Cell lines

The K562, Raji, JeKo-1, MDA-MB-231, MDA-MB-468, and 293T cell lines were obtained from the American Type Culture Collection. Dr. Edus H. Warren (FHCRC) kindly provided the renal cell cancer lines FARP, TREP and RWL. K562/ROR1 and Raji/ROR1 were generated by lentiviral transduction with the full-length ROR1-gene. JeKo-1/ffluc were derived by lentiviral transduction with the firefly luciferase (ffluc)-gene.

Immunophenotyping

Immunophenotyping was performed with conjugated mAbs: CD3, CD4, CD5, CD8, CD19, CD28, CD45RO, CD62L, CD314 (NKG2D), MICA/B and matched isotype controls (BD Biosciences). Propidium iodide (PI) staining was performed for live/dead cell discrimination. Cell surface expression of ROR1 was analyzed using a polyclonal goat anti-human-ROR1 antibody (R&D Systems) (10). Surface expression of 2A2 ROR1-CARs was analyzed using a polyclonal goat anti-mouse-IgG antibody (Fab-specific) (Jackson ImmunoResearch). Flow analyses were done on a FACSCanto, sort-purifications on a FACSAriaII (Becton Dickinson) and data analyzed using FlowJo software (Treestar).

Vector construction and preparation of CAR-encoding lentivirus

ROR1-specific and CD19-specific CARs were constructed using VL and VH segments of the 2A2 and R12 (ROR1) (24, 25), and FMC63 mAbs (CD19) (26). Each scFV was linked by a (G₄S)₃ peptide to a spacer domain derived from IgG4-Fc (Uniprot Database: P01861) comprising either ‘Hinge-CH2-CH3’ (229 AA), ‘Hinge-CH3’ (119 AA) or ‘Hinge-only’ (12 AA) sequences. All spacers contained a S→P substitution within the ‘Hinge’ domain located at position 108 of the native IgG4-Fc protein, and were linked to the 27 AA transmembrane domain of human CD28 (Uniprot: P10747) and to a signaling module comprising either (i) the 41 AA cytoplasmic domain of human CD28 with an LL→GG substitution located at positions 186-187 of the native CD28 protein (27) or (ii) the 42 AA cytoplasmic domain of human 4-1BB (Uniprot: Q07011), each of which was linked to the 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Uniprot: P20963). The construct encoded a T2A ribosomal skip element (28) and a tEGFR sequence downstream of the CAR (29). Codon-optimized nucleotide sequences encoding each transgene were synthesized (Life Technologies) and cloned into the epHIV7 lentiviral vector (29, 30). ROR1-CAR, CD19-CAR or tEGFR-encoding lentiviruses were produced in 293T cells using the packaging vectors pCHGP-2, pCMV-Rev2 and pCMV-G.

Generation of T-cell lines expressing ROR1 and CD19-CARs

CD8⁺ CD45RO⁺ CD62L⁺ central memory T-cells (T_CM) or bulk CD4⁺ T-cells were sorted from PBMC of normal donors, activated with anti-CD3/CD28 beads (Life Technologies), and transduced on day 3 after activation by centrifugation at 800 g for 45 min at 32°C with lentiviral supernatant (MOI = 3) supplemented with 1 μg/mL polybrene (Millipore). T-cells were expanded in RPMI with 10% human serum, 2 mM L-glutamine and 1% penicillin-streptomycin (CTL medium), supplemented with recombinant human IL-2 to a final concentration of 50 U/mL. The tEGFR⁺ subset of each T-cell line was enriched by immunomagnetic selection with biotin-conjugated anti-EGFR mAb (ImClone Systems) and streptavidin-beads (Miltenyi). ROR1-CAR and tEGFR control T-cells were expanded using a rapid expansion protocol (31), and CD19-CAR T-cells were expanded by stimulation with irradiated (8,000 rad) B-LCL at a T-cell:LCL ratio of 1:7. T-cells were cultured in CTL medium with 50 U/mL IL-2.

