Contents
Synthetic gene construction
The designed protein sequences were codon optimized for Escherichia coli expression and ordered as synthetic genes in pET29b + E. coli expression vectors (Genscript). Each synthetic gene was inserted at the NdeI and XhoI sites of the vector, including an N-terminal hexahistidine tag followed by a TEV protease cleavage site and the addition of a stop codon at the C terminus. VHH fusion proteins were inserted in pCMV/R for mammalian cell expression cloned between NheI and AvrII sites and including an N-terminal signal peptide (MDSKGSSQKGSRLLLLLVVSNLLLPQGVLAGSDG) (Genscript). Amino acid sequences are listed in Supplementary Tables 1–3.
Protein expression and purification in E. coli
All DARPin protein fusions H132′ and H32’4 were expressed in E. coli strain Lemo21(DE3) (NEB) and VHH fusion proteins in E. coli strain SHuffle T7 (NEB). Bacteria were transformed with a pET29b+ plasmid encoding the synthesized gene of interest. Cells were grown for 24 h in lysogeny broth medium supplemented with kanamycin. Cells were inoculated at a ratio of 1:50 ml in Studier TBM-5052 autoinduction medium supplemented with carbenicillin or kanamycin, grown at 37 °C for 2–4 h and then grown at 18 °C for an additional 18 h. Cells were harvested by centrifugation at 4,000g and 4 °C for 15 min and resuspended in 30 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.02 mg ml–1 DNAse). Cell resuspensions were lysed by sonication for 2.5 min (5-s cycles). Lysates were clarified by centrifugation at 24,000g at 4 °C for 20 min and passed through 2 ml of Ni-NTA nickel resin (Qiagen, no. 30250) pre-equilibrated with wash buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole). The resin was washed twice with ten column volumes (CV) of wash buffer and then eluted with 3 CV of elution buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 300 mM imidazole). Eluted proteins were concentrated using Ultra-15 Centrifugal Filter Units (Amicon) and further purified with a Superdex 75 Increase 10/300 GL (GE Healthcare) size-exclusion column in Tris-buffered saline (25 mM Tris-HCl pH 8.0, 150 mM NaCl). Fractions containing monomeric protein were pooled and concentrated, concentration measured by absorbance at 280 nm (NanoDrop), snap-frozen in liquid nitrogen and stored at −80 °C. Proteins used in animal studies were further purified to remove endotoxin using NoEndo High Capacity Spin Columns (Protein Ark). Endotoxin levels were measured with an Endosafe LAL Cartridge, PTS201F 0.1 EU ml–1 sensitivity (Charles River) in an Endosafe nexgen-MCS (Charles River).
Protein production in 293-6E cells
Nanobody (VHH) fusion proteins used in animal studies were expressed in 293-6E cells. Four liters of 293-6E cells were transfected with pCMV/R plasmids encoding the gene of interest using linear polyethylenimine. Cultures were harvested when average viability was ≤90% (4–6 days). Protein expression was determined in the culture supernatant, and total cell lysate was analyzed by reducing and nonreducing SDS–polyacrylamide gel electrophoresis (SDS–PAGE). His-tagged proteins were purified from clarified culture supernatant via Ni-NTA affinity chromatography. Proteins were then dialyzed overnight into PBS pH 7.4, concentrated using Ultra-15 Centrifugal Filter Units (Amicon) and analyzed by reducing and nonreducing SDS–PAGE and analytical size-exclusion chromatography. Finally, concentration was measured by absorbance at 280 nm (NanoDrop) and aliquots were snap-frozen in liquid nitrogen and stored at −80 °C. Expression and purification were performed by Proteos.
Peptide synthesis
Peptides H1 (Neo2A), H13, H2’4 and H4 were synthesized to >85% purity by Genscript. For Biolayer interferometry assays measuring the affinity between Neo2A and Neo2B, the Neo2A peptide was biotinylated at the N terminus and a flexible linker added to avoid steric hindrance (Biotin-GGGSGGGSPKKKIQLHAEHALYDALMILNIVKTNS).
