapeutically Applicable Research To Generate Effective Treatments (TARGET; https://ocg.cancer.gov/programs/target). Informed consent from each research participant or legal guardian was obtained for each deidentified tumour and blood sample used in this study through the COG neuroblastoma biobanking study ANBL00B1.

Human-derived neuroblastoma cell lines, including SKNAS, SKNFI and NBSD, were obtained from the Maris Laboratory cell line bank. Neuroblastoma cell lines were cultured in RPMI supplemented with 10% FBS, 100 U ml^–1 penicillin, 100 µg ml^–1 streptomycin and 2 mM l-glutamine. Other human cancer cell lines, including Jurkat, SW620, HEPG2 and KATO III, were obtained from the American Type Culture Collection (ATCC). Jurkat cells were cultured in IMDM supplemented with 10% FBS, 100 U ml^–1 penicillin, 100 µg ml^–1 streptomycin and 2 mM l-glutamine. SW620 cells were cultured in RPMI supplemented with 10% FBS, 100 U ml^–1 penicillin, 100 µg ml^–1 streptomycin and 2 mM l-glutamine. HEPG2 cells were cultured in EMEM supplemented with 10% FBS, 100 U ml^–1 penicillin, 100 µg ml^–1 streptomycin and 2 mM l-glutamine. KATO III cells were cultured in IMDM supplemented with 20% FBS, 100 U ml^–1 penicillin, 100 µg ml^–1 streptomycin and 2 mM l-glutamine. Packaging cell lines, including platinum-A cells and HEK293T cells were obtained from Cell BioLabs and ATCC, respectively. Both packaging cell lines were cultured in DMEM supplemented with 10% FBS, 100 U ml^–1 penicillin, 100 µg ml^–1 streptomycin and 2 mM l-glutamine. All cell lines were grown under humidified conditions in 5% CO₂ at 37 °C, and samples were regularly tested for mycoplasma contamination.

Primary human T cells

Primary human T cells were obtained from anonymous donors through the Human Immunology Core at the University of Pennsylvania (Philadelphia, Pennsylvania) under a protocol approved by the Children’s Hospital of Philadelphia Institutional Review Board. Cells were cultured using AIM-V (Thermo Fisher Scientific) supplemented with 10% FBS, 100 U ml^–1 penicillin, 100 µg ml^–1 streptomycin and 2 mM l-glutamine under humidified conditions in 5% CO₂ at 37 °C. T cell donors provided informed consent through the University of Pennsylvania Immunology Core.

Isolation of HLA ligands by immunoaffinity purification

HLA class I molecules were isolated using standard immunoaffinity purification methods as previously described^1,2. In brief, cell pellets were lysed in 10 mM CHAPS/PBS (AppliChem/Lonza) containing 1× protease inhibitor (Complete; Roche). Mouse MHC molecules were removed using a 1 h immunoaffinity purification method with H-2K-specific monoclonal antibody 20-8-4S, covalently linked to CNBr-activated sepharose (GE Healthcare). Remaining HLA molecules were purified overnight using the pan-HLA class I-specific monoclonal antibody W6/32 or a mix of the pan-HLA class-II-specific monoclonal antibody Tü39 and the HLA-DR-specific monoclonal antibody L243, covalently linked to CNBr-activated. pMHC complexes were eluted by the repeated addition of 0.2% trifluoroacetic acid (Merck). Elution fractions E1–E4 were pooled, and free MHC ligands were isolated by ultrafiltration using centrifugal filter units (Amicon, Merck Millipore). MHC ligands were extracted and desalted from the filtrate using ZipTip C18 pipette tips (Merck Millipore). Extracted peptides were eluted in 35 µl acetonitrile (Merck)/0.1% trifluoroacetic acid, centrifuged to complete dryness and resuspended in 25 µl 1% acetonitrile/0.05% trifluoroacetic acid. Samples were stored at −20 °C until analysis by LC–MS/MS.

