Human cell lines

Cytotoxicity target cells were Jurkat cells (ATCC, Manassas, VA) maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Damstadt, Germany) containing 10% fetal bovine serum (FBS; R10 medium).

Isolation of primary human cells

Collection of anonymous human cord blood units from delivery rooms at UCLA (Los Angeles, CA) and use of human peripheral blood cells were deemed exempt from need for formal approval by the Institutional Review Board at UCLA. After FiColl Hypaque (Stem Cell Technologies, Vancouver, Canada) isolation of mononuclear cells from fresh umbilical cord blood, human CD34+ cells were isolated using immunomagnetic beads (MACS CD34 MicroBead Cell Separation Kit; Miltenyi, Auburn, CA) at enrichment >70% and stored in liquid nitrogen. Anonymous human peripheral blood samples were obtained from the UCLA CFAR Virology Core Laboratory. Whole leukocytes were isolated using Hetasep (Stem Cell Technologies). Samples were enriched for natural killer (NK) cells by negative selection using the RosetteSep system (Stem Cell Technologies). Enriched samples were immediately used for cytotoxicity experiments.

Vector production and transduction of cell lines and primary human cells

Vector-containing supernatant was harvested from transfected HEK293T cells treated with sodium butyrate, and large-scale preparations (2–5L) were concentrated by tangential flow filtration, with titers measured by vector copy number (VCN) assessment in transduced HT29 cells, as previously described.²⁷

To generate stably transduced target cells, lentiviral vectors were added to 1×10⁵ Jurkat cells at a concentration of >1×10⁸ TU/mL and allowed to incubate for 24h, expanding cells in R10 medium.

For transduction of human CD34+ cells,^10–13 thawed cells were pre-stimulated for 14–18h in X-Vivo15 medium (Lonza, Basel, Switzerland) containing 1×L-glutamine/penicillin/streptomycin (L-Glut/Pen/Strep; Gemini BioProducts, West Sacramento, CA), enriched with recombinant human (rhu) SCF (50ng/mL), rhuFlt-3 ligand (50ng/mL), and rhuThrombopoietin (50ng/mL; cytokines from R&D Systems, Minneapolis, MN). Transduction was performed for 24h with the addition of lentiviral vectors at a concentration of 5.5×10⁷ TU/mL onto 10⁵ cells in 1mL transduction medium in RetroNectin-precoated 48-well plates, as previously described.^10–13

Proliferation and differentiation cultures of primary human HSC

Myeloid differentiation was performed in culture for 12–14 days in Iscove's modified Dulbecco's medium (IMDM; Mediatech, Inc., Manassas, VA) containing L-Glut/Pen/Strep and 10% FBS, enriched with cytokines rhuSCF (100ng/mL) and rhuIL-3 (100ng/mL) from day 1, with the addition of rhuGM-CSF (10ng/mL) from day 3.^10,28 Short-term proliferation of human HSC was performed for 9 days and cultured in basal bone marrow medium (BBMM): IMDM enriched with 20% FCS, 0.5% bovine serum albumin, 5ng/mL rhuIL-3, 10ng/mL rhuIL-6, and 25ng/mL rhuFlt-3 ligand.²⁷ Cell concentrations were maintained at <5×10⁵ per well.

Clonogenic assays

Erythro-myeloid colony formation was assayed by culturing human HSC immediately after transduction in duplicate 35mm gridded cell culture dishes, using complete methylcellulose (MethoCult™ H4435 Enriched, Stem Cell Technologies) for 14 days. Colony-forming units (CFU) on plates were then counted under microscopy, and colony type was scored based on morphology. Plating efficiency was calculated as 100%×(total number CFU counted/total number cells plated).²⁹ Colonies were then collected for genomic DNA extraction and quantitative droplet digital PCR (ddPCR) analysis.

