Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors
© Hammill et al. 2015
Received: 29 May 2015
Accepted: 23 October 2015
Published: 15 December 2015
Adoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain.
We prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity.
The DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs.
These findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.
KeywordsCancer Immunotherapy Chimeric antigen receptor CAR Designed ankyrin repeat protein DARPin
Cancer immunotherapy aims to treat tumors by engaging the patient’s immune system. One form of immunotherapy, called adoptive cell transfer, infuses cancer patients with a bolus of tumor-specific T lymphocytes (T cells), and is proving to be an effective treatment for a variety of malignancies [1–3]. In adoptive cell transfer, T cells isolated from a tumor-bearing patient are grown to large numbers ex vivo and are administered back into the patient to induce a robust anti-tumor immune response. Tumor specificity can be achieved by either i) isolating naturally occurring tumor-specific T cells from the patient, or ii) engineering bulk T cells from the peripheral blood to express tumor-specific receptors on their surface. Naturally-occurring tumor-specific T cells are rare and expanding them from a cancer patient is typically a laborious procedure. In contrast, it is becoming relatively easy to engineer readily-available peripheral T cells with tumor-specific receptors through genetic manipulation. Techniques have been developed for this engineering process which are clinically-viable and multiple clinical trials have demonstrated feasibility and efficacy of genetically-engineered T cells for the treatment of cancer [1, 3–9].
Chimeric antigen receptors (CARs), recombinant proteins designed for expression on the surface of T cells, offer one way to engineer T cells with anti-tumor functionality. CARs are composed of an extracellular antigen recognition domain linked to intracellular signaling domains derived from the T cell receptor and co-receptors (including combinations of the signaling regions of CD3ζ, CD28, and/or 4-1BB, for example) such that the T cells become activated following binding of tumor antigen by the CAR. Depending upon the nature of the intracellular signaling domains, this activation event can lead to cytokine production, cytotoxic attack of the tumor, and proliferation of the T cells.
Most CARs developed to date, including those specific for the tumor associated antigens human epidermal growth factor receptor 2 (HER2) [4, 10] and CD19 [3, 7, 8], utilize a single-chain variable fragment (scFv), derived from an antibody, to enable antigen recognition. However, scFvs do not represent the sole or, necessarily, the optimal option for antigen targeting of CARs.
Ankyrin repeats (ARs), one of the most common protein motifs found in nature, are 33 amino acid long sequences composed of a β-turn followed by two anti-parallel α-helices and a loop [11, 12]. Various numbers of these individual ARs stack together to form ankyrin repeat proteins which function as protein binders [11, 13]. Recognizing the potential of these natural ankyrin repeat proteins as alternative target-binding domains, libraries of artificial stacked ARs, called designed ankyrin repeat proteins (DARPins) were developed to allow for the generation of repeat protein binders against a defined target of interest [14, 15]. Each DARPin in these libraries typically consists of between 2 and 6 repeating units; 2–4 repeats containing both fixed (framework sites required for correct AR folding) and variable (randomized sites leading to a diversity of target-binding capacity within the library) amino acid positions sandwiched between non-variable N-terminal and C-terminal capping repeats (essential for correct DARPin folding) [16, 17]. Expression of these genetic DARPin libraries using ribosome or phage display systems allows for the selection of DARPins with the capacity to bind a defined target of interest as well as refine binding affinity for that target .
DARPins offer a number of features which make them attractive for use in the CAR field: 1) they are more compact than scFvs and, thus, take up less space in the genetic transfer vectors typically used for engineering T cells (ex. retrovirus and lentivirus), 2) they are very thermodynamically stable, and 3) they do not require pairing of separate binding domains (e.g. VH and VL of the scFv), allowing the facile linkage of multiple DARPins, with different specificities, which could be used to create a multi-specific CAR.
We tested the utility of DARPins to target CARs using a DARPin specific for the tumor associated antigen HER2 . As a gold standard, we employed an scFv against HER2 . Both targeting elements were incorporated into murine and human CAR scaffolds to rigorously test the suitability of DARPins. Our results demonstrated that targeting CARs with DARPins is as efficacious as targeting CARs with scFvs and supports the further investigation of DARPin CARs.
