Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2

Background Cancer immunotherapy with monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies can benefit from selection of new targets with high levels of tumor specificity and from early assessments of efficacy and safety to derisk potential therapies. Methods Employing mass spectrometry, bioinformatics, immuno-mass spectrometry and CRISPR/Cas9 we identified the target of the tumor-specific SF-25 antibody. We engineered IgE and CAR T cell immunotherapies derived from the SF-25 clone and evaluated potential for cancer therapy. Results We identified the target of the SF-25 clone as the tumor-associated antigen SLC3A2, a cell surface protein with key roles in cancer metabolism. We generated IgE monoclonal antibody, and CAR T cell immunotherapies each recognizing SLC3A2. In concordance with preclinical and, more recently, clinical findings with the first-in-class IgE antibody MOv18 (recognizing the tumor-associated antigen Folate Receptor alpha), SF-25 IgE potentiated Fc-mediated effector functions against cancer cells in vitro and restricted human tumor xenograft growth in mice engrafted with human effector cells. The antibody did not trigger basophil activation in cancer patient blood ex vivo, suggesting failure to induce type I hypersensitivity, and supporting safe therapeutic administration. SLC3A2-specific CAR T cells demonstrated cytotoxicity against tumor cells, stimulated interferon-γ and interleukin-2 production in vitro. In vivo SLC3A2-specific CAR T cells significantly increased overall survival and reduced growth of subcutaneous PC3-LN3-luciferase xenografts. No weight loss, manifestations of cytokine release syndrome or graft-versus-host disease, were detected. Conclusions These findings identify efficacious and potentially safe tumor-targeting of SLC3A2 with novel immune-activating antibody and genetically modified cell therapies.

Sciences, King's College London. The rat basophilic leukemia RBL SX-38 cells transfected to stably express the human FcεRI αβγ2 was kindly provided by Professor J.P. Kinet (Harvard University, Boston, USA). The PC3-LN3 (PL) cell line was kindly provided by Professor Sue Eccles (Institute of Cancer Research, Sutton, UK). All other cancer cell lines were sourced from ATCC. Expi 293-F human embryonic kidney cells were from ThermoFisher. Cell culture media for cell lines were supplemented with 10% fetal calf serum (FCS) (v/v), 100 U/mL penicillin and 100 U/mL streptomycin. PBMCs and T cells were cultured in RPMI1640 plus 5% human serum. Adherent cell lines were passaged once they reached 80-90% confluence by detachment in 0.5% Trypsin/0.53 M EDTA at 37°C for 5 minutes, washed and plated in fresh media.

SF-25 antigen immunoprecipitation
Pellets containing 20 to 50x10 6 target expressing MDA-MB-231, A2058 or MDA-MB-468 cells were resuspended in 1.75mL lysis buffer (PBS, 0.1% Tween20, 1X Halt TM protease inhibitors cocktail -Thermo Scientific) in a 15mL tube, incubated at 4⁰C on a roller and vortexed for 20 minutes. 300µL ProteinA Dynabeads® (Invitrogen) were prepared with 100µg SF-25 humanized IgG1 in 800µL Binding Buffer as per manufacturer's protocol. Cell lysates were centrifuged for 10 minutes at 4500rcf at 4⁰C and supernatants were transferred to the washed SF-25 Dynabeads®. Beads with immunoprecipitated fraction were placed in 30µL of Elution Buffer and 10µL of 4X LDS sample buffer (Invitrogen) and stored at -20⁰C. Samples were thawed, beads were concentrated on a magnet, supernatant were transferred to a 1.5mL microcentrifuge tube and β-mercapto-Ethanol was added to a final concentration of 5% (v/v). Samples were incubated for 10 minutes at 95⁰C before being resolved on a 4-12% gradient NuPAGE TM gel (Invitrogen) at 200 Volts in MOPS buffer. Migration was stopped when Coomassie G250 reached the bottom of the gel. The gel was fixed in 7% Acetic Acid/40% methanol (v/v) for 30 minutes at room temperature and stained with a 1X solution of colloidal Brilliant Blue G with 20% methanol for 1 hour at room temperature. The gel was first destained for 5 minutes with 7% Acetic Acid and BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) 25% methanol and then overnight with 2% acetic Acid and 25% methanol and stored in distilled water.
Bands of interest were cut out and sent to Aulesa Biosciences for mass spectrometry analysis.