Cytotoxicity assays

Direct antibody cytotoxicity was assessed by culturing EGFRt-transduced and non-transduced Jurkat cells with different concentrations of cetuximab antibody—0.3, 1, and 10μg/mL—or vehicle alone. Complement-dependent cytotoxicity was assessed by culturing EGFRt-transduced and non-transduced Jurkat cells with different dilutions of rabbit complement, with 1μg/mL of cetuximab or vehicle (phosphate-buffered saline [PBS]) alone. Target cell cytotoxicity was measured by incubating cells with 4′,6-diamidino-2-phenylindole (DAPI; BD Biosciences, San Jose, CA) at room temperature for 15min. Cells positive for DAPI by flow cytometry were counted as nonviable.

Assays for ADCC were performed using the flow-cytometry-based Live/Dead Cell Mediated Cytotoxicity Kit (Invitrogen),¹⁰ with 2×10⁴ EGFRt-transduced or non-transduced Jurkat target cells/well pre-labeled with 3,3′-dioctadecyloxacarbocyanine (DiOC). Effector cells were added at different effector:target (E:T) ratios, with or without cetuximab at a concentration of 1μg/mL. To determine maximal lysis, 20% ethanol³⁰ was added to target cells. Cell mixtures were incubated in propidium iodide (PI) containing R10 for 4–6h. DiOC+ cells were counted as target cells, and cells double positive for DiOC and PI were counted as nonviable target cells. All samples were loaded in triplicate, and all assays had controls with isolated effectors and targets.

Determination of VCN

Genomic DNA of cell samples was extracted using NucleoSpin Tissue XS (Macherey-Nagel, Düren, Germany) or a PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA). VCN was determined by multiplex ddPCR of the HIV-1 packaging signal sequence in the provirus and normalized to the cellular autosomal gene SDC4 to calculate the average VCN.^9,31

Flow cytometry

To evaluate the cell expression of huEGFRt, transduced cells were washed with blocking solution and incubated with biotinylated cetuximab antibody. Pharmaceutical-grade cetuximab (Erbitux, Eli Lilly and Company, Indianapolis, IN) was biotinylated by using the One-Step Antibody Biotinylation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Primary human cells were incubated with 1×Human TruStain FcX Fc Receptor Blocking Solution (BioLegend, San Diego, CA) before incubation with biotinylated cetuximab, followed by incubation with streptavidin-R–phycoerythrin conjugate (BD Biosciences) after washing with blocking solution. Transduced Jurkat cells with >95% EGFRt expression served as positive controls for EGFRt detection.

NK cell markers were assessed with fluorescent-labeled murine monoclonal antibodies to human CD16-PE, CD56-APC, and CD3-PerCP. After washing, cells were analyzed on an LSR II flow cytometer (BD Biosciences).

Flow cytometry was used to analyze the cell target lysis percentage based upon the following formula: % specific lysis=100%×[(# of nonviable target cells in co-culture with antibody, complement, or effector cells/# of target cells in co-culture) – (# of nonviable targets cultured alone/# of target cells cultured alone)]/[(# of nonviable target cells cultured in maximal lysis conditions/# of target cells cultured in maximal lysis conditions) – (# of nonviable targets cultured alone/# of target cells cultured alone)].¹⁰ DIVA analysis software (BD Biosciences) and FlowJo software (FlowJo, LLC, Ashland, OR) were used for data evaluation.

In vivo studies

NOD/SCID/γ-chain^null (NSG) mice (NOD.Cg.Prkdc^scid Il2rg^tm1Wjl/SzJ; stock no. 005557; Jackson Laboratory, Bar Harbor, ME) were housed in accordance with approved protocol. Newborn pups aged 3–7 days were injected with 3×10⁵ cells/pup via intrahepatic injection of transduced or non-transduced human CD34+ cells 1 day after conditioning with 125 cGy of sub-lethal body irradiation from a ¹³⁷Ce source with attenuator and allowed to engraft over 12 weeks.¹⁰ Engraftment was assessed by flow cytometry and VCN assessment from retro-orbital venous plexus blood sampling and harvested bone marrow and spleen.