Biolayer interferometry
Binding data were collected in an Octet RED96 (ForteBio) and processed using ForteBio Data Analysis Software v.9.0.0.10. Biotinylated human IL-2Rγ (Acro Biosystems, no. ILG-H85E8) was immobilized on streptavidin-coated biosensors (SA ForteBio) at 5 μg ml–1 in binding buffer HBS:EP+ (Cytiva) + Blotting Grade Blocker Non Fat Dry Milk (BioRad) until the signal reached 0.5 nm. After loading the IL-2Rγ receptor onto the biosensor, baseline measurement was performed by dipping the biosensors in binding buffer only (60 s), followed by monitoring of binding kinetics by dipping the biosensors in wells containing the target analyte protein (association step) and then dipping them back into baseline/buffer (dissociation). For the association step, analyte proteins were diluted from concentrated stocks into binding buffer to the indicated final concentration. Human IL-2Rβ (Acro Biosystems, no. CD2-H5221) was added in solution at the indicated concentration. The experiments using Biotin-Neo2A were performed following the same method, except that Biotin-Neo2A was loaded on the streptavadin sensors and human IL-2Rγ-Fc (Acro Biosystems, no. ILG-H5256) was also added in solution during association.
Circular dichroism
Far-ultraviolet circular dichroism measurements were carried out with AVIV spectrometer model 420 in PBS (pH 7.4) in a cuvette of 1-mm path length at a protein concentration of ~0.20 mg ml–1 (unless otherwise mentioned in the text). Temperature melts were performed at 25–95 °C and monitored absorption signal at 222 nm (steps of 2 °C min–1, 30 s of equilibration by step). Wavelength scans (195–260 nm) were collected at 25 and 95 °C, and again at 25 °C after rapid refolding (~5 min).
Cell lines and cell culture
YT-1 cells were provided by Yodoi31 and cultured in RPMI complete medium (Gibco): RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, 15 mM HEPES and 1% penicillin/streptomycin. WT K562 cells were purchased from American Type Culture Collection (ATCC), and engineered K562 cell lines expressing EGFR-iRFP, Her2-eGFP or both (provided by Lajoie30) were cultured in RPMI complete medium. WT B16F10 melanoma cells (purchased from ATCC), PD-L1-overexpressing B16 melanoma cells35 and Her2+/PD-L1Hi B16F10 melanoma cells were cultured in RPMI medium supplemented with 10% FBS, 2 mM l-glutamine, 1% penicillin/streptomycin, 1% nonessential amino acids, 1 mM sodium pyruvate (Gibco) and 0.1 mM β-mercaptoethanol. Cell lines SKOV3, JIMT-1 and OVCAR8 were purchased from ATCC and cultured in RPMI complete medium. NCI-H1975 cells were purchased from ATCC and cultured in RPMI + 5% FBS + 10 U ml–1 penicillin/streptomycin. Cells were trypsinized and split every 3 days to avoid confluency. Cell line 293-6E (human embryonic kidney) was provided by Proteos and cultured in F17 supplemented with 0.1% Pluronic F-68, 4 mM GlutaMAX and 25 μg ml–1 G418. All cells were maintained at 37 °C in a humidified incubator with 5% CO2. Murine IFN-ɣ was purchased from Peprotech and used at the indicated concentrations to induce PD-L1 expression on B16F10 cell lines. Cells used for in vivo experiments had been passaged for <2 months, were negative for known mouse pathogens and were implanted at >95% viability.
YT-1 cell STAT5 phosphorylation studies
Approximately 2 × 105 YT-1 cells were plated in each well of a 96-well plate and resuspended in RPMI complete medium containing serial dilutions of targeted or untargeted intact or split Neo-2/15 proteins. Cells were stimulated for 20 min at 37 °C and immediately fixed by the addition of formaldehyde to 1.5% and 10 min incubation at room temperature. After fixation, cells were permeabilized by resuspension in ice-cold 100% methanol for 30 min on ice. Fixed and permeabilized cells were washed twice with PBSA buffer (PBS pH 7.2 containing 0.1% bovine serum albumin) and then incubated with Alexa Fluor 647-conjugated anti-STAT5 pY694 antibody (BD Biosciences) diluted 1:50 in PBSA buffer for 2 h at room temperature. Cells were washed twice in PBSA buffer, and Alexa Fluor 647 was measured on a CytoFLEX flow cytometer (Beckman Coulter). Dose–response curves were fitted to a logistic model and half-maximal effective (EC50) concentrations calculated using GraphPad Prism data analysis software, after subtraction of mean fluorescence intensity (MFI) of unstimulated cells and normalization to maximum signal intensity. Experiments were conducted in triplicate and performed three times, with similar results. The gating strategy is shown in Supplementary Fig. 6.