Cytotoxicity, cytokine secretion and proliferation assays

Cytotoxicity: Target cells were labeled with ⁵¹Cr (PerkinElmer), washed and incubated in triplicate at 1-2×10³ cells/well with effector T-cells at various effector to target (E:T) ratios. Supernatants were harvested for γ-counting after a 4-hour incubation and specific lysis calculated using the standard formula. Cytokine secretion: 5×10⁴ T-cells were plated in triplicate with target cells at an E:T ratio of 1:1 (primary CLL), 2:1 (Raji/ROR1; JeKo-1), 4:1 (K562/ROR1, K562/CD19 and K562) or 10:1 (MDA-MB-231), and IFN-γ, TNF-α and IL-2 measured by ELISA or multiplex cytokine immunoassay (Luminex) in supernatant removed after 24-h incubation. In experiments blocking NKG2D signaling, anti-NKG2D (clone 1D11), anti-MICA/B (clone 6D4, all from BD) and anti-ULBP (kindly provided by Dr. Veronika Groh, FHCRC) were used at saturating concentrations. Proliferation: T-cells were labeled with 0.2 μM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), washed and plated in triplicate with stimulator cells in medium without exogenous cytokines. After 72-h incubation, cells were labeled with anti-CD8 mAb and PI, and analyzed by flow cytometry to assess cell division of live CD8⁺ T-cells.

Experiments in NOD/SCID/γc^-/- (NSG) mice

The Institutional Animal Care and Use Committee approved all mouse experiments. Six-to 8-week old female NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice were obtained from the Jackson Laboratory or bred in-house. Mice were injected with 0.5×10⁶ JeKo-1/ffluc tumor cells via tail vein and received a subsequent tail vein injection of CAR-modified or control T-cells.

For bioluminescence imaging of tumor growth, mice received intraperitoneal injections of luciferin substrate (Caliper Life Sciences) resuspended in PBS (15 μg/g body weight). Mice were anesthetized with isoflurane and imaged using an Xenogen IVIS Imaging System (Caliper) 10, 12 and 14 minutes after luciferin injection in small binning mode at an acquisition time of 1 s to 1 min to obtain unsaturated images. Luciferase activity was analyzed using Living Image Software (Caliper) and the photon flux analyzed within regions of interest that encompassed the entire body or the thorax of each individual mouse.

ROR1-CARs derived from a mAb R12 with higher affinity than 2A2 mediate superior anti-tumor reactivity

We next examined whether increasing the affinity of the scFV used to construct the ROR1-CAR might influence tumor recognition and T-cell function. We generated ROR1-specific CARs from the mAb R12 that like 2A2, binds to an epitope in the NH2-terminal Ig-like/Frizzled domain of ROR1 but with >50-fold higher monovalent binding affinity (24, 25). R12 ROR1-CARs were constructed with both long and short IgG4-Fc spacers to determine whether the optimal spacer design for this higher affinity scFV differed from that for a lower affinity scFV. We found that similar to 2A2, the short spacer R12 ROR1-CAR conferred improved cytolytic activity, cytokine secretion and proliferation (data not shown), suggesting that the shorter spacer length provides superior spatial engagement of the T-cell and ROR1⁺ target cell for T-cell activation.

We then designed R12 and 2A2 ROR1-CARs that contained an optimal (short) extracellular spacer, and either a CD28 or 4-1BB costimulatory domain in tandem with CD3ζ (4 constructs) for comparison (Suppl. Fig. 1B). These ROR1-CAR constructs were expressed in purified CD8⁺ T_CM of healthy donors, and we confirmed equivalent transgene expression by tEGFR staining (Fig. 2A). T-cells modified with each of the 2A2 and R12 ROR1-CARs specifically lysed K562/ROR1 and Raji/ROR1 tumor cells with approximately equivalent efficiency (Fig. 2B). However, analysis of cytokine production showed that the high affinity R12 ROR1-CARs that contained CD28 or 4-1BB conferred significantly higher IFN-γ, TNF-α and IL-2 production compared to the corresponding 2A2 constructs (Fig. 2C). Consistent with previous work, we found that T-cells expressing CARs with a CD28 costimulatory domain produced more IFN-γ, TNF-α and IL-2 compared to those with 4-1BB (32-34). Experiments to analyze the proliferation of ROR1-CAR T-cells showed a higher percentage of proliferating T-cells and a higher number of cell divisions in T-cells expressing the high affinity R12 ROR1-CARs with CD28 and 4-1BB domain compared to T-cells expressing the respective 2A2 counterparts (Fig. 2D). There was more vigorous proliferation in T-cells that expressed CARs with a CD28 domain, consistent with higher IL-2 production induced by these receptors. There was a lower frequency of AICD as measured by PI staining in T-cell lines modified with R12 compared to 2A2 ROR1-CARs after stimulation with Raji/ROR1 and JeKo-1 tumor cells respectively (R12: 5.6%/6.9% vs. 2A2: 10%/9.65%), and T-cell lines that expressed CARs with CD28 domain had lower AICD compared to 4-1BB (R12: 16.4%/18.4% vs. 2A2 38.1%/39.6%).