Humanized mice received tumor challenges with subcutaneous injection of 1×10⁶ CD19+ human non-Hodgkin lymphoma Raji cells on week 13 after injection of human HSC, followed by tumor size measurements every other day, as previously published.^10,13 Euthanasia was performed when tumor interfered with ambulation or nutrition, obstructed any orifice opening, developed skin ulceration, or otherwise achieved a size that interfered with normal activity or body functions.

Cohorts of mice were treated with cetuximab at 1mg/mouse intraperitoneally daily for 12 days before euthanasia, blood sampling, and spleen and bone marrow harvest, as described.²¹

Conjugation of cetuximab

As previously published,^32,33 the pH of 75μL (150μg) of cetuximab was adjusted with Na₂CO₃, and molar excess of p-isothiocyanatobenzyl-desferrioxamine (SCN-DFO) was added for incubation at 37°C for 30min. During incubation, a Bio-Rad Micro Bio-Spin Chromatography column was prepped per manufacturer instructions. After incubation, the sample was added to the column and placed in the centrifuge at 1,000 g for 4min. This sample was then measured in Nanodrop to determine concentration and recovery, and run on a sodium dodecyl sulfate polyacrylamide gel with the unconjugated cetuximab in non-reducing and reducing conditions to confirm conjugation.

Cetuximab radiolabeling with ⁸⁹zirconium

A previously published protocol was followed.^32,33 The activity and volume of [89Zr]Zr-oxalic were measured, and 40% of 2M Na₂CO₃ was added, with pH adjustment with HEPES. The desired amount of Zr-89 was added to obtain approximately 500μCi per 100μg of cetuximab, and then incubated for 1h at room temperature. To determine radiolabeling efficiency, instant thin-layer chromatography (ITLC) was done using chromatography strips and citric acid 20mM, and samples were run through a gamma counter. Radiolabeling efficiency was determined by calculating the cpm in section #1 on the strip, representing the conjugated antibody, divided by the cpm of section #1 plus the cpm of section #2. To determine radiochemical purity, a BioRad Micro Bio-Spin Chromatography column was prepped as described above. ITLC was repeated with this sample, and the purity was determined by calculating the cpm of section #1 divided by the cpm of section #1 plus cpm of section #2.

Positron emission tomography/computed tomography imaging of gene-modified NSG mice

All mice for imaging were injected via the tail vein with 10μg ⁸⁹Zr-cetuximab and imaged on Inveon positron emission tomography (PET) and CrumpCAT computed tomography (CT) at the California NanoSystems Institute at 24 and 120h after injection. The images were processed and analyzed using Amide's Medical Imaging Data Examiner. Immediately after animal imaging, mouse tissues were dissected and imaged for biodistribution calculations, defined as percentage of injected dose per gram of tissue (% ID/g).

Gene modification of human HSC did not impair proliferation or differentiation

Transduced HSC and their progeny were evaluated for proliferation adjusted for the starting cell population. There was no significant difference in proliferation of eGFP-, EGFRt-, or EQ-transduced cells using either the short-term BBMM culture or myeloid differentiation cultures (Fig. 1F). The mean (SEM) fold proliferation after 9 days in short-term BBMM culture was 95.9 (27.7) for eGFP (n=5), 61.1 (15.2) for EGFRt (n=8), and 85.5 (31.7) for EQ (n=6) compared to 77.6 (16.3) for mock-transduced cells (n=11, p=0.93). The mean (SEM) fold proliferation after 14 days in myeloid differentiation culture was 210.8 (68.9) for eGFP (n=4), 149.5 (85.9) for EGFRt (n=5), and 230.1 (87.2) for EQ (n=4) compared to 207.8 (54.9) for mock-transduced cells (n=7, p=0.66; Fig. 1F). These results are similar to previously reported findings on human HSC transduced with CD19-specific CAR.¹⁰