Analysis of HLA ligands by LC–MS/MS

Peptide samples were separated by reverse-phase LC (nanoUHPLC, UltiMate 3000 RSLCnano, Dionex) and subsequently analysed in an online coupled Orbitrap Fusion Lumos (Thermo Fisher Scientific). Samples were analysed in three technical replicates. Sample volumes of 5 µl (sample shares of 20%) were injected onto a 75 µm × 2 cm trapping column (Acclaim PepMap RSLC, Dionex) at 4 µl min^–1 for 5.75 min. Peptide separation was subsequently performed at 50 °C and a flow rate of 300 nl min^–1 on a 50 µm × 25 cm separation column (Acclaim PepMap RSLC, Dionex), applying a gradient ranging from 2.4 to 32.0% acetonitrile over the course of 90 min. Eluting peptides were ionized by nanospray ionization and analysed in a mass spectrometer implementing the TopSpeed method. Survey scans were generated in the Orbitrap at a resolution of 120,000. Precursor ions were isolated in quadrupole, fragmented by collision induced dissociation (CID) in the dual-pressure linear ion trap for MHC class I-purified peptides. Finally, fragment ions were recorded in the Orbitrap. An AGC target of 1.5 × 10⁵ and a maximum injection time of 50 ms was used for MS1. An AGC target of 7 × 10⁴ and a maximum injection time of 150 ms was used for MS2. The collision energy for CID fragmentation was 35%. For fragmentation, mass ranges were limited to 400–650 m/z with charge states 2+ and 3+ for MHC class I.

Synthetic peptides were analysed using a 30-min gradient owing to the simplicity of the sample. Full scans were acquired in the Orbitrap with a scan range of 300–1,200 at 120,000 resolution. The AGC target was 5.0 × 10⁵ with a maximum injection time of 50 ms. Precursor ions were isolated in quadrupole, fragmented (CID, HCD and ETD) and analysed in the Orbitrap. MS2 were also acquired in the Orbitrap with 30,000 resolution, collision energy of 35%, AGC of 5 × 10⁴ and maximum injection time of 150 ms. As the discovery analysis was completed using CID, synthetic peptides fragmented with CID were compared for validation.

The MS proteomics data have been deposited into the ProteomeXchange Consortium through the PRIDE⁴⁰ partner repository with the dataset identifier PXD027182.

HLA typing

FASTQ files from TARGET DNA and RNA sequencing data from cell lines were processed using the PHLAT algorithm, as previously validated on 15 HLA alleles⁸.

Database search and spectral annotation

Data were processed against the human proteome per the Swiss-Prot database (https://www.uniprot.org, release 27 May 2021; 20,395 reviewed protein sequences contained) using the SequestHT algorithm⁴¹ in Proteome Discoverer (v.2.1; Thermo Fisher Scientific) software. Precursor mass tolerance was set to 5 ppm and the fragment mass tolerance to 0.02 Da. The search was not restricted to an enzymatic specificity. Oxidized methionine was allowed as a dynamic modification. FDR was determined using the Percolator algorithm based on processing against a decoy database consisting of shuffled sequences. FDR was set to 1%. Peptide lengths were limited to 8–14 amino acids for MHC class I. HLA annotation was performed using NetMHC-4.0 for HLA class I. For peptide matching, data were reprocessed using Proteome Discoverer (v.2.4; Thermo Fisher Scientific) using the same parameters but with the addition of the feature mapper node to allow peptide matching between samples. Synthetic peptides were searched using a similar approach, but Percolator was replaced with the fixed value PSM validator owing to the simplicity of the synthetic peptide sample. Gene ontology analyses were performed using PANTHER⁴² (http://geneontology.org/), and P values were calculated using Fisher’s exact test.