In vitro reconstitution of split Neo-2/15 on target cells
The four K562 cell lines (engineered K562 tumor cell lines transduced for expression of EGFR-iRFP, HER2-eGFP, both or neither) were mixed in equivalent ratios. Cell mixtures were washed with flow buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 and 1% BSA) and aliquoted into V-bottom plates with 200,000 cells per well. Serially diluted split Neo-2/15 fusion proteins were made from concentrated stocks, added to cells in a 50-μl volume and incubated for 30 min. Subsequently, cells were washed with 150 μl of flow buffer and incubated with a mixture of biotinylated human IL-2Rγ (Acro Biosystems, no. ILG-H85E8), human IL-2Rβ (Acro Biosystems, no. CD2-H5221) and a fluorescent streptavidin–phycoerythrin conjugate (SA-PE, Invitrogen) for 15 min. Data were acquired on a LSRII cytometer (BD Biosciences). The gating strategy is shown in Supplementary Fig. 7. The same protocol was used to measure targeted split Neo-2/15 activity on cell lines SKOV3, JIMT-1, OVCAR8 and B16F10 acquired on a fluorescent activated cell sorter (FACS) Celesta cytometer (BD Biosciences).
YT-1:K562 cell trans-activation assays
Approximately 2 × 105 YT-1 cells at the indicated ratio (K562:YT-1 20:1, 6:1 or 2:1) (either untransduced HER2–/EGFR– K562 cells or transduced double-positive HER2+/EGFR+ K562 cells) were used in each well for trans-activation studies. K562 cells were plated in each well of a 96-well plate and resuspended in RPMI complete medium containing serial dilutions of split Neo-2/15 proteins. Cells were incubated for 30 min at 37 °C and then washed once with PBSA buffer. YT-1 cells were added to each well and cocultured with K562 cells for 30 min at 37 °C. Cells were stained with BV421-conjugated anti-CD132 antibody (BD Biosciences, no. 566222; Clone AG184, 1:50 dilution) for 30 min at 4 °C and washed once with PBSA buffer. After washing, these were immediately fixed by the addition of formaldehyde to 1.5% and 10-min incubation at room temperature. After fixation, cells were permeabilized by resuspension in ice-cold 100% methanol for 30 min on ice. Fixed and permeabilized cells were washed twice with PBSA buffer then incubated with Alexa Fluor 647-conjugated anti-STAT5 pY694 antibody (BD Biosciences) diluted 1:50 in PBSA buffer for 2 h at room temperature. Alexa Fluor 647 fluorescence on YT-1 cells (gated based on CD132 expression) was measured on a CytoFLEX flow cytometer (Beckman Coulter). Dose–response curves were fitted to a logistic model and EC50 values calculated using GraphPad Prism data analysis software after subtraction of MFI of unstimulated cells and normalization to maximum signal intensity. Experiments were conducted in duplicate and performed twice, with consistent results. The gating strategy is shown in Supplementary Fig. 8.
In vitro T cell:B16 trans-activation experiments
B16 cells overexpressing PD-L1 were treated overnight with 20 ng ml–1 murine IFN-γ (Preprotech), washed, coated with 1.0 μM desired protein (that is, OVA, Neo-2/15, Ctrl-Neo2A and so on) for 10 min, washed and resuspended repeatedly and replated in a U-bottom plate. Mouse trp1-specific CD8 T cells (JAX Stock, no. 030958) were isolated via a negative isolation kit (Stemcell, no. 19853) and plated under the listed conditions either alone or in coculture with B16 cells at a 1:1 ratio for 1 day. To confirm trans-activation, 1.0 μM αPD-L1 or Ctrl VHH was added at the start of coculture. T cells were then harvested 24–36 h later and analyzed by flow cytometry for expression of activation markers CD25 (Biolegend, no. 102017) and CD69 (Biolegend, no. 104508). T cells were gated separately from B16 cells via FSC/SSC gating and CD8 staining (Biolegend, no. 100728). All antibodies were used at 1:100 dilution.