Similar to published evaluations of clonogenic assays of CAR-modified HSC,¹⁰ gene-modified HSC were plated at 100 cells/plate in methylcellulose for clonogenic assays, and then evaluated by optic microscopy 14 days later. Once again, there was no significant difference in the plating efficiency between the different transduced human HSC (Fig. 1G, left panel). The percentages (SEM) of plating efficiency in eGFP- (n=2), EGFRt- (n=2), and EQ-transduced (n=2) cells were 37.7% (4.9), 61% (8), and 43.3% (9.3), respectively, compared to the mock-transduced cells (47.7 [4.8]; p=0.28; Fig. 1G, left panel). There was a similar proportion of CFU-monocyte, CFU-granulocyte, CFU-granulocyte/monocyte (CFU-GM), CFU-erythroid, burst forming unit-erythroid (BFU-E), and CFU-granulocyte/erythroid/monocyte/megakaryocyte in non-transduced and transduced cells (Fig. 1G, left panel). The plating efficiency of BFU-E in EGFRt-transduced cells was 1.5×higher than in mock-transduced cells. However, statistical analysis showed no significant difference (p=0.146; Fig. 1G, left panel). To demonstrate that transduced cells contributed to overall plating efficiency counted on clonogenic assay, colonies were individually selected and evaluated for VCN analysis, and the results confirm that gene-modified cells are well represented in the CFU counted on the clonogenic assay, with a similar pattern to the copy numbers found on cells cultured in BBMM and myeloid differentiating conditions (Fig. 1G, right panel). The overall mean VCN of colonies arising from EGFRt-transduced HSC was 0.59, and the mean VCN of colonies from EQ-transduced HSC was 0.49 (Fig. 1G, right panel).

As seen with human T cells transduced with CD19RCD28 and EQ (Fig. 1B), myeloid cells differentiated from gene-modified HSC had engineered antigen-specific cytotoxicity (Fig. 1H), confirming previously published results.^10,21,22 In the experiments, the myeloid cells had lower cytotoxicity compared to T cells due to lower transduction efficiency and because unsorted cells were used on the cytotoxicity assays against the CD19+ Raji cell line. CAR expression in myeloid cells was 5.3% in EQ-transduced cells and 16.9% in CD19RCD28-transduced cells, but these populations determined statistically significant antigen-specific killing compared to non-transduced (mock) cells (Fig. 1H),

Direct antibody cytotoxicity of cetuximab on EGFRt-transduced cells

To evaluate direct antibody toxicity of the commercially available monoclonal antibody cetuximab, mock-transduced Jurkat cells and Jurkat cells transduced with CD19RCD28, EGFRt, and EQ with >95% of transgene expression were used as targets for in vitro cytotoxicity analysis (Fig. 2A). There was no significant increase in cytotoxicity with different concentrations (0, 0.3, 1, and 10μg/mL) of cetuximab in any of the Jurkat cells (p=0.38). There was an isolated significant increase in cytotoxicity in Jurkat-EGFRt cells with the supra-therapeutic cetuximab concentration (10μg/mL) compared to CD19RCD28 (p=0.0235) or EQ (p=0.0165), but not with any of the other cetuximab concentrations (Fig. 2A). These findings support the conclusion that cetuximab is not expected to exert direct cytotoxicity on targets expressing the truncated EGFRt antigen (solely expressed on the cell membrane without EGFR signaling transduction in the cells transduced with EQ and EGFRt constructs used in this study) binding the antibody at concentrations up to 10μg/mL.^21,22,34

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Figure 2.