HLA binding predictions

HLA binding predictions were performed using NetMHC (v.4.0)⁴³, NetMHCpan (v.4.1)⁴⁴ and HLAthena⁴⁵ on HLA class I.

scFv biopanning and CAR design

scFv binders against MHC-presented peptides were retrieved from a large (2 × 10¹⁰) naive phage display scFv library⁴⁶. A competitive panning process was developed to identify specific binders targeting the pMHC complex based on previous protocols⁴⁷. Biotinylated pMHC monomers (target antigens) and non-biotinylated tetramers (decoy competitors) were obtained from the NIH tetramer core facility. A total of 10¹² copies of phages were depleted against magnetic beads (Invitrogen Dynabeads MyOne Streptavidin T1) for 30 min before incubation for 1.5 h with 5 µg biotinylated pMHC-conjugated beads in the presence of 20 µg irrelevant decoy competitors. After incubation, the beads were washed with PBS containing 0.05% Tween-20 (PBST buffer) 5 times followed by 2 PBS washes. The remaining bound phage were recovered by log-phase TG1 and rescued using the M13KO7 helper phage. The amplified phage was collected the next day by PEG–NaCl precipitation and used for the next round of panning. The target antigen input was decreased from 5 µg for the first round of panning to 2 µg and 0.5 µg for the second and third rounds, respectively, and the washing conditions were more stringent along with the panning rounds. After three rounds of panning, polyclonal phage ELISA was performed to evaluate enrichment. TG1 cells from the second and third rounds were randomly spotted into 96-well plates for soluble expression-based monoclonal ELISA as previously described^47,48. Clones producing signals binding to target antigens but not the decoy competitors were amplified and sequenced. For protein preparation, these clones were transformed into HB2151 cells for expression, and proteins were purified by one-step Ni-NTA resin. Protein purity and homogeneity were analysed by SDS–PAGE. Protein concentration was measured spectrophotometrically (NanoVue, GE Healthcare). Second-generation CAR constructs were synthesized using scFv sequences with 4-1BB and CD3ζ co-stimulatory domains and cloned into a pMP71 vector for screening.

ReD library panning

The Ruby scFv library (>10¹¹ diversity) was constructed using fully germline IGLV3-1 and IGLV6-57 scaffolds paired with the IGHV3-23 scaffold, as previously described³³, with fully synthetic amino acid diversity in both V_L and V_H CDR3 loops.

The Ruby library was panned for two rounds using PHOX2B(43–51)–MHC complex bound to MyOne Streptavidin C1 Dynabeads (Thermo Fisher, 65002). Panned library outputs were transferred into the ReD cell-display platform³³ and cells were permeabilized using 0.5% n-octyl β-d-thioglucopyranoside (Anatrace, 0314) and labelled using recombinant PHOX2B pMHC complex ligated to fluorophores excitable by 405 nm and 488 nm lasers. Cells that were positive for target binding were isolated using a FACSMelody sorter (Becton-Dickinson).

After two rounds of positive selection for binding to the PHOX2B–MHC complex, two further FACS rounds were conducted using counter-labelled A*24:02–MHC complexes with unrelated peptides. After four rounds of FACS, individual colonies were picked and grown in 96-well plates before scFv induction, cell permeabilization and PHOX2B–MHC labelling and detection by CytoFLEX (Beckman Coulter).

Clones that were identified as binding specifically to the PHOX2B(43–51)–MHC complex were sequenced, and unique scFvs were expressed as fusions to the AviTag biotinylation motif in Escherichia coli. Biotinylated scFv protein was released through permeabilization with 0.5% n-octyl β-d-thioglucopyranoside and purified to about 90% purity on Nickel NTA agarose resin (ABT, 6BCL-NTANi).

Binding kinetics

Affinity measurements were performed using a BLItz system (ForteBio) and analysed using BLItz Pro software. Streptavidin biosensors (ForteBio, 18–5019) were loaded with AviTag-biotinylated scFv, blocked with biotin, washed in PBS and then associated with pMHC ligand in PBS.