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Figure 2

Anti-tumor reactivity of T-cells modified with ROR1-CARs derived from mAb R12 with higher affinity than 2A2

(A) tEGFR expression on purified polyclonal CD8⁺ T_CM-derived T-cell lines modified with each of the R12 and 2A2 ROR1-CARs with short IgG4-Fc ‘Hinge’ spacer, and CD28 or 4-1BB costimulatory domain. (B) Cytotoxicity against ROR1⁺ and control target cells by T-cells expressing R12 and 2A2 ROR1-CARs or a tEGFR control vector. (C) Multiplex cytokine assay of supernatants obtained after 24 hours from co-cultures of 5×10⁴ T-cells expressing the various ROR1-CARs with Raji/ROR1 tumor cells. The middle/right bar diagrams show normalized multiplex data from 3 independent experiments (cytokine release by ROR1-CAR 2A2 = 1) analyzed by Student's t-test. (D) Proliferation of ROR1-CAR T-cells and tEGFR control T-cells 72 hours after stimulation with Raji/ROR1 cells and without addition of exogenous cytokines was assessed by CFSE dye dilution. Numbers above each histogram indicate the number of cell divisions the proliferating subset underwent, and the fraction of T-cells in each gate that underwent ≥4/3/2/1 cell divisions is provided above each plot.

To determine if the enhanced function observed with R12 ROR1-CARs in CD8⁺ T-cells extended to CD4⁺ T-cells, we transduced bulk CD4⁺ T-cells with the 2A2 and R12 ROR1-CARs containing the short spacer and CD28 costimulatory domain. In response to Raji/ROR1⁺ tumor cells, CD4⁺ T-cells that expressed the high affinity R12 scFV produced higher levels of IFN-γ, TNF-α, IL-2, IL-4, and IL-10, and underwent greater proliferation than CD4⁺ T-cells that expressed 2A2 (Suppl. Fig. 2A, B). Both cytokine production and proliferation was superior in CD4⁺ compared to CD8⁺ T-cells modified with the same ROR1-CARs. In summary, our data demonstrate that tailoring both the length of the non-signaling extracellular CAR spacer domain and scFV affinity are independent parameters that affect the function of ROR1-CAR T-cells.

CD8⁺ T-cells modified with a high affinity ROR1-CAR have comparable activity to a CD19-CAR against primary CLL in vitro

ROR1 and CD19 are both uniformly expressed on all primary CLL (Fig. 3A), however the absolute number of ROR1-molecules per tumor cell is estimated to be 10-fold lower than that of CD19, which has been successfully targeted in clinical trials with CD19-CAR T-cells (1, 5, 10, 14). We compared recognition of primary CLL by CD8⁺ T-cells expressing the optimized R12 and 2A2 ROR1-CARs, and a CD19-CAR derived from the FMC63 scFV (1-3, 5, 35). We used purified CD8⁺ T_CM for CAR-modification to provide a uniform cell product and each CAR contained a short IgG4-Fc ‘Hinge-only’ spacer and 4-1BB costimulatory domain. We confirmed our CD19-CAR (IgG4 Hinge) was at least equivalently effective in recognizing CD19⁺ tumors as a CD19-CAR with CD8a Hinge spacer and 4-1BB costimulatory domain that is being used in ongoing clinical trials (Suppl. Fig. 3A-C) (1, 5). The cytolytic activity of R12 ROR1-CAR T-cells against primary tumor cells from multiple CLL patients (n=4) was higher compared to T-cells modified with the lower affinity 2A2 ROR1-CAR, and equivalent to the lysis observed with CD19-CAR T-cells (Fig. 3B). Multiplex cytokine analysis showed nearly equivalent production of IFN-γ and TNF-α, but less IL-2 production by CD8⁺ T-cells expressing the R12 ROR1 compared with those expressing the CD19-CAR after co-culture with primary CLL (Fig. 3C). 2A2 ROR1-CAR T-cells produced lower amounts of all cytokines than R12 ROR1-CAR T-cells as noted previously. Cytokine production by all of the CAR-transduced T-cells after stimulation with CLL was substantially less than with Raji/ROR1, which unlike CLL expresses both CD80 and CD86 that can engage CD28 expressed on CAR T-cells (Fig. 3A, C).