Results and discussion
Expression of DARPin28z on murine and human T lymphocytes
DARPin28z induces cytokine production by CAR-T cells upon antigen binding
Observed differences in the magnitudes of response may be explained by variances in target binding by the anti-HER2 DARPin vs anti-HER2 scFv. For example, the anti-HER2 DARPin utilized here has an affinity for HER2 of 0.070nM  while the scFv has an affinity of 7.2nM . In addition, the scFv used to generate our scFv28z CAR (FRP5 [20, 22]) binds to the distal-most extracellular loops of HER2 , whereas the anti-HER2 DARPin used to generate our DARPin28z CAR binds HER2 proximal to the cell membrane , which may influence epitope availability. While these differences negate any direct comparisons between DARPin28z and scFv28z efficacy, the above data reveals that the DARPin28z CAR demonstrates an equivalent capacity to activate T cell effector functions, specifically cytokine production, as the scFv28z CAR.
DARPin28z induces CAR-T cell cytotoxicity against HER2-positive tumor cells
These experiments position DARPin-targeted CARs as a suitable alternative to their scFv-targeted counterparts and strongly support the further investigation of DARPins for use targeting CARs; by all measures investigated, DARPin28z performed similarly to scFv28z as a mechanism for targeting T cells against a HER2-positive target.
In our opinion, DARPins offer a number of attractive features as an antigen-targeting domain. First, DARPins are smaller than scFvs; our anti-HER2 scFv is 739 bp  compared to 408 bp for the anti-HER2 DARPin . Since lentivirus titers are often inversely correlated with the size of the lentiviral insert [25, 26], the ability to conserve coding sequence in the lentivirus insert is a desirable feature afforded by using DARPins. Second, it has been argued that DARPins are poorly immunogenic [16, 17], a useful property for the safety and longevity of DARPin-based CAR-T cell therapies. Finally, ARs are amenable to stacking; the number of ARs stacked consecutively in a single protein ranges from one to 33 . As such, we postulate that several DARPin molecules, each with unique antigen-binding properties, could be stacked consecutively to generate a single CAR with the capacity to identify multiple tumor-targets.
Parental D2F2 and human HER2 expressing D2F2/E2 murine mammary tumor cell lines (provided by Dr. Wei-Zen Wei, Barbara Ann Karmanos Cancer Institute, Detroit, MI) were cultured in hi-glucose DMEM supplemented with 5 % FBS (Gibco; Life Technologies, Grand Island, NY), 5 % cosmic calf serum (Fisher Scientific, Waltham, MA), 2.4 mM L-glutamine (BioShop Canada Inc., Burlington, ON), 0.12 mM non-essential amino acids (Gibco), 120U/mL penicillin (Gibco), 120 μg/mL streptomycin (Gibco), 55 μM β-mercaptoethanol (Gibco), and 0.6 mM sodium pyruvate (Sigma-Aldrich Canada Co., Oakville, ON). D2F2/E2 media contained 800 μg/mL geneticin (Gibco). The human tumor cell lines SKBR3, HCC1954, and LOX-IMVI (provided by Dr. Karen Mossman, McMaster University, Hamilton, ON) were cultured in RPMI 1640 supplemented with 10 % FBS, 2 mM L-glutamine, 10 mM HEPES (Roche Diagnostics, Laval, QC), 100U/mL penicillin, 100 μg/mL streptomycin, and 55 μM β-mercaptoethanol. All cell lines were grown at 5 % CO2, 95 % air, and 37 °C.
Generation of CAR retroviruses
Amino acid sequences of CAR domains. The ten n-terminal and ten c-terminal amino acids which flank the protein regions utilized as CAR domains (with the exception of c-myc for which the sequence is listed in its entirety)
Murine CAR sequences:
CD28 transmembrane and cytoplasmic
Human CAR sequences:
CD28 transmembrane and cytoplasmic
Generation of CAR lentiviruses
Human variants of scFv28z and DARPin28z were constructed in which the membrane proximal CD8 hinge region, the transmembrane and cytoplasmic regions of CD28, and the cytoplasmic region of CD3ζ of the murine CARs were replaced with the corresponding regions of their human counterparts (Table 1) (again, the cDNA sequence was synthesized at Genscript). Human scFv28z and DARPin28z CAR sequences were cloned into the lentiviral vector pCCL ΔNGFR (used for the generation of CAR –‘ve transduced human T cells)  (kindly provided by Dr. Megan Levings, University of British Columbia, Vancouver, BC). CARs were cloned downstream of the human EF1α promoter leaving ΔNGFR intact downstream of the minimal cytomegalovirus promoter in a bicistronic vector, pCCL. Third generation lentiviruses were made for each CAR construct; 8 × 106 HEK 293 T cells in 15 cm tissue culture treated dishes (NUNC) (cultured in DMEM supplemented with 10 mM HEPES, 2 mM L-glutamine, 10 % FBS, and 0.1 mg/mL normocin (Invivogen, San Diego, CA)) were transfected with plasmids pRSV-Rev (6.25 μg), pMDLg-pRRE (12.5 μg), pMD2.G (9 μg), and CAR-encoding pCCL (30 μg) (kindly provided by Dr. Megan Levings, University of British Columbia, Vancouver, BC) using Lipofectamine 2000. After overnight incubation, media was replaced and supplemented with 1 mM sodium butyrate (Sigma-Aldrich). 30 h later, supernatants were harvested, filtered (0.45 μm), and concentrated (4 °C, 1 hr 40 min, 1.3 × 105rcf). Viral stocks were resuspended in PBS and stored at −80 °C. Thawed virus aliquots were titrated by serial dilution and transduction of HEK 293 T cells to determine transduction units per milliliter.