Steady-state binding assay

An equilibrium binding assay to target pMHC complexes was also established using MyOne Streptavidin C1 Dynabeads. In brief, 50 µg of Streptavidin C1 Dynabeads were incubated with excess biotinylated scFv before being blocked with free biotin and washed in PBS. Fluorophore-labelled pMHC complex was added to a concentration of 3.5 nM and incubated for 1 h at 4 °C followed by 10 min at 25 °C. Binding of the free MHC complex to the beads was quantitated using CytoFLEX at 488 nm (excitation) and 525 nm (emission). Binding was normalized to beads without scFv and with unrelated control MHC complex.

This bead-binding assay was used to quantitate the binding of scFv to MHC complexes with alanine-scan substitutions of the PHOX2B peptides and to a plate of 95 unrelated 9-amino-acid peptide A*24:02–MHC complexes. The degree of cross-reactivity of binding of MHC complexes with peptides identified as having high homology to the PHOX2B peptide was analysed using eXpitope 2.0.

Viral production and transduction of Jurkat and primary T cells

Retrovirus for transduction of Jurkat cells and primary CD4/8T cells was produced using platinum-A cells, a retroviral packaging cell line. Cells were plated in 6-well plates at 7 × 10⁵ cells per well and transfected with 2.5 µg of the appropriate TCR or CAR construct in the retroviral vector pMP71 using Lipofectamine 3000 (Life Technologies, Invitrogen). After 24 h, medium was replaced with IMDM-10% FBS or AIM-V-10% FBS for Jurkat cells or primary cells, respectively. Supernatants were collected and filtered through 0.2 μm filters after 24 h of incubation.

A second-generation lentiviral system was used to produce replication-deficient lentivirus. The day preceding transfection, 15 million HEK293T cells were plated in a 15-cm dish. On the day of transfection, 80 µl Lipofectamine 3000 (Life Technologies, Invitrogen) was added to 3.5 ml room-temperature Opti-MEM medium (Gibco). Concurrently, 80 µl P3000 reagent (Thermo Fisher Scientific), 12 µg psPAX2 (Gag/Pol), 6.5 µg pMD2.6 (VSV-G envelope) and a matching molar quantity of transfer plasmid were added to 3.5 ml room-temperature Opti-MEM medium. Virus supernatant was collected after 24 and 48 h and briefly centrifuged at 300g and passed through a 0.45 µm syringe.

Jurkat cells were plated in 6-well plates pre-treated with 1 ml well/retronectin (20 mg ml^–1, Takara) at 1 × 10⁶ cells per well and spinoculated with 2 ml retroviral supernatant at 800g for 30 min at room temperature. After 24 h, cells were collected and grown in IMDM with 10% FBS.

Primary T cells were thawed and activated in culture for 3 days in the presence of 100 U ml^–1 IL-2 and anti-CD3/CD28 beads (Dynabeads, Human T-Activator CD3/CD28, Life Technologies) at a 3:1 bead:T cell ratio. On days 4 and 5, activated cells were plated in 6-well plates pre-treated with 1 ml well/retronectin (20 mg ml^–1, Takara) at 1 × 10⁶ cells per well and spinoculated with 2 ml retroviral supernatant at 2,400 r.p.m. for 2 h at 32 °C. On day 6, cells were collected and washed, beads were magnetically removed, and cells were expanded in AIM-V and 10% FBS supplemented with 25 U ml^–1 IL-2.

Primary human T cells were thawed and activated in culture for 1 day in the presence of 5 ng ml^–1 recombinant IL-7, 5 ng ml^–1 recombinant IL-15 and anti-CD3/CD28 beads (Dynabeads, Human T-Activator CD3/CD28, Life Technologies) at a 3:1 bead:T cell ratio in G-Rex system vessels (Wilson Wolf). On day 2, thawed lentiviral vector was added to cultured T cells with 10 µg ml^–1 polybrene (Millipore Sigma), and 24 h later, vessels were filled with complete AIM-V medium supplemented with indicated concentrations of IL-7 and IL-15. On day 10, cells were collected and washed. Activation beads were magnetically removed, and cell viability was determined before freezing.