Engineering of B16F10 melanoma cells overexpressing mouse PD-L1 and human HER2
B16F10 PD-L1hi cells were transduced with retrovirus to express human HER2 (extracellular domain IV and transmembrane domain). HER2+ cells were identified by staining with anti-HER2 antibody (24D2, Biolegend, no. 324405, 1:200 dilution) and FACS sorted twice to achieve a pure population. The gating strategy is shown in Supplementary Fig. 4.
Safety study in murine models
As shown in Fig. 3a and Supplementary Fig. 2: Immunocompetent female 6–8-week-old C57BL/6J mice (n = 5 per group) were treated daily with the targeted Neo-2/15 and targeted split Neo-2/15 molecules at equivalent doses (2.6 nmol, equivalent to 30 µg per mouse of Neo-2/15). Weight change and survival were monitored to evaluate toxicity of the dosed molecules. Mice were euthanized if they lost 10% of body weight. Spleen and lungs were harvested following euthanasia. Organs from mice in cohorts Neo-2/15, Ctrl-Neo2/15 and αPD-L1-Neo2/15 were obtained and analyzed on different days, when the euthanasia criteria were met. Organs from mice in the PBS and split Neo-2/15 cohorts that did not lose weight and did not meet euthanasia criteria were harvested on day 60 at the conclusion of the study. Spleens and lungs were weighed to assess the potential toxicity of treatment. Spleens were subjected to immunophenotyping by flow cytometry to quantify expansion of CD8+ T cells using the following antibodies: Alexa Fluor 488 anti-CD25 (clone PC61, Biolegend, no. 102018); PE anti-CD69 (H1.2F3, Biolegend, no. 104508); Pacific Blue anti-CD4 (GK1.5, Biolegend, no. 100428); Brilliant Violet 711 anti-CD45 (30-F11, Biolegend, no. 103147); and Brilliant Violet 785 anti-CD8a (53-6.7, Biolegend, no. 100749). All antibodies were used at 1:100 dilution. All animal protocols were approved by the Dana-Farber Cancer Institute Committee on Animal Care (protocol nos. 14-019 and 14-037) and are in compliance with the NIH/NCI ethical guidelines for tumor-bearing animals.
Syngeneic murine melanoma model experiments
All animal protocols were approved by the Dana-Farber Cancer Institute Committee on Animal Care (protocol nos. 14-019 and 14-037) and are in compliance with the NIH/NCI ethical guidelines for tumor-bearing animals. At day 0, 6–8-week-old C57BL/6 J mice (JAX Stock, no. 000664) were inoculated with 80–90% confluent WT or engineered B16F10 cells (500,000 cells per mouse). Starting at day 5, mice were treated daily with the listed test items. Neo2A and Neo2B fusion proteins were injected individually following one of two regimens as indicated per experiment. Mice were monitored for survival, weight change and symptoms of toxicity including pallor, notable weight loss and fatigue. Mice were euthanized if they lost 10% of body weight or their tumors ulcerated or reached 2,000 mm3 in volume, which is the maximal permitted tumor size for these studies.