In vitro ablation of gene-modified cells by cetuximab. (A) Jurkat cell line non-modified or transduced with CD19RCD28, EGFRt, or EQ, treated in culture with cetuximab for assessment of direct antibody-mediated cytotoxicity at different concentrations. Statistical analysis: two-way ANOVA for antibody concentration (p=0.38 comparing the different cetuximab concentrations) and among groups. There was isolated significant cytotoxicity increase in Jurkat-EGFRt cells with the supra-therapeutic cetuximab concentration (10μg/mL) when compared to CD19RCD28 (p=0.0235) or EQ (p=0.0165), but not on any of the other cetuximab concentrations. (B) Complement-dependent cytotoxicity on non-modified Jurkat cells or transduced with CD19RCD28, EGFRt, or EQ in the presence or absence of cetuximab. Statistical analysis: two-way ANOVA for the different complement doses and among groups. (C) ADCC assay of non-transduced and EGFRt-transduced Jurkat cells using primary human leukocytes in the presence or absence of cetuximab. Statistical analysis: two-way ANOVA for the different vector constructs (p<0.0001) and the presence of cetuximab (p<0.0001). (D) ADCC assay of non-transduced and EGFRt-transduced Jurkat cells using primary human NK cells in the presence or absence of cetuximab. Statistical analysis: two-way ANOVA for the different vector constructs (p<0.0001) and the presence of cetuximab (p<0.0001). Representative of four experiments. Data are presented as the mean±SEM; *p<0.03, and NS indicates statistically not significant. ADCC, antibody-dependent cell-mediated cytotoxicity; ANOVA, analysis of variance; HI, heat inactivated; NK, natural killer.

Complement-dependent cytotoxicity of cetuximab on EGFRt-transduced cells

To evaluate direct complement toxicity on target cells, non-transduced Jurkat cells and Jurkat cells transduced with CD19RCD28, EGFRt, and EQ with >95% of transgene expression were incubated in medium with different concentrations of rabbit complement. All cells incubated at 2% dilution of complement revealed a significant increase of cytotoxicity compared to complement doses of 0.5% or 1% and to medium only (p<0.0001). However, there was no difference in cytotoxicity between transduced Jurkat cells versus non-transduced Jurkat, or between cells treated with 1μg/mL cetuximab versus non-treated cells, except for the EQ-transduced cells in both non-treated (p=0.0025) and cetuximab-treated (p<0.0001) conditions at 2% complement (Dunnett's multiple comparisons test against non-transduced, non-treated target cells). These findings are consistent with a direct dose-dependent cytotoxicity of complement on all target cells, which is not specific to the presence of cetuximab or EGFRt expression on cells transduced with EQ or EGFRt constructs (Fig. 2B).

To confirm whether the addition of complement determined direct cytotoxicity, the same Jurkat target cells, with or without cetuximab treatment at a dose of 1μg/mL, were exposed to 2% complement inactivated at 56°C for 60min. With heat-inactivated (HI) complement, cytotoxicity decreased to 0–16% across all groups, showing that complement is required for cytotoxicity and supporting the assertion the direct cytotoxicity by complement is not antigen-specific nor antibody-mediated at the cetuximab dose of 1μg/mL (Fig. 2B).

The findings described in Fig. 2B reflect the published literature suggesting that complement is not a relevant mediator for cetuximab cytotoxicity on cancer cells, as complement-dependent cytotoxicity by cetuximab has only been described on cell lines with very high expression of EGFR.³⁴

ADCC of cetuximab on EGFRt-transduced cells

Having evaluated the direct cytotoxicities of cetuximab and complement on cells expressing EGFRt, the next experiments addressed ADCC. Total human leukocytes were isolated from whole donor blood, with a yield of 2–5×10⁵ cells/mL of blood for in vitro cell-mediated cytotoxicity assays. By flow cytometry analysis, a typical composition of leukocyte phenotype subsets was 7.5% CD8+, 7.9% CD4+, 5.3% CD14+, 14.6% CD3+, 6.2% CD33+, 5.1% CD19+, and 16.2% CD56+ (data not shown).

After 4h of incubation with target cells and cetuximab, increased cytotoxicity was significant (p<0.0001) only in leukocytes incubated with EGFRt-expressing or EQ-expressing Jurkat cells with cetuximab at 1μg/mL. This was true for E:T ratios of 2:1, 5:1, and 10:1 at p<0.01, and only for EQ-transduced cells at 1:1 (p=0.0210), and only for EGFRt-transduced cells at 20:1 (p<0.0001; Fig. 2C).