Transduction of murine T cells
Female BALB/c and C57BL/6 mice were purchased from Charles River Breeding Laboratory (Wilmington, MA). All of our investigations have been approved by the McMaster Animal Research Ethics Board. To generate murine T lymphoctes for retroviral transduction, 3 × 106 freshly isolated splenocytes were cultured in 1 mL T cell media (RPMI supplemented with 10 % FBS, 2 mM L-glutamine, 10 mM HEPES, 0.5 mM sodium pyruvate, 0.1 mM non-essential amino acids, 55 μM β-mercaptoethanol, and 0.1 mg/mL normocin or 100U/mL penicillin + 100 μg/mL streptomycin) supplemented with 0.3 μg/mL α-CD3e (clone 145-2C11, Cat No. 553057, BD Pharmingen, San Diego, CA) and 400U/mL rhIL-2 (Cat No. 200–02, Peprotech, Rocky Hill, NJ). Twenty-four hours after activation, 600 μL of media were removed from T cell cultures and 100 μL of the concentrated retroviral supernatant was added, along with 2 μg/mL Lipofectamine 2000 and 1.6 μg/mL Polybrene (Sigma-Aldrich). Cultures were spun at 2000 rpm, 32 °C for 90 min, allowed to rest for 1–4 h, and were supplemented with 0.5 mL T cell media + 400U/mL rhIL-2. This process was repeated at 48 h after activation with the 72 h retroviral concentrates. Seventy-two hours after activation, retrovirally transduced T cell cultures were expanded into 30 mL of DC media + 400U/mL rhIL-2. Six to eight days after activation, resultant CAR-T cells were enumerated for use in vitro.
Transduction of human T cells
Peripheral blood mononuclear cells from healthy donors were obtained using Ficoll-Paque-Plus (GE Healthcare, Baie d’Urfe, QC) separation. This research was approved by the McMaster Health Sciences Research Ethics Board that operates in compliance with the ICH Good Clinical Practice Guidelines, the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans, Division 5 Health Canada Food and Drug Regulations, and the Helsinki Declaration. All donors in this study provided informed written consent. 1 × 105 cells were activated with anti-CD3/CD28 beads at a 1:1 ratio (Dynabeads, Cat No. 11131D, Life Technologies) in a 96-well round bottom plate (cultured in T cell media) with 100U/mL rhIL-2 and 10 ng/mL rhIL-7. Twenty-four hours after activation, T cells were transduced with lentivirus at an MOI of 1:1. CAR-T cell cultures were expanded into fresh media (T cell media supplemented with 100U/mL rhIL-2 and 10 ng/mL rhIL-7) as required for a period of 10–15 days prior to enumeration and use in vitro.
Detection of CAR constructs on the surfaces of murine or human T lymphocytes was determined by indirect immunofluorescence with HER2Fc chimeric protein (Cat No. 1129-ER-050, R&D Systems, Minneapolis, MN) followed by a phycoerythrin (PE)-conjugated goat anti-human IgG (Cat No. 109-115-098, Jackson ImmunoResearch, West Grove, PA) or anti-myc-tag (clone 9B11, Cell Signaling Technology, Danvers, MA) followed by a PE-conjugated goat anti-mouse IgG (Cat No. 115-116-146, Jackson ImmunoResearch). Cell surface phenotyping of murine CAR-T cells was determined by direct staining with AlexaFluor(AF)700-conjugated anti-CD8a (clone 53–6.7, eBioscience Inc., San Diego, CA), PerCP-Cy5.5-conjugated anti-CD8a (clone 53–6.7, BD Pharmingen), PE-conjugated anti-CD8b (clone H35-17.2, BD Pharmingen), PE-conjugated anti-CD90.1 (Thy1.1, clone OX-7, BD Pharmingen), and/or fluorescein isothiocyanate (FITC)-conjugated anti-CD90.1 (clone HIS51, eBioscience Inc.). Cell surface phenotyping of human CAR-T cells was determined by indirect staining with HER2Fc chimeric protein (as for murine T cells) and direct staining with PE-CF594-conjugated anti-CD271 (NGFR, clone C40-1457, BD Biosciences), and AF700-conjugated anti-CD8a (clone OKT8, eBioscience Inc.). All flow cytometry was conducted on a BD FACSCanto or BD LSRII cytometer (BD Bioscience) and analyzed using FlowJo vX.0.7 software (FlowJo, LLC, Ashland, OR, USA).
Functional analysis of CAR-T cells following stimulation with recombinant protein
106 murine or human CAR-T cells were stimulated in round bottom tissue culture treated 96-well plates coated with 200 ng HER2Fc chimeric protein (Cat No. 1129-ER-050, R&D Systems) or 200 ng KDRFc chimeric protein (Cat No. 443-KD, R&D Systems) for 4 h at 37 °C. Protein transport was inhibited according to the BD Golgi Plug protocol (Cat No. 555029 BD Biosciences, San Diego, CA). Production of activation cytokines was determined by flow cytometry. Cells were stained for surface phenotype markers as above. To permit intracellular cytokine staining (ICS), CAR-T cells were fixed and permeabilized according to BD Cytofix/Cytoperm Fixation/Permeabilization Kit protocol (Cat No. 554714, BD Biosciences). ICS of murine CAR-T cells was conducted by direct staining with allophycocyanin (APC)-conjugated anti-IFN-γ (clone XMG1.2, BD Pharmingen) and PE-cyanine(Cy)7-conjugated anti-TNF (clone MP6-XT22, BD Pharmingen). ICS of human CAR-T cells was conducted by direct staining with APC-conjugated anti-IFN-γ (clone B27, BD Pharmingen), FITC-conjugated anti-TNF (clone MAb11, BD Pharmingen), PE-conjugated anti-IL-2 (clone MQ1-17H12, BD Biosciences), and FITC-conjugated anti-CD107a (clone H4A3, BD Pharmingen). Analysis and presentation of distributions was performed using SPICE version 5.1, downloaded from . Comparison of distributions was performed using a Student’s T test and a partial permutation test as described , with a threshold of 0.09.
In vitro cytotoxicity assay
Adherent tumor cell lines were plated at 1.25 × 104 cells/well (D2F2, D2F2/E2, HCC1954, or LOX-IMVI) or 1.25 × 104 (human CAR-T cell cytotoxicity assay) or 2.5 × 104 cells/well (murine CAR-T cell cytotoxicity assay) (SKBR3) overnight in 96-well flat bottom tissue culture treated plates. Transformed murine or human T cell cultures were added to wells of tumor cells at various E:T ratios (from 0.1:1 to 6:1) and co-incubated together at 37 °C for 6 h. Murine T cells were added based on effectors defined as cells from day 6 to 8 transduced murine T cell cultures. Human T cells were added based on effectors defined as CAR-positive cells from day 15 transduced human T cell cultures. Wells were washed 3× with warmed PBS to remove any non-adherent cells. 100 μL of a 10 % solution of AlamarBlue cell viability reagent (Life Technologies) in T cell media was added and wells were incubated at 37 °C overnight. Colour change, indicative of live cells, was measured by fluorescence (excitation 530 nm, emission 595 nm) on a Safire plate reader (Tecan, Maennendorf, Switzerland). Tumor cell viability was calculated as the loss of fluorescence in experimental wells compared to untreated target cells. Each condition was tested in triplicate.
Student’s t tests, two-tailed, type two, were used to compare data between two groups. Results were prepared using Microsoft Excel 2010. Significant differences were defined as: * = p < 0.05, ** = p < 0.01, *** = p < 0.001; NS = not significant.
chimeric antigen receptor
- CAR-T cell:
chimeric antigen receptor transduced T cell
designed ankyrin repeat protein
single-chain variable fragment
This work was supported by funds from the Terry Fox Foundation and the Canadian Institutes for Health Research. JAH and HV were supported by scholarships from the Canadian Institutes for Health Research and the Government of Ontario. JAH was also supported by a scholarship from the Canadian Breast Cancer Foundation - Ontario Region.
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