SF-25 antigen: Mass spectrometry analysis
Proteins in gel slices were reduced (DTT), alkylated (iodoacetamide) and digested overnight with trypsin. Peptides within the tryptic digests were fractionated using an Ultimate 3000 nano-LC system in line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). In brief, peptides in 1% (v/v) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Scientific).
After washing with 0.5% (v/v) acetonitrile 0.1% (v/v) formic acid peptides were resolved on a 250 mm x 75 μm Acclaim PepMap C18 reverse phase analytical column (Thermo Scientific) over a 150 min using 7 gradient segments (1-6% solvent B over 1min, 6-15% B over 58min, 15-32%B over 58min, 32-40%B over 5min, 40-90%B over 1min, held at 90%B for 6min and then reduced to 1%B over 1min) with a flow rate of 300 nL.min −1 . Solvent A was 0.1% formic acid and Solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.2 kV using a stainlesssteel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary temperature of 250°C. All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by Xcalibur 2.0 software (Thermo Scientific) and operated in data-dependent acquisition mode. FTMS1 spectra were collected at a resolution of 120 000 over a scan range (m/z) of 350-1550, with an automatic gain control (AGC) target of 400 000 and a max injection time of 100ms. The Data Dependent mode was set to Cycle Time with 3s between master scans. Precursors were filtered according to charge state (to include charge states 2-7), with monoisotopic precursor selection and using an intensity range of 5E3 to 1E20. Previously interrogated precursors were excluded using a dynamic window (40s +/-10ppm). The MS2 precursors were isolated with a quadrupole mass filter set to a width of 1.6m/z. ITMS2 spectra were collected with an AGC target of 5000, max injection time of 50ms and HCD collision energy of 35%. LC-MS/MS data was processed using Proteome Discoverer BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) (ThermoFisher Scientific) with database searching against a downloaded FASTA file originating from Uniprot_SwissProt_2019_02. Results were initially visualized within the software and then exported to Excel for further review.

SLC3A2 differential expression study
Data for normal and associated tumor tissues were retrieve from the UCSC Toil RNA-seq Recompute dataset. [1] TPM values data from TARGET were filtered out and statistical analysis of SLC3A2, HER1 and HER2 differential expression was assessed by Mann-Whitney test in Graphpad Prism. All tumor samples were paired with the associated normal tissue samples from TCGA and the GTex normal The SF-25 scFv was subcloned into a myc-tag containing 28ζ construct downstream of the 4αβ chimeric cytokine receptor (IL4/2R). [2] The truncated CAR version 4SFm28Tr was generated by PCR by introducing a stop codon and a cloning site after the Lysine in position 3 in the cytoplasmic CD28 fragment. RD114 viral particles were produced by transiently transfecting HEK-293T cells. 1.5x10 6 cells were plated in a 10cm diameter culture dish in IMDM medium without antibiotics and allowed to grow overnight. Transfection reagents were prepared by gently pipetting 30µL GeneJuice® (Novagen) mammalian cell transfection reagent into 470µL plain IMDM medium (no serum) and incubated for 5 minutes at room temperature. Plasmids were gently added and incubated for 15 minutes before GalV virus containing supernatants were aliquoted, snap frozen and stored at -80⁰C.

Human PBMCs transduction and CAR T cell expansion
Isolated PBMCs were counted and placed in 6 well non-tissue culture plates at a concentration of 3x10 6 cells per mL at a maximum of 3mL per well. Polyhydroxyalcanoate (PHA) was used to activate the PBMCs at a final concentration of 5µg/mL. Plates were incubated for 24 hours at 37⁰C and 5% CO2.
Interleukin-2 is added at 24 hours to a final concentration of 100U/mL. RetroNectin® (Takara) coating 24 hours after Interleukin-2 treatment, 1x10 6 activated PBMCs were added to each well in 500µL RPMI 5% human serum. Human interleukin-2 and interleukin-4 (R&D systems) were added at a final concentration of 100U/mL and 30ng/mL respectively. Fresh RPMI with 5% human serum containing interleukin-2 (untransduced cells) or interleukin-4 (4αβ transduced cells) was added every 2 days during the expansion phase. Enrichment of CAR positive T cells was assessed by Flow Cytometry on a Fortessa cytometer at days 10 using the 9E10 anti-myc antibody. Analysis of flow cytometry data was performed by FlowJo (TreeStar Inc) software.

Tumor cell and CAR T cell coculture assays
Tumor cells were seeded at 5.10 4 tumor cells per well in 48-well plate in 200µL culture medium without antibiotics and allowed to form monolayers over 24 hours at 37°C in 5% CO2. CAR T cells were washed in PBS and 25.10 4 , 5.10 4 or 1.10 4 T cells were added in 100µL culture medium to the monolayers respectively resulting in a 5:1, 1:1 and 1:5 Effector T cell:Target cell ratio. After a 24-hour coculture, 200µL of supernatant was harvested and stored at -20°C for further cytokine measurement.
Monolayers viability was assessed immediately as detailed in the main text Methods.