Experiments shown in Fig. 3b and Supplementary Fig. 3: Five mice per group were included in these two studies. For the study shown in Fig. 3b, all groups were cotreated biweekly with TA99 starting on day 3 (150 µg per mouse). Mice in the study shown in Supplementary Fig. 3 were not cotreated with TA99. The two studies were carried out in parallel. Starting on day 5 after tumor cell inoculation, mice were administered daily with therapeutic doses of αPD-L1-Neo2/15 and Ctrl-Neo2/15 at 430 nmol (12 µg per mouse) (intraperitoneally), or targeted split Neo-2/15 fragments were administered at 8 nmol (200 µg per mouse) (Neo2A fusions given intraperitoneally and Neo2B fusions given subcutaneously in the opposite flank to the tumor). Mice in the αPD-L1-Neo2/15 treatment cohort showed symptoms of toxicity (pallor, notable weight loss, fatigue) at day 12 and so their treatment schedule was modified. They then received treatment daily from days 5–12, no treatment on days 13 and 14 and then treatment every other day starting at day 15. Peripheral blood was collected at day 15 of treatment and analyzed by flow cytometry using the following antibodies: Alexa Fluor 488 anti-CD25 (clone PC61, Biolegend, no. 102018); PE anti-CD69 (H1.2F3, Biolegend, no. 104508); Pacific Blue anti-CD4 (GK1.5, Biolegend, no. 100428); Brilliant Violet 711 anti-CD45 (30-F11, Biolegend, no. 103147); and Brilliant Violet 785 anti-CD8a (53-6.7, Biolegend, no. 100749). All antibodies were used at 1:100 dilution.
Experiments shown in Fig. 3c,d: Five mice were used in the PBS and TA99 groups, ten mice in all other groups. Starting on day 5 after tumor cell inoculation, mice were administered daily with each test item at therapeutic doses (Neo-2/15 at 2.6 nmol (30 µg per mouse) and split Neo-2/15 fusions at 8 nmol (~200 µg per mouse)). The indicated groups were cotreated with Ta99 mAb administered biweekly starting on day 3 (150 µg per mouse).
Rechallenge experiments shown in Fig. 3e: Five surviving, tumor-free mice from the αPD-L1 split Neo-2/15 + TA99-treated cohorts in several experiments (including the studies shown in Fig. 3b,c) were pooled into one group. The mice had cleared their tumors 2–5 months before the start of the experiment, as shown in Fig. 3e. Five age-matched B16 naïve C57BL/6J mice were designated as controls. On day 0, mice were inoculated with B16 F10 cells overexpressing PD-L1 (1,000,000 cells per mouse, twice the amount of a usual primary challenge). On day 5, mice were bled and peripheral blood analyzed by flow cytometry for the presence of tumor-specific Trp1+ CD8 T cells via anti-Trp1-MHCI tetramer staining as previously described34.
Experiments shown in Fig. 3f and Extended Data Fig. 6c: At day 0, mice were inoculated in the flank with 80–90% confluent B16F10 cells overexpressing both PD-L1 and HER2 (500,000 cells per mouse). Twelve mice were included in the PBS control group, five in the Neo-2/15-treated group and seven each in groups ɑPD-L1-Neo2A + ɑPD-L1-Neo2B and ɑHER2-Neo2A + ɑPD-L1-Neo2B. Starting on day 5 after tumor cell inoculation, mice were administered daily with each test item at therapeutic doses (Neo-2/15 at 2.6 nmol (30 µg per mouse) and split Neo-2/15 fusions at 8 nmol (~200 µg per mouse)). Neo2A fusions were administered subcutaneously interscapularly while PBS, Neo-2/15 and Neo2B were injected intraperitoneally. The mice in these treatment cohorts did not receive TA99 mAb. Mice in the Neo2/15 treatment cohort showed symptoms of toxicity (pallor, notable weight loss, fatigue) after 1 month of daily dosing, and thus surviving mice were euthanized when their body condition score was less than or equal to 2, in accordance with IACUC Standard Procedure.