Similarly, human NK cells were isolated from whole donor blood, with a yield of 0.4–0.5×10⁶ cells/mL of blood. A typical flow cytometry analysis of a NK cell suspension was 0.1% CD14+, 1.4% CD33+, 95.5% CD56+, 0.2% CD19+, and 0.3% CD3+ (data not shown). Cytotoxicity assays of NK cells incubated for 4h at different E:T ratios with target cells and cetuximab (1μg/mL) showed significantly higher NK-mediated cytotoxicity (p<0.0001) against EGFRt-expressing or EQ-expressing Jurkat cells compared to non-transduced cells (Fig. 2D). This was true for all E:T ratios at p<0.0001.

The results described on Fig. 2C and D provide evidence that cytotoxicity by cetuximab is dependent on ADCC mediated by neutrophils and NK cells, in accordance with previous publications.³⁴

CAR-modified HSC successfully engraft in vivo, establish survival advantage to tumor challenge, and are amenable to ablation

In vivo assessment of the engraftment, proliferation, and differentiation of modified human HSC was performed by the intrahepatic injection of mock-transduced, EGFRt-transduced, and EQ-transduced human HSC into sub-lethally irradiated NSG pups. Human hematopoiesis in transplanted mice was analyzed through harvest of bone marrows and spleens at 16 weeks post transplant. Human hematopoietic engraftment (as detected by the percentage of human CD45+ cells by flow cytometry) for both compartments was not significantly different between the EQ-, EGFRt-, and mock-transduced arms (Fig. 3A), confirming that gene modification did not impair HSC proliferation.

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Figure 3.

In vivo engraftment, anti-CD19 activity and ablation of gene-modified human HSC. (A) Humanization of NSG mice injected with non-transduced and gene-modified HSC in the bone marrow (left panel) and spleen (right panel), defined by the detection of the human CD45 marker as a percentage of all viable cells. (B) Kaplan–Meier curve of humanized mice challenged with subcutaneous injection of CD19+ Raji cell line and followed over 80 days; Log-rank test with p=0.0003 for the comparison among the different mouse populations. (C) Comparison of flow cytometry expression of EGFRt after treatment with vehicle (PBS; black circles) versus cetuximab (white squares) in the bone marrow and spleen of humanized NSG. (D) Box and whiskers graphical representation of the data presented in (C). Each box extends from the 25th to 75th percentiles, and the whiskers depict minimum to maximum values. (E) Comparison of lentiviral VCN after treatment with vehicle (PBS; black circles) versus cetuximab (white squares) in the bone marrow and spleen of humanized NSG. (F) Box and whiskers graphical representation of the data presented on (E). Each box extends from the 25th to 75th percentiles, and the whiskers depict minimum to maximum values. Representative of four experiments. Data are presented as the mean±SEM; *p<0.05 by Student's t-test (A and C–F), and NS indicates statistically not significant. In (C) and (E), each symbol represents an individual mouse for each tissue. PBS, phosphate-buffered saline.

Humanized mice were challenged with a subcutaneous injection of CD19-expressing Raji human cell line to evaluate engineered antigen-specific cytotoxicity using previously published methods.^10,13 All wild-type non-humanized NSG mice injected with Raji cells did not survive the tumor challenge, similar to the NSG humanized with non-transduced HSC or EGFRt-transduced HSC (Fig. 3B). Similar to previously published data,¹⁰ NSG mice humanized with CAR-modified HSC had a survival advantage facing the tumor challenge, with 60% of mice engrafted with HSC transduced with CD19RCD28 and 28.5% of mice engrafted with HSC transduced with EQ surviving (p=0.0003; Fig. 3B).