Human neuroblastoma cell lines were plated in 6-cm dishes, and 2 ml of thawed lentiviral vector produced with transfer plasmid pLenti-CMV-eGFP-Puro (Addgene, plasmid 17448) was added with 10 µg ml^–1polybrene (Millipore Sigma). Cells were selected for eGFP expression using flow-assisted cell sorting (BD FACSJazz, BD Biosciences) followed by 10 µg ml^–1 puromycin selection.

sCRAP prediction

Tumour antigens were compared against the entire normal human proteome on the matched HLA (85,915,364 total normal peptides among HLA 84 HLAs). Each residue in the same position of the tumour and human peptides was assigned a score for perfect match, similar amino acid classification or different polarity, scoring 5, 2 or –2, respectively (Extended Data Fig. 12). Similarity scores were calculated based on amino acid classification and hydrophobicity was determined using residues one and three until eight and excluding MHC anchor residues. Next, the maximum normal tissue RPKM values were identified from 1,643 normal tissues in GTEx. Normal peptides were compared with a database of normal tissue immunopeptidomes⁴⁹. The overall cross-reactivity score for each normal peptide was then calculated using the following equation:

where n is the peptide length, P is the score of each amino acid of the normal peptide compared to the tumour antigen, b is the pMHC binding affinity of the normal peptide, and E_max is the maximum normal tissue expression. The algorithm is available at https://marisshiny.research.chop.edu/sCRAP.

Tetramer and dextramer staining and flow cytometric analysis

Surface expression and binding of TCR-transduced and CAR-transduced Jurkat cells and primary T cells was measured by staining with PE-conjugated or APC-conjugated dextramers carrying NB antigen pMHC (Immudex). Cells were collected from culture, washed with 2 ml PBS at 800g for 5 min, incubated with 1 µl dextramer for 10 min in the dark, washed again and resuspended in 300 µl PBS for analysis. Typically, 5 × 10⁵ cells were used for staining and analysed on a BD LSR II (BD Biosciences) or an Attune Acoustic Focusing cytometer (Applied Biosystems, Life Technologies). Flow cytometry data were collected using CytExpert (Beckman Coulter) and FACSDiva (BD Biosciences). The gating strategy for all tetramer and dextramer staining is shown in Extended Data Fig. 9f.

Cross-reactivity pMHC screen

Potential cross-reactive peptides (GenScript) were suspended at a 200 µM working concentration. For each test, 0.5 µl of peptide was added to 5 µl HLA-A*24:02 empty loadable tetramer (Tetramer Shop) before incubating on ice for 30 min or using TAPBR peptide exchange as previously described⁵⁰. Following preparation, pMHC tetramers were used to stain cells (described above). CAR construct cross-reactivity values were determined using Jurkat cells transduced with CAR clones followed by staining with HLA-A*24:02 tetramers loaded with cross-reactive peptides. Mean fluorescent intensity was compared across peptides to determine cross-reactivity.

Antigen-specific CD8 T cell enrichment and expansion

Normal donor monocytes were plated on day 1 in 6-well plates at 5 × 10⁶ per well in RPMI-10 FBS supplemented with 10 ng ml^–1 IL-4 (Peprotech) and 800 IU ml^–1 GM-CSF (Peprotech) and incubated at 37 °C overnight. On day 2, fresh medium supplemented with 10 ng ml^–1 IL-4 and 1,600 IU ml^–1 GM-CSF was added to the monocytes and incubated at 37 °C for another 48 h. On day 4, non-adherent cells were removed, and immature dendritic cells washed and pulsed with 5 µM peptide in AIM-V-10% FBS supplemented with 10 ng ml^–1 IL-4, 800 IU ml^–1 GM-CSF, 10 ng ml^–1 LPS (Sigma-Aldrich) and 100 IU ml^–1 IFNγ (Peprotech) at 37 °C overnight. Day 1 was repeated on days 4 and 8 to generate dendritic cells for the second and third stimulations on days 8 and 12, respectively.