Experiments shown in Extended Data Fig. 7d: Five mice per group were included in this study. Starting on day 5 after tumor cell inoculation, mice were administered daily with therapeutic doses of mIL-2 (13 µg per mouse) (Preprotech, no. 212-12), Neo-2/15 (10 and 30 µg per mouse) and αPD-L1-Neo2A + αPD-L1-Neo2B at 8 nmol (200 µg per mouse). Neo2B fusions were given intraperitoneally and Neo2A fusions subcutaneously (in the scruff of the neck). All groups were cotreated with TA99 αTrp-1 melanoma-specific antibody, administered biweekly starting on day 3 (150 µg per mouse). Three early ulcerated tumors (<500 mm3) were removed from this analysis. Experiments shown in Fig. 4c: Starting on day 5 after tumor cell inoculation, mice were administered daily with: PBS (five mice), Ctrl-Neo-2/15 (500 pmol per mouse, 5 mice), Ctrl-Neo2A + Ctrl-Neo2B (500 pmol per mouse, 5 mice) and αCD8-Neo2A + αCD8-Neo2B (500 pmol per mouse, 10 mice). Neo2B fusions were given intraperitoneally and Neo2A fusions subcutaneously (in the scruff of the neck). All groups were cotreated with TA99 αTrp-1 melanoma-specific antibody, administered biweekly starting on day 3 (150 µg per mouse).
Quantibrite surface receptor quantification
The antibody-binding capacity of molecules EGFR (AY13, Biolegend, no. 352903), EpCAM (9C4, Biolegend, no. 324205), HER2 (24D2, Biolegend, no. 324405) and murine PD-L1 (MIH7, Biolegend, no. 155404) on the surface of K562, B16F10, SKOV3, JIMT-1 and OVCAR8 cells was determined using Quantibrite beads (BD Biosciences) according to the manufacturer’s protocols. Flow cytometry data were analyzed using FlowJo v.10. All antibodies were used at 1:200 dilution. The gating strategy is shown in Supplementary Fig. 9.
In vitro CD8 cis-targeting experiments with splenocytes
Splenocytes were harvested from a WT C57BL/6 J mouse and plated together with split Neo-2/15 fusion test items at the indicated concentrations (negative-control, single-split construct wells were plated at 50 nM) in a 96-well plate in RPMI complete medium. After 4 days, cells were harvested and stained for flow cytometry. The antibodies used for staining were: APC anti-CD45 (clone 30-F11, Biolegend, no. 103111); Zombie NIRTM Fixable Viability Kit (Biolegend, no. 423105); Pacific Blue anti-B220 (RA3-6B2, Biolegend, no. 103227); and Brilliant Violet 421 anti-CD4 (GK1.5, Biolegend, no. 100443). All antibodies were used at 1:100 dilution.
In vivo T cell cis-targeting in Foxp3-GFP mice
Split Neo-2/15 fusion proteins (or intact Neo2 constructs) were administered daily to nontumor-bearing Foxp3-GFP mice (JAX Stock, no. 006772) for 5 days. Neo2A fusions were injected subcutaneously in the scruff of the neck while Neo2B fusions, PBS and intact Neo-2/15 constructs were injected intraperitoneally. Intact Neo-2/15 fusions were dosed at 500 pmol (12 µg per mouse per day). Split Neo-2/15 fusions were dosed at 500 pmol (10 µg per mouse per day) and 250 pmol (4 µg per mouse per day). On day 6 mice were euthanized, spleen and both inguinal lymph nodes collected and homogenized into single-cell suspensions (and spleen resuspended briefly in ACK lysis buffer to lyse red blood cells) and cell populations investigated by flow cytometry using the following antibodies: APC anti-CD45 (clone 30-F11, Biolegend, no. 103111); Brilliant Violet 421 anti-CD4 (GK1.5, Biolegend, no. 100443); and PE anti-CD8a (53-6.7, Biolegend, no. 100707). All antibodies were used at 1:100 dilution.
CAR-T cell in vitro STAT5 phosphorylation assay
Primary human CD8 T cells were obtained from healthy donors, with written informed consent for research protocols approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center (FHCRC). Peripheral blood mononuclear cells were isolated using lymphocyte separation medium (Corning). T cells were isolated using EasySep CD8 negative isolation kits (Stemcell Technologies). CAR-T cells were generated by stimulation of thawed CD8 with anti-CD3/CD28 Dynabeads (Gibco) at a ratio of three beads to one T cell, in medium supplemented with 50 IU ml–1 (3.1 ng ml–1) IL-2. The following day, T cells were transduced with lentivirus encoding anti-CD19 CAR by spinoculation for 90 mins at 800g or left untransduced. Beads were removed 5 days post stimulation. CAR+ cells were FACS sorted by HER2 transduction marker on day 8. On day 10, CAR-T cells or untransduced T cells were cultured at the indicated concentrations of Neo2 variants in 96-well plates for 12 min. T cells were then methanol fixed, permeabilized and stained for pSTAT5 for flow cytometry using pSTAT5-APC antibody (A17016B.Rec, Biolegend, no. 936902) at 1:200 dilution. The gating strategy is shown in Supplementary Fig. 10.