To evaluate the ablation in vivo of gene-modified cells, additional cohorts of mice humanized with EQ-, EGFRt-, or mock-transduced human HSC were then given either an intraperitoneal injection of cetuximab 1mg/mouse or PBS for 12 consecutive days. On day 13, cells harvested from the bone marrow and spleen were evaluated by flow cytometry detection of EGFRt expression and with VCN assessment by ddPCR.

When evaluated for EGFRt expression by flow cytometry, human cells from spleens of EGFRt-HSC-transplanted mice were on average 28.02% positive for EGFRt if treated with vehicle, and 9.51% positive for EGFRt if treated with cetuximab (p<0.001). When comparing human cells from cetuximab-treated versus vehicle-treated mice in bone marrow, there was 34.18% EGFRt positivity in the vehicle group versus 7.60% in the cetuximab group (p=0.003; Fig. 3C). For EQ-HSC-transplanted mice, evaluation of EGFRt expression by flow cytometry from spleens was 15.54% if treated with vehicle compared to 10% if treated with cetuximab (p=0.046). For bone marrow, there was 24.75% EGFRt positivity in the mice in the vehicle group compared to 10.43% in mice treated with cetuximab (p<0.001). The significant decrease in EGFRt-expressing cells in the spleen and bone marrow after cetuximab treatment demonstrate the elimination of gene-modified cell progeny in mice humanized with EGFRt- or EQ-transduced HSC. Mice harvested up to 5 weeks after cetuximab treatment showed similar levels of expression of EGFRt, suggesting long-lasting effect of the ablation. Figure 3D presents the same data in Fig. 3C in a box and whiskers graphic representation, allowing clear observation of the significant drop in EGFRt expression in the bone marrow and spleen of humanized mice from both EGFRt- and EQ-transduced HSC groups.

When evaluated for lentiviral transduction by VCN, cells from the spleen of EGFRt-HSC-transplanted mice had an average VCN of 2.21 if treated with vehicle compared to 0.40 if treated with cetuximab (p<0.001). In the bone marrow, the average VCN was 3.48 in the vehicle-treated group, which decreased to 0.59 in the cetuximab-treated group (p<0.001; Fig. 3E). Cells from the spleen of EQ-HSC-transplanted mice had an average VCN of 0.18 if treated with vehicle compared to 0.10 if treated with cetuximab (p=0.034). In the bone marrow, the average VCN was 0.54 in the vehicle group and 0.20 in the cetuximab-treated group (p=0.005). Consistent with the flow cytometry analysis, in the spleen and bone marrow, there were significant differences in VCN between the vehicle group and the cetuximab-treated group (Fig. 3E and F). Mice harvested up to 5 weeks after cetuximab treatment showed similar levels of VCN, suggesting a long-lasting effect of the ablation.

Support was provided by the UCLA Department of Pediatrics 3K12HD034610 and the NIH/NHLBI 2T32HL086345-06 Training Grant in Developmental Hematology, NCI 1K23CA222659, American Society of Hematology Scholar Award, Hyundai Hope on Wheels Scholar Award, Miranda D. Beck Pediatric Cancer Research Foundation, Gwynne Hazen Cherry Memorial Fund, the Pediatric Cancer Research Fund, UCLA Children's Discovery and Innovation Institute, UCLA Jonsson Comprehensive Cancer Center, UCLA Cancer Research Coordinating Committee, UCLA Clinical and Translational Science Institute Grant UL1TR000124, and the St. Baldrick's Foundation. All the authors acknowledge the collaboration with Drs. Stephen Forman and Christine Brown, from City of Hope, who provided the EGFRt and EQ CAR constructs, and the training and technical support of the Flow Cytometry Core of the UCLA Broad Stem Cell Research Center (BSCRC), where all flow cytometry experiments were performed. The authors also acknowledge the valuable scientific advice and technical support from Dr. Gay Crooks, MBBS, and Donald B. Kohn, MD, and the help on data acquisition and analyses from Felix Bergara Salazar, Andy Tu, Tulika Tyagi, Therese Dinoso, and Nezia Rahman.