On day 5, normal donor-matched CD8⁺ T cells were enriched using the protein kinase inhibitor dasatinib (Sigma-Aldrich), dextramers and anti-PE or anti-APC beads (Miltenyi Biotec) as previously described⁵¹. Enriched T cells were co-incubated with the appropriate pulsed dendritic cells in AIM-V-10% FBS. Day 5 protocol was repeated on day 8 and day 12 using dendritic cells generated on days 4 and 8 for the second and third stimulation, respectively. Expanded T cells were validated for antigen-specificity by staining with the appropriate dextramers and for activation marker 41BB/CD137 (BioLegend).

Antigen-specific T cell sorting, sequencing and cloning

Expanded T cells were stained with CD3, CD8, CD14, CD19, live/dead, and matched and mismatched dextramer, and single-antigen-specific T cells were sorted using a FACSAria Fusion (BD Biosciences).

Sorted cells were loaded onto 10x Genomics 5′ V(D)J chips and libraries prepared according to manufacturer’s protocols. TCRα/β amplicons were run on MiSeq using 5,000 reads per cell. Sequencing data were processed using Cell Ranger and analysed using Loupe VDJ Browser. TCRα and β chains were codon optimized and synthesized into bicistronic expression cassettes using engineered cysteine residues in the TCR constant domains, using F2A ribosomal skip sites and furin cleavage sites⁵². TCR cassettes were cloned into pMP71 retroviral vector.

Incucyte cytotoxicity assay

A total of 0.5 × 10⁵ tumour cell targets were incubated with varying ratios of transduced primary cells (5 × 10⁵, 2.5 × 10⁵, 1 × 10⁵, 0.5 × 10⁵ and 2.5 × 10⁴ for 10:1, 5:1, 2:1, 1:1 and 1:2 effector-to-target ratios, respectively) in 96-well plates at 37 °C in the presence of 0.05 µM caspase-3/7 red (Incucyte, Essence BioScience). Plates were run on an Incucyte Zoom or S3 for 24–72 h and measured for apoptosis activity through caspase cleavage and comparison of relative confluency. Following the assay, supernatants were collected for ELISA. Total GFP integrated intensity (total GCU × μm² per image) was assessed as a quantitative measure of live, GFP⁺ tumour cells. Values were normalized to the t = 0 measurement.

Cytokine secretion assays

Cell supernatant collected from cell cytotoxicity assays was thawed and plated in triplicate for each condition. IFNγ and IL-2 levels were determined using ELISA kits according to the manufacturer’s protocol (BioLegend).

Antigen processing and presentation

Neuroblastoma cell lines were titrated with H1N5 influenza virus and infectivity was measured by flow cytometry using virus nucleoprotein (NP) antibody. HLA-A2 neuroblastoma cell lines were cultured with either 5 μM CEF1 or 50 HAU of H1N5 virus then cultured with M1 antigen-specific T cell hybridoma provided by D. Canaday⁵³. T cell activation was measured using IL-2 ELISA (Abcam).