Repeated tumor-killing assay by CAR-T cells
NCI-H1975 cells were seeded at 1.1 × 105 per well in octuplet for each condition in 96-well E-plates (Agilent) and incubated overnight in the xCelligence Esight instrument (Agilent). Tumor cell growth was monitored in real time using electric impedance measurement. The following day, medium was decanted from the E-plate and 2.2 × 105 anti-ROR1 CAR-T cells with either a HER2 or CD19 transduction marker were plated in medium or medium supplemented with 30 nM ɑHER2-Neo2A + ɑHER2-Neo2B, 30 nM ɑHER2-Neo-2/15 or 50 IU of hIL-2. Anti-ROR1 CAR-T cells (Clone R12), with IgG4 hinge CD28 transmembrane domain and 41BB CD3z costimulatory domains, were generated as previously described54. Forty-eight hours post CAR-T cell addition, CAR-T cells were removed from the E-plate by vigorous pipetting and transferred to an Eppendorf tube. Cells were counted, pelleted by centrifugation and resuspended in medium or medium supplemented with 30 nM ɑHER2-Neo2A + ɑHER2-Neo2B, 30 nM HER2-Neo-2/15 or 50 IU of hIL-2. CAR-T cells were then transferred to a fresh E-plate seeded with NCI-H1975 tumor cells. Replating was performed for a total of four times.
In vivo lymphoma xenograft treatment with CAR-T cell study
Six-to-eight-week-old female NSG mice were obtained from the Jackson Laboratory, with five mice housed per cage. The temperature of the housing room was 68–75 °F and humidity 40–60%. Mice were provided with cellulose pellet bedding (Performance BioFresh), irradiated diamond twists as enrichment and nesting material (Envigo) and irradiated laboratory diet no. 5058. Water bottles were filled with acidified water. Raji tumor cells (0.5 × 106) transduced with a lentiviral vector encoding the (ffLuc)-eGFP fusion gene were injected into the tail veins of NSG mice. CAR-T cells were produced as described above and expanded as previously described55 post FACS sorting. Seven days after tumor injection, lentiviral-transduced anti-CD19 HER2+ CAR-T cells (0.8 × 106) were infused intravenously into mice. Either ɑHER2-Neo2A + ɑHER2-Neo2B or ɑEpCam-Neo2A + ɑEpCam-Neo2B, at 7.5 mg kg–1, was injected into the peritoneum on days 1–3, 6–10 and 13–15 post T cell injection. The FHCRC Institutional Animal Care and Use Committee approved all mouse experiments.
Statistical analysis
Comparison of fitted curves in cellular phospho-STAT5 signaling assays was performed by measuring differences in EC50 values, which were considered statistically significant if their 95% confidence intervals did not overlap. For all bar plots, error bars represent ±s.d. and individual data points are shown (as dots) for experiments where n < 5. Comparisons of weight loss and tumor growth in tumor-bearing mice were performed using unpaired two-tailed Student’s t-test. Comparisons of the survival of tumor-bearing mice were performed using the log-rank Mantel–Cox test (95% confidence interval). Unless otherwise noted, the remaining results were analyzed by one-way ANOVA; if significant (95% confidence interval), post hoc t-tests were performed comparing groups and P values adjusted for multiple comparisons are reported. Statistical tests were performed with GraphPad Prism 9.0 data analysis software.
Software
Protein visualization and protein structure images were generated using PyMOL 2.0. All flow cytometry data were analyzed using FlowJo v.9 and v.10. Statistical tests were performed with GraphPad Prism 8.0 and 9.0 data analysis software.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