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Figure 3

Recognition of primary CLL by T-cells modified with 2A2 and R12 ROR1-CARs with optimal short spacer and 4-1BB costimulatory domain or with a CD19-specific CAR

(A) Expression of ROR1/CD19 on primary CLL, and CD80/86 on primary CLL and Raji/ROR1 tumor cells (black dot plots) that can engage CD28 on CAR T-cells (black histograms). Staining with matched isotype control mAbs is shown as grey dot plots/histograms. (B) Cytolytic activity of T-cells expressing the 2A2 and R12 ROR1-CAR, a CD19-specific CAR and T-cells modified with a tEGFR control vector against primary CLL (left diagram) and normal B cells (right diagram) analyzed by chromium release assay. Cytotoxicity data against primary CLL from 4 independent experiments (E:T = 30:1) were normalized (cytolytic activity by ROR1-CAR 2A2 = 1) and analyzed by Student's t-test (bar diagram). (C) Multiplex cytokine analysis after a 24 hour stimulation of 5×10⁴ CAR T-cells with primary CLL cells. Cytokine release of unstimulated CAR T-cells was below 3.6 pg/mL (detection limit) (left bar diagram). ELISA for IFN-γ production by 5×10⁴ 2A2 and R12 ROR1-CAR T-cells after a 24-hour co-culture with primary CLL. O.D. of 1 corresponds to approximately 250 pg/mL (right bar diagram). (D) Proliferation of CD8⁺ T-cells modified with the 2A2 ROR1, R12 ROR1 and a CD19-CAR, 72 hours after stimulation with primary CLL cells. Numbers above each histogram indicate the number of cell divisions, and the fraction of T-cells in each gate that underwent ≥3/2/1 cell divisions is provided next to each plot.

We observed less proliferation of T-cells expressing the R12 and 2A2 ROR1-CAR compared to the CD19-CAR after stimulation with CLL (CD19>R12>2A2) (Fig. 3D). We hypothesized that proliferation of CD8⁺ ROR1-CAR T-cells in response to CLL may be augmented in the presence of CAR-modified CD4⁺ T-cells because of their higher secretion of IL-2 compared to CD8⁺ T_CM (Suppl. Fig. 2A; Suppl. Fig. 4A). To test this possibility, we performed in vitro co-culture experiments where CD4⁺ and CD8 T_CM were separately modified with the R12 ROR1, 2A2 ROR1 and CD19-CARs respectively, enriched for CAR expression, and combined at a 1:1 ratio to ensure equivalent proportions of CD8⁺ and CD4⁺ T-cells modified with each of the vectors. These cells were CFSE-labeled and stimulated with primary CLL. We observed a dramatic increase in proliferation of CD8⁺ R12 ROR1-CAR T-cells after addition of CAR-transduced, but not untransduced CD4⁺ T-cells (Suppl. Fig. 4B). Notably, when provided with CD4-help, we observed equivalent proliferation of R12 ROR1 and CD19-CAR CD8⁺ T-cells in response to CLL, whereas proliferation of CD8⁺ T-cells expressing the lower affinity 2A2 ROR1-CAR remained less. Collectively, our data show that the high affinity R12 ROR1-CAR confers superior reactivity compared to 2A2 against primary CLL cells in vitro.

ROR1-CAR T-cells mediate in vivo anti-tumor activity in a mouse model of systemic mantle cell lymphoma

It remained uncertain whether the superior in vitro activity of T-cells modified with the higher affinity R12 CAR would translate into improved anti-tumor activity in vivo, and how targeting ROR1 would compare to CD19. To address these questions, we inoculated cohorts of immunodeficient NSG mice with the human MCL line JeKo-1/ffluc by tail vein injection, and seven days later when tumor was disseminated, treated the mice with a single intravenous dose of R12 ROR1, 2A2 ROR1 or CD19-CAR CD8⁺ T-cells. Control mice were treated with tEGFR T-cells or untreated. All CARs had the optimal short spacer and the 4-1BB costimulatory domain. Untreated NSG/JeKo-1 mice developed a rapidly progressive systemic lymphoma necessitating euthanasia approximately 4 weeks after tumor inoculation (Fig. 4A-C). We observed tumor regression and improved survival in all mice treated with R12 ROR1, 2A2 ROR1 and CD19-CAR T-cells. Mice treated with R12 ROR1-CAR T-cells had a superior anti-tumor response and survival compared to mice treated with 2A2 ROR1-CAR T-cells (p<0.01), and comparable anti-tumor activity to mice treated with CD19-CAR T-cells (Fig. 4A-C).