Expression, refolding and purification of recombinant pHLA molecules

HLA-A*02:01, HLA-A*24:02:01, HLA-A*23:01, HLA-B*14:02 and HLA-C*07:02 constructs for bacterial expression were cloned into pET24a+ plasmids. DNA plasmids encoding HLA heavy chain and human β2M (light chain) were transformed into E. coli BL21-DE3 (Novagen), expressed as inclusion bodies and refolded using previously described methods⁵⁴. E. coli cells were grown in autoinduction medium for (16–18 h)⁵⁵. Afterwards, the E. coli cells were collected by centrifugation and resuspended with 25 ml BugBuster (Milipore Sigma) per litre of culture. The cell lysate was sonicated and subsequently pelleted by centrifugation (5,180g for 20 min at 4 °C) to collect inclusion bodies. The inclusion bodies were washed with 25 ml of wash buffer (100 mM Tris pH 8.0, 2 mM EDTA and 0.01% v/v deoxycholate), sonicated and pelleted by centrifugation. A second wash was done using 25 ml Tris-EDTA buffer (100 mM Tris pH 8.0 and 2 mM EDTA). The solution was once again resuspended by sonication then centrifuged. The inclusion bodies were then solubilized by resuspending in 6 ml of resuspension buffer (100 mM Tris pH 8.0, 2 mM EDTA, 0.1 mM DTT and 6 M guanidine-HCl). Solubilized inclusion bodies of the heavy and light chain were mixed in a 1:3 molar ratio and then added dropwise over 2 days to 1 litre of refolding buffer (100 mM Tris pH 8.0, 2 mM EDTA, 0.4 M arginine-HCl, 4.9 mM l-glutathione reduced, and 0.57 mM l-glutathione oxidized) containing 10 mg of synthetic peptide at >98% purity confirmed by MS (Genscript). Refolding was allowed to proceed for 4 days at 4 °C without stirring. Following this incubation period, the refolding mixture was dialysed into size-exclusion buffer (25 mM Tris pH 8.0 and 150 mM NaCl). After dialysis, the sample was concentrated first using a Labscale Tangential Flow Filtration system and then using an Amicon Ultra-15 Centrifugal 10 kDa MWCO Filter Unit (Millipore Sigma) to a final volume of 5 ml. Purification was performed using size-exclusion chromatography on a HiLoad 16/600 Superdex 75 column. The purified protein was exhaustively exchanged into 20 mM sodium phosphate pH 7.2 and 50 mM NaCl. The final sample was validated using SDS–PAGE to confirm the formation of a pMHC complex containing both the heavy and light chains.

Differential scanning fluorimetry

To measure the thermal stability of the pMHC class I molecules, 2.5 μM protein was mixed with 10× Sypro Orange dye in matched buffer (20 mM sodium phosphate pH 7.2, 100 mM NaCl) in MicroAmp Fast 96well plates (Applied Biosystems) at a final volume of 50 μl. Differential scanning fluorimetry was performed using an Applied Biosystems ViiA qPCR machine with excitation and emission wavelengths at 470 nm and 569 nm, respectively. Thermal stability was measured by increasing the temperature from 25 °C to 95 °C at a scan rate of 1 °C min^–1. Melting temperatures (T_m) were calculated in GraphPad Prism 7 by plotting the first derivative of each melt curve and taking the peak as the T_m.

Protein crystallization

Purified HLA-A*02:01–LLLPLLPPL, HLA-A*02:01–LLPLLPPLSP, HLA-A*02:01–LLPLLPPLSPS, HLA-A*02:01–LLPRLPPL and HLA-A*24:02–QYNPIRTTF complexes were used for crystallization. Proteins were concentrated to 10–12 mg ml^–1 in 50 mM NaCl, 25 mM Tris pH 8.0, and crystal trays were set up using a 1:1 protein-to-buffer ratio at room temperature. Optimal crystals for HLA-A*02:01–LLLPLLPPL, HLA-A*02:01–LLPLLPPLSP and HLA-A*02:01–LLPLLPPLSPS were obtained with 1 M sodium citrate dibasic and 0.1 M sodium cacodylate pH 6.5. For HLA-A*02:01–LLPRLPPL, diffracting crystals were obtained with 0.2 M magnesium chloride, 0.1 M HEPES pH 7.0 and 20% PEG 6000. HLA-A*24:02–QYNPIRTTF diffracting crystals were obtained with 0.1 M HEPES pH 7.0 and 10% PEG 6000. Diffraction-quality crystals were collected and incubated from the above conditions plus glycerol as a cryoprotectant and flash-frozen in liquid nitrogen before data collection. All crystals used in this study were grown using the hanging drop vapour diffusion method. Data were collected from single crystals under cryogenic conditions at Advanced Light Source (beam lines 8.3.1 and 5.0.1). Diffraction images were indexed, integrated and scaled using MOSFLM and Scala in CCP4 Package⁵⁶. Structures were determined by Phaser⁵⁷ using previously published structures of HLA-A*02:01 (PDB identifier 5C07)⁵⁸ and HLA-A*24:02 (PDB identifier 3VXN)⁵⁹. Model building and refinement were performed using COOT⁶⁰ and Phenix⁶¹, respectively.