Cytokine detection after coculture
Supernatants from cocultures were thawed and analyzed using human Interferon-γ (R&D systems) and human Interleukin-2 (Invitrogen) ELISA kits as described by manufacturer. Supernatants were diluted from 15 to 45-fold to fit within the standard curves. Cytokine levels were plotted using Graphpad Prism. wells/construct, pooled and distributed into 3 FACS tubes (2/5 th for phenotype labelling, 2/5 th for activation labelling and 1/5 th for isotype labelling). Same staining, gating strategy and analysis were used in post-coculture conditions as per pre-coculture staining. Analysis of flow cytometry data was performed by FlowJo (TreeStar Inc) software and data were plotted using Graphpad Prism.

CD98hc Expression on human PBMCs
Human PBMCs were isolated by density centrifugation as describe in the Methods. Basal expression level was assessed on 1.10 5 cells stained with the 1µg anti-CD98hc antibody MEM-108 (Biolegend) for 20 minutes at 4⁰C, washed with 2mL cold FACS buffer, stained again with goat anti-mouse IgG AlexaFluor647 (Jackson Labs), washed again prior to analysis. Isotype control staining was utilized. Cell surface staining was assessed by Flow Cytometry on a BD LSRFortessa cytometer. PBMCs were then activated or not with PHA as described above. Over 15 days, 1x10 5 PBMCs were retrieved from the cell BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) culture and stained as described above. Analysis of flow cytometry data was performed by FlowJo (TreeStar Inc) software and data were plotted using Graphpad Prism.

Immunofluorescence study of antigen expression
SF-25 IgG1 and NIP-IgG1 were directly labelled with Alexa Fluor 488 fluorophore using the Alexa Fluor™ 488 Antibody Labeling Kit (ThermoFisher). Frozen tissues were thawed at room temperature for 10 minutes, allowed to dry and fixed with 4% paraformaldehyde (PFA) for 10 minutes. Once PFA was removed, tissues were washed 3 times with PBS. Sections were covered with Human AB serum for 1 hour at room temperature. Tissues were incubated overnight with SF-25 IgG1-AF488 or NIP-IgG1-AF488 and kept at 4°C in the dark. Sections were washed 3 times in PBS and allowed to dry at RT.
ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher) was added, coverslip were applied and slides solidified overnight at room temperature in the dark.

In vivo human cancer xenograft model to study SF-25 IgE
On day 0, NSG mice were injected simultaneously with 5x10 5 LS-180 colorectal cancer cells, 5x10 6 PBMCs from a healthy human volunteer and 10mg/kg of SF-25 IgE or 50µl PBS, to a final volume of 200µl. Subsequent injections of antibody/PBS were performed on days 2 and 3. Mice were sacrificed on day 21 by CO2 asphyxiation. Lungs were analyzed for tumor growth in the lungs using the following protocol: the trachea was exposed by performing a mid-line incision from below the diaphragm to the throat and the chest activity opened. An intravenous cannula was inserted into the trachea, and a 10 mL syringe was attached to it. Approximately 3 mL of a solution of 15% (v/v) Indian Ink was then injected into the lungs. The ink-stained lungs were then removed from the thoracic cage, placed in MilliQ water to remove the excess ink and then transferred to Fekete solution for 48 hours. The number of metastatic nodules/lung lobe and the proportional surface occupancy of tumor nodules were calculated. Images were acquired using a Nikon SMZI500 Stereo Microscope (Nikon UK Ltd) with 0.75x BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) and 1.0x objective. The NIS Elements Basic Research software (Nikon UK Ltd) was used to determine the total surface area of the lung section and to distinguish the white lung metastasis nodules. Data were acquired from two independent experiments using PBMCs from two human donors. week for the rest of the experiment.

Lectin blots
Purified IgE samples (150ng) were reduced with 50mM dithiothreitol and boiled at 95°C for 5 minutes.

Transcript-to binding-level correlation analysis
This section is divided into three steps, an example of the code used is located at: https://github.com/ramipod/Antigen-Identification-by-Binding-and-Transcriptomic-Comparison -TPM dataset generation (TPM averaging) -Spearman score calculation for a specific binding dataset and the TPM counts (spearman AVG1) -Spearman scores compilation (Spearman-AVGs)

Statistical methods
All statistical analyses were performed using GraphPad TM Prism Software (version 6.0). Error bars represent SD and SEM in in vitro and in ex vivo evaluations.         BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)