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Figure 4

In vivo anti-tumor efficacy of 2A2 ROR1, R12 ROR1 and CD19-CAR T-cells

Cohorts of mice were inoculated with 0.5×10⁶ JeKo-1/ffluc MCL via tail vein injection, and 5×10⁶ 2A2 ROR1, R12 ROR1 or CD19-CAR T-cells, or T-cells expressing a tEGFR control vector were administered 7 days after tumor inoculation. All CAR constructs had the short IgG4 ‘Hinge-only’ spacer and a 4-1BB costimulatory domain. (A, B) Serial bioluminescence imaging of tumor in cohorts of mice treated with T-cells expressing the 2A2 ROR1-CAR, the high affinity R12 ROR1-CAR, a CD19-specific CAR, with T-cells transduced with tEGFR alone, and untreated mice. Bioluminescence imaging showed tumor manifestations in the bone marrow and thorax and thus, signal intensity was measured in regions of interest that encompassed the entire body and thorax of each individual mouse. (C) Kaplan-Meier analysis of survival in individual treatment and control groups. Statistical analyses were performed using the log-rank test. The data shown in A-C are representative of results obtained in 2 independent experiments. (D) Proliferation of 2A2 ROR1, R12 ROR1 and CD19-CAR T-cells in vivo. Tumor bearing NSG/JeKo-1 mice received a single dose of 5×10⁶ CFSE-labeled 2A2 ROR1, R12 ROR1 or CD19-CAR T-cells on day 7 after tumor inoculation, and 72 h later peripheral blood, bone marrow and spleen were collected from each individual mouse. The frequency and proliferation of live (PI^-), CD45⁺ CD8⁺ tEGFR⁺ T-cells was analyzed. The frequency of 2A2 ROR1, R12 ROR1 and CD19-CAR T-cells respectively is provided on the left of each histogram as percentage of live cells, and the fraction of T-cells that underwent ≥4/3/2/1 cell divisions is provided above each plot.

We analyzed the frequency of CAR T-cells in the peripheral blood following adoptive transfer and detected higher numbers of tEGFR⁺ T-cells in mice treated with the R12 CAR compared to the 2A2 ROR1-CAR, suggesting more vigorous proliferation in vivo improved tumor control. To confirm this, we administered CFSE-labeled CD19-CAR, R12 and 2A2 ROR1-CAR T-cells to cohorts of NSG mice bearing JeKo-1/ffluc, and analyzed T-cell proliferation in the peripheral blood, bone marrow and spleen 72 hours after transfer. A higher percentage of the R12 and CD19-CAR T-cells proliferated and underwent a greater number of cell divisions compared to 2A2 ROR1-CAR T-cells (Fig. 4D). The JeKo-1 tumor eventually recurred in all mice treated with ROR1 or CD19-CAR T-cells (Fig. 4A-C). Tumor recurrence was not a result of the selection of ROR1 or CD19 loss variants, as recurrent tumors were positive for both molecules. For comparison, we analyzed anti-tumor efficacy of CD19-CAR T-cells in NSG mice engrafted with ROR1^- Raji tumors and observed complete tumor eradication, indicating the recurrence of JeKo-1 reflects difficulty eradicating this tumor (data not shown). In summary, this data is the first to show that ROR1-CAR T-cells have anti-tumor efficacy in vivo, and suggest that for B-cell malignancies, an optimized ROR1-CAR such as R12 may be effective and spare normal CD19⁺ B-cells that lack ROR1 expression (10, 14).

The authors thank Cynthia Nourigat, Melissa Comstock and LaKeisha Perkins (Shared Resources, FHCRC) for expertise in performing the mouse experiments. We thank Edus H. Warren and Veronika Groh for cells lines and reagents.

Grant support: This work was supported by the Lembersky family and by grants from the National Institutes of Health CA136551 (S.R.R., M.C.J.), CA114536 and P50CA138293-01 (S.R.R.) and the University of Würzburg (Interdisziplinäres Zentrum für Klinische Forschung, IZKF, Z-4/109 and D-244). M.H. was supported by the Leukemia and Lymphoma Society (LLS). M.H. and D.S. were supported by the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft).

Author contributions: M.H. designed and performed research, analyzed data, and wrote the manuscript. M.T.L.S. and D.S. designed and performed research and analyzed data. P.L.K. performed research and analyzed data. M.C.J. and C.R. provided key reagents and expert advice, and designed experiments. S.R.R. designed and analyzed experiments and wrote the manuscript.