Homology modelling of pHLA complexes using RosettaMHC

Three-dimensional structural models of HLA-A*23:01 and HLA-C*07:02 bound to the peptide QYNPIRTTF were generated using RosettaMHC, an in-house method for modelling the α₁/α₂ peptide binding domains of pMHC class I molecules³⁶. In brief, the amino acid sequences of HLA-A*23:01 and HLA-C*07:02 were first obtained from the IPD-IMGT/HLA Database⁶². The sequence of HLA alleles of interest was aligned against the sequences of 318 HLA curated template structures available in RosettaMHC. For each allele, all candidate templates were selected according to a 70% sequence identity criterion between aligned residues within the peptide-binding groove (within 3.5 Å of any peptide heavy atom). Generation of 3D models was performed using a Monte Carlo sampling of sidechain rotamer conformations, followed by gradient-based optimization of all backbone and side chain degrees of freedom. For each pHLA complex, the top five models with the lowest Rosetta binding energy were selected as the final structural ensemble. The quality of the final models was assessed using the Molprobity webserver⁶³. Analysis of polar contacts and surface area were performed using the PyMOL Molecular Graphics System (v.2.4.1).

Immunohistochemistry

CD3 (Dako A0452), PHOX2B (Abcam ab183741) and HLA-ABC (Abcam ab70328) antibodies were used to stain formalin-fixed paraffin-embedded tissue slides. Staining was performed on a Bond Max automated staining system (Leica Biosystems). A Bond Refine polymer staining kit (Leica Biosystems, DS9800) was used. The standard protocol was followed with the exception of the primary antibody incubation, which was extended to 1 h at room temperature. CD3, PHOX2B and HLA-ABC antibodies were used at 1:100, 1:500 and 1:1,200 dilutions, respectively. Antigen retrieval was performed with E1 (Leica Biosystems) retrieval solution for 20 min (E2 for PHOX2B). Slides were rinsed, dehydrated through a series of ascending concentrations of ethanol and xylene, then coverslipped. Stained slides were then digitally scanned at ×20 magnification on an Aperio CS-O slide scanner (Leica Biosystems).

Mouse PC-CAR T cell preclinical trials

NOD SCID Gamma (NSG) female (6–8 weeks of age) mice from Jackson Laboratory (stock number 005557) were used to propagate subcutaneous xenografts. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the Institutional Animal Care and Use Committee at the Children’s Hospital of Philadelphia. Treatment was initiated through lateral tail intravenous injection. The dose administered was 100 µl per animal of vehicle or CAR T cells as a single treatment. Treatment was administered at weeks 8–10 when tumour volumes reached 150–250 mm³. Six mice were enrolled per group based on previous experience and randomized based on tumour size. The mouse technician was blinded to T cell engineering. Tumour volume and survival were monitored through bi-weekly measurements until the tumours reached a size of 2.0 cm³ or mice showed signs of graft versus host disease (GVHD). Animals were removed from study and studies terminated following onset of GVHD when animals display hunched posture, rapid breathing, urine staining, weight loss and a body condition score of 2, as determined by visual inspection. Onset of GVHD defined as urine staining and weight loss of 20% or weight loss of 10–15% if accompanied by hunched posture, laboured breathing or poor body condition.

Statistics and reproducibility

Box and whisker plot representations of data show the median as centre, 25th percentile and 75th percentile as bounds of boxes for plots shown in Fig. 1e and Extended Data Figs. 1 and 13